( 12 ) united states patent ( 10 ) patent no . : us 10 ...clip.korea.ac.kr/uspatent.pdf · ( 12 )...

US010227592B2

( 12 ) United States Patent Chi et al .

( 10 ) Patent No . : US 10 , 227 , 592 B2 ( 45 ) Date of Patent : Mar . 12 , 2019

WO Wo

2009 / 044392 A2 2010 / 145778 A1 2011 / 085056 Al

4 / 2009 12 / 2010

7 / 2011 WO

OTHER PUBLICATIONS Cottonham et al ( J . Biol . Chem . 285 ( 46 ) : 35293 - 35302 , 2010 ) . * International Searching Authority , International Search Report of PCT / KR2014 / 011994 dated Mar . 13 , 2015 . Aimee L Jackson et al . , Expression profiling reveals off - target gene regulation by RNAi , Nature Biotechnology , Jun . 2003 , vol . 21 , No . 6 .

( 54 ) NUCLEIC ACID INDUCING RNA INTERFERENCE MODIFIED FOR PREVENTING OFF - TARGET , AND USE THEREOF

( 71 ) Applicant : ENCODEGEN CO . , LTD , Seoul ( KR ) ( 72 ) Inventors : Sung Wook Chi , Seoul ( KR ) ;

Eun - Sook Jang , Seoul ( KR ) ( 73 ) Assignee : ENCODEGEN CO . , LTD . , Seoul ( KR ) ( * ) Notice : Subject to any disclaimer , the term of this

patent is extended or adjusted under 35 U . S . C . 154 ( b ) by 0 days .

( 21 ) Appl . No . : 15 / 104 , 454 ( 22 ) PCT Filed : Dec . 8 , 2014 ( 86 ) PCT No . : PCT / KR2014 / 011994

$ 371 ( c ) ( 1 ) , ( 2 ) Date : Jun . 14 , 2016

( 87 ) PCT Pub . No . : W02015 / 093769 PCT Pub . Date : Jun . 25 , 2015

( 65 ) Prior Publication Data US 2016 / 0304878 A1 Oct . 20 , 2016

( 30 ) Foreign Application Priority Data

Aimee L . Jackson et al . , Widespread siRNA “ off - target ” transcript silencing mediated by seed region sequence complementarity , RNA , 2006 , pp . 1179 - 1187 , Cold Spring Harbor Laboratory Press . Amanda Birmingham et al . , 3 ' UTR seed matches , but not overall identity , are associated with RNAi off - targets , Nature Methods , Mar . 2006 , pp . 199 - 204 , vol . 3 , No . 3 . Xiaoyu Lin et al . , siRNA - mediated off - target gene silencing trig gered by a 7 nt complementation , Nucleic Acids Research , 2005 , pp . 4527 - 4535 , vol . 33 , No . 14 . Emily M . Anderson et al . , Experimental validation of the impor tance of seed complement frequency to siRNA specificity , RNA , 2008 , pp . 853 - 861 , vol . 14 , No . 5 , Cold Spring Harbor Laboratory Press . David P . Bartel , “ MicroRNAs : Target Recognition and Regulatory Functions " , Cell , Jan . 23 , 2009 , pp . 215 - 233 , vol . 136 , Elsevier Inc . Nitin Puri et al . , “ LNA incorporated siRNAs exhibit lower off - target effects compared to 2 - OMethoxy in Cell Phenotypic Assays and Microarray Analysis ” , Nucleic Acids Symposium Series , Sep . 8 , 2008 , pp . 25 - 26 , No . 52 , Oxford University Press . Jesper B . Bramsen et al . , A screen of chemical modifications identifies position - specific modification by UNA to most potently reduce siRNA off - target effects , Nucleic Acids Research , 2010 , pp . 5761 - 5773 , vol . 38 , No . 17 . Pooja Dua et al . , “ Modified siRNA Structure With a Single Nucleo tide Bulge Overcomes Conventional siRNA - mediated Off - target Silencing " , Molecular Therapy , Sep . 2011 , pp . 1676 - 1687 , vol . 19 , No . 9 . Sung Wook Chi et al . , An alternative mode of microRNA target recognition , Nat Struct Mol Biol . , Jan . 10 , 2013 , pp . 321 - 327 .

( Continued )

Dec . 17 , 2013 ( KR ) . . . . . . . . . . . . . . . . . . . . . . 10 - 2013 - 0157498 Dec . 4 , 2014 ( KR ) . . . . . . . . . . . . . . . . . . . . . . . 10 - 2014 - 0173038

( 51 ) Int . CI . C12N 15 / 113 ( 2010 . 01 ) A61K 31 / 712 ( 2006 . 01 ) A61K 31 / 7115 ( 2006 . 01 ) C12N 15 / 11 ( 2006 . 01 )

( 52 ) U . S . CI . CPC . . . . . . . . C12N 15 / 1137 ( 2013 . 01 ) ; C12N 15 / 111

( 2013 . 01 ) ; C12N 15 / 113 ( 2013 . 01 ) ; CI2N 2310 / 14 ( 2013 . 01 ) ; C12N 2310 / 141 ( 2013 . 01 ) ;

C12N 2310 / 31 ( 2013 . 01 ) ; C12N 2310 / 332 ( 2013 . 01 ) ; C12N 2310 / 344 ( 2013 . 01 ) ; CI2N

2310 / 531 ( 2013 . 01 ) ; CI2N 2310 / 533 ( 2013 . 01 ) ; CI2N 2320 / 53 ( 2013 . 01 )

( 58 ) Field of Classification Search None See application file for complete search history .

Primary Examiner — Richard A Schnizer ( 74 ) Attorney , Agent , or Firm — Sughrue Mion , PLLC

( 56 ) References Cited

U . S . PATENT DOCUMENTS

( 57 ) ABSTRACT Provided is an RNA interference - inducing nucleic acid comprising at least one nucleic acid strand , the at least one nucleic acid strand comprising a modification substituted to a spacer , which is unable to form a base pair , in the 5 ' end or the 3 ' end region . The RNA interference - inducing nucleic acid is a modified form of nucleotide provided to prevent off - target effects , offering a method to selectively repress target gene expression . The RNA interference - inducing nucleic acid provides modified forms with target selectivity and specificity as a method to block the off - target effects while silencing the target gene expression , whereas the usage of conventional RNA interference - inducing nucleic acids cause inaccuracy and adverse effects through off targets , thereby the RNA interference - inducing nucleic acid was offered to solve the problem , wherein it will be widely used as a method for repressing gene expression in research and for gene therapy without concerning the off - target effects .

7 , 595 , 387 B2 2007 / 0203084 A1 *

9 / 2009 Leake et al . 8 / 2007 Weiler . . . . . . . . . . . . . . . . C12N 15 / 111

514 / 44 A C12N 15 / 111

435 / 6 . 12 2012 / 0142011 A1 * 6 / 2012 Hahn . . . . . . . . . . . .

FOREIGN PATENT DOCUMENTS

CN 103140582 A 2007 - 531520 A 2011 - 500003 A 2013 - 511990 A

10 - 2011 - 0049733 A

6 / 2013 11 / 2007

1 / 2011 4 / 2013 5 / 2011

?? 16 Claims , 46 Drawing Sheets Specification includes a Sequence Listing . KR

US 10 , 227 , 592 B2

( 56 ) References Cited

OTHER PUBLICATIONS Nicole T . Schirle et al . “ The crystal structure of human Argonaute2 ” , Science , May 25 , 2012 , ( 6084 ) , pp . 1037 - 1040 , vol . 336 . Maria Frank - Kamenetsky et al . , “ Therapeutic RNAi targeting PCSKO acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates ” , PNAS , Aug . 19 , 2008 , pp . 11915 - 11920 , vol . 105 , No . 33 . Christopher D Armour et al . , “ Digital transcriptome profiling using selective hexamer priming for cDNA synthesis ” , Nature Methods , Sep . 2009 , pp . 647 - 649 , vol . 6 , No . 9 . Da Wei Huang et al . , " Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources ” , 2009 , Nature Protocols , pp . 44 - 57 , vol . 4 , No . 1 . Rui Shi et al . , “ Facile means for quantifying microRNA expression by real - time PCR ” , BioTechniques , pp . 519 - 524 , 2005 , vol . 39 , No . 4 .

Korea Intellectual Property Office , Office Action of Korean Appli cation No . 10 - 2014 - 0173038 dated Oct . 21 , 2015 . Korea Intellectual Property Office , Office Action of Korean Appli cation No . 10 - 2014 - 0173038 dated May 26 , 2016 . Japanese Patent Office ; Communication dated Jul . 11 , 2017 in counterpart Japanese application No . 2016 - 541157 . European Patent Office ; Communication dated Jun . 19 , 2017 in counterpart European application No . 14872036 . 0 . Australian Patent Office ; Communication dated Jun . 5 , 2017 in counterpart Australian application No . 2014367550 . State Intellectual Property Office of People ' s Republic of China , Communication dated Oct . 15 , 2018 in Counterpart Application No . 201480068785 . 4 .

* cited by examiner

FIG . 1

atent

No off - target effect

( 55 - SÍRNA , . . ) - 6pi

( SHRNA , DSIRNA , ISIRNA . . . ) - 6pi Dicer

Ago - SIRNA - 6pi

Mar . 12 , 2019

ACUCQUA

passenger strand

SS WXXE Seed region

* *

* * * *

*

* * * * *

Sheet 1 of 46

Ago ( Argonaute )

* * *

dSpacer ( @ , Pi )

US 10 , 227 , 592 B2

U . S . Patent Mar . 12 , 2019 Sheet 2 of 46 US 10 , 227 , 592 B2

FIG . 2a

1001 RL * Relative activity ( % )

D

NT WT 1pi 2pi 3pi 4pi 5pi 6pi 7pi Spi 9pi 10pilipi

( SEQ ID no . 3 , 4 ) ( SEQ ID no . 1 ) ( SEQ ID no . 2 )

FIG . 2b RL - Seed

Relative activity ( % ) +

Známsminnin

NT , WT 1pi 2pi 3pi 4pi 5pi 6pi 7pi 8pi 9pi 10pillpi ,

( SEQ ID no . 3 , 4 ) ( SEQ ID no . ) ( SEQ ID no . 2 )

FIG . 2c RL - Nuc Relative activity ( % ) L L . . . LI

* * * *

NT WT 1pi 2pi 3pi 4pi Spi 6pi 7pi 8pi



FIG . 2d

+ ( SEQ ID no . 1 ) ( SEQ ID no . 2 ) parvi * * * 3 * *

S?RL ~ ~ ~ ~ WT Relative activity ( % )

( SEQ ID no . 1 )

?????????????? - -

( NM ) 0 . 001 0 . 01 0 . 1 i 10 100 FIG . 2e

( SEQ ID 10 . 1 ) ( SEQ ID 110 , 2 )

SÍRLICOM ) Lx { % 6 ) WT . 0 . 012100 loi 0 . 035 95 2pi 0 . 42477 3pi 0 . 508 43 4pi 1120 68 Spi 0 . 382 years Opi 0 . 36178 70i NO 43 Bpi ND 55 Spi 0 . 175 42 10pi NID 137 Ilpi 0 . 212 45 |


FIG . 2f

SIRL Transitional nucleation Seed Nuc

8 . 6 2 86 2 3 ' w . QUGAGGAUG - - - 5 ' 3 ' waUGAGGAUG ~ ~ - 5 5 - - - ACUCCUAC - - - 3 ' 5 ' - - - ACUUCCUAC - - - 3 '

Pivot pairing ( Stable duplex )

siR - 6pi No nucleation 8 6 2

3 - aUGOGGAUG - 5 5 - UACUCCUAC 3 Unstable duplex

* * * * * * - - - - - - - - - - -

Nonproductive complexes ( No off - target effect )

3 ' - naUGAGGAUG - 5 ' 3 ' - AUGAGGAUG - - 5 EN 5 - - - ACUCCUAC - - - 3 S - - AQUCCUAC - - - 3 "

Post - transcriptional inhibition ( Off - target effect )

FIG . 2g

RL - Seed * * * *

* * I Relative activity ( % )

17 +

NT WT 1pi 2pi 3pi 4pi 5pi 6pi 7pi Spi


FIG . 3a

U . S . Patent

R1 - 0

Oznamy OH ( SEQ ID no . 1 ) OH Oy

rSpacer ( pi - r )

( SEQ ID no . 2 )

R2 - 0 OH

with more than onde en hom * *

* * * * * * * *

Mar . 12 , 2019

AS JANUARY Hodine

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Relative activity ( % )

1 .

www . . . WT van wis um 3pi - r em v am - 4pi - r

5pi - r - - - - 7pi - r Opi

M24

Sheet 5 of 46

-

elem

. - A

wel

- 3

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Concentration ( log 10 ( M ) )

2

US 10 , 227 , 592 B2

FIG . 36

U . S . Patent

( SEQ ID no . 1 ) ( SEQ ID no . 2 )

100

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SiRL - - - - - - WT - - - - - - - 2pi - r

* * *

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Mar . 12 , 2019

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Concentration ( log 10 ( nM ) )

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US 10 , 227 , 592 B2


FIG . 3c 4994999

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MW

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| siR ICsonM ) Inax ( % ) WT330 . 013 1005 2pi - r 0 . 41 1 94 3pi - NA 57 4pi - r 0 . 99 68 5pi - r 4 . 01 T 54 Opi - r 3 . 17 T 70 7pi - 1 . 91 T 58 6pi 0 . 4178


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FIG . 3d

Luc - Seed

THAI Relative activity ( % ) hacen memand

207 WT ( 1C5o = 0 . 10nM ) www . mn - - - 2pi - r ( 1C50 = 0 . 50 nM )

Concentration ( log 10 nM ) ) ( SEQ ID no . 1 ) SEO ID no . 2 )


FIG . 3e

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FIG . 4a

70 2 SiRNA ( SiRL ) - 6pi - I 3 ' dTdTCGGAAAGUGAUGA GGAUG 5

( SEQ No . 1 ) : : : : : : : : : : | | | IIT : 5 - - - - - - - - - - GGCCUUUCACUACU CCUAC - - - - 3 ' RL target mRNA ( on - target effect )

FIG . 45

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FIG . 40

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US 10 , 227 , 592 B2


FIG . 4d



XXXW3 - ~ ~ ~ ~ - WT ww ws - 2pi - I

- 3pi - 1 Har * * * *

# VA !

Opi

PU Concentration ( log 10 ( M ) )


FIG . 4e

RL - Seed

- - * *

wirinin Relative activity ( % ) Med . pw to

w what - ~ ~ ~ - - WT - ~ ~ - - - 2pi - I vevo . . . 3pi - I

- 6pi + ( SEQ ID no . 1 )

( SEO ID no . 2 ) www

- 2 1 - 1 Concentration ( log ( nM ) )


FIG . 4f

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FIG . 5a

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- 6pi

2oT Concentration ( log ( nM ) )


FIG . 5b

apparemmesenger ( SEQ ID no . 1 ) ( SEQ ID no . 2 ) - * * * * * * * * 019

z esss to rescue Relative activity ( % ) * * * * * *

WT - 2pi - r1 3pi - ri

- 6pi . . .

- 2 - 1 0 Concentration ( log ( nM ) )


FIG . 5c

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0 . 38 3pi - 0 . 23 88 4pi - r 5 . 194 9 Spi - r N / D 36 6pi - 0 . 49 55 7pi - r 0 . 35 26 6pi X 0 . 41 78


FIG . 5d

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1 Concentration ( log ( nM ) )

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Relative activity ( ratio )

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FIG . 5e

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FIG . 6a


lete Luc - Seed

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a RAI Relative repression ( % ) miR - 124 80 | 100

| o iz opi ?????????????????? ????????????? muri

vi

It VY 124 6pi ( IC50

- 5 . 0nM )

i 10 Sonm )

( nm ) 0 . 01 0 . 1 100


FIG . 6b


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( P < 0 . 01 ) Control

( SEQ ID No . 3 , 4 ) Number of transcripts thote

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5 - 2 0 1 Fold change ( log2 )


FIG . 6c .

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FIG . 6d

Ago - miR - 124 ( Seed ) - - - - - . .

. . .

-

miR - 124 Ago / seed

. Total ( P < 0 . 01 )

7 Cumulative fraction - - - - -

S

.

( SEQ ID no . 5 ) ( SEQ ID no . 6 ) miR - 124 - 6pi

- - - Ago / seed - - - - - - - - Total

- -

-

- omnin o e arrepentimiento amamos M

- 1 . 5 0 0 . 5 Fold change ( log )


FIG . 6e

* * * * * * * * * * * * * * * * ' 111

* * * * * * * * * * * * * *

2 ' OME * - - - - RL www . mm Opi


: # 444444 : ? * * 4544

* * * * * * * * * * * * from

mwen log ( nM ) - 3 - 2 - 1 0 1 2


FIG . 6f : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

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: : : : : : : : : : : : : : : : : : : : : : : : : : : : : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

: : : : : : : : : : : : : : : : i . . . . . . . . . . . . . . . . i iii . . . . . . . . . . . . . . : : . : . . . .

i iiiiiii . . . . . . . . . : : : : : : : : : : : : : : H : : : : : : : : : : : : : : : : : : : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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. . : : : : : : : : : : : : : : : : : : : : :

: : : : :

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: : : : : : : : : : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . .

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: : : : : : : : : : : : : : : : : :

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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : : : : : : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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. . . .

: : :

DOOD . .

: : . : : : : : : : : : : : : : :

: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

( SEQ ID no . 1 ) ( SED ID no . 2 ) o

: : : : : : : : : : :

: : : : : : : : : : : : : : : : : : : : :

2

: : : : : : : : : :

( SEQ ID no . 3 , 4 ) : : : : : :

: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

:

: : :

: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

* * * : : : : : : * * *

: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

tent Mar . 12 , 2019 Sheet 24 of 46 US 10 , 227 , 592 B2

: : : :

miR - 124 - 2 ' 0Me PES . . . . . . . .

: .

: : : : : : :

:

miR - 124 - 6pi ( SEQ ID no . 5 ) ( SEQ ID no . 6 )

0 : : :

: :

FIG . 6g . . . . . . . . . .

. . .

. . . . .

. . . . .

miR - 124

soom2 Control ( NT ) ( SEQ ID no . 3 , 4 )

31111111


FIG . 7a


+ + + + + + + + + + + + + + + + + + + RL - PCSK9

VVVV Relative activity www WWW

A Al - 2pi A A1 - 3pi I A1 - 4pi • Al - 5pi O A1 - 6pi

- -

. - - * -

( nm ) 0 . 0001 0 . 001 0 . 01 FIG . 75


Al - 5pi

1 Relative activity .

RL - Seed 20

( nM ) 0 . 001 0 . 1


FIG . 7c

( SEQ ID no . 9 ) ( SEQ ID no . 10 ) A2 - 6pi WA

W

V V V V V V V

V V V V V

V V V Relative activity V V V

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T U VIVAVAVAVAVAVAVVIVIVIV V V V V V V

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HAAHHHHHHH www .

Relative activity O A2 - 6p O A2

H L 1

SNEY RL - PCSK9 D

( nM ) 0 . 0001 0 . 01 ( SEQ ID no . 9 ) ( SEQ ID no . 10 )


FIG . 7e

HepG2 I PCSK9

Relative exp MY

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FIG . 7f : : : : : : : : : : : : : : : : : :

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: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

w : : : : : : :

( Log ratio )

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: :

: : : : : : : : : : : : :

: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

HepG2 ( SEQ ID no . 9 ) ( SEQ ID no . 10 ) ( SEQ ID no . 3 , 4 )

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FIG . 7g Control www

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A2

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FIG . 71

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FIG . 8a

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FIG . 8c


Total cholesterol

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FIG . 8d ( SEQ ID no . 3 , 4 ) ( SEQ ID no . 9 ) ( SEQ ID no . 10 )

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om o ( Log ratio )

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: : : : : : : : : : : : : : : : : : : Liver : : : : : : : 15


FIG . 8e

30 ( ug / dl )

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( SEQ ID no . 10 )


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FIG . 8f intendente en intendente de

: : : : : : : : : : : : : : de

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letteralmente dalla

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intervallen in den lettere delle tradition de la . .

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tendente dentro delle interior de ( SEQ ID no . 3 , 4 )

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FIG . 9a

we

( SEQ ID no . 11 ) ( SEQ ID no . 12 ) miR - 124 5 ' - UAAGGCACGCGGUGAAUGCDTDT - 3 '

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MULTUMITTUNT hect 3 ' - AUUCCGUGCGCCACUUACG - 5 ' match target

AAAAAAAA

FIG . 9b

Perfect match W

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( SEQ ID no . 13 ) ( SEQ ID no . 14 )

nergie

( nm ) 0 . 01 0 . 1 i 10 100

atent Mar . 12 , 2019 Sheet 35 of 46 US 10 , 227 , 592 B2

FIG . 9c

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FIG . 9d

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100 + 15

Relative activity ( % ) I ( SEQ ID no . 11 )

( SEQ ID no . 12 )

w . 1 - - - * * miR - 124

miR - 124 - 6mm ( nM ) Ó 0 . 01 0 . 1 40

i 10 100 ( SEQ ID no . 13 ) ( SEQ ID no . 14 )


FIG . 9e

( SEQ ID no . 13 ) miR - 124 - 6mm 5 - UAAGGGACGCGGUGAAUGCDTDT - 3 ' miR - 124 - 6mm seed target 3 . UUCCCUG - - - - - - - - - - - - - - - 5 % * * * * * * * * W

FIG . 91

miR - 124 - 6mm seed target

?

Relative activity ( % ) ( SEQ ID no . 11 ) ( SEQ ID no . 12 )

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FIG . 9g

44 me ( SEQ IDNO : 11 )

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W

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20 MAMLAKAMLAR A MAMAMAN LA L LATARAAMAAAAAMA internet * * * * MAMA * * * * * * * * * * * * * * * * * *

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FIG . 9i

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Relative activity ( % ) miR - 124

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7UNA 1 * I * ( NM ) Ó 0 . 01 0 . 1 i 10 100


FIG . 9j

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VIRAL


X

Y w

PCSK9 - A2 * *

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( nM ) • 0 . 01 0 . 1 i 10 100

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FIG . 10

Complete elimination of off - target effect ( No off - target effect )

SIRNA - 6Spacer

Mar . 12 , 2019

passenger strand

( SEQ D 00 . 2 )

( SEQ ID no1 ) wwwwwwwww

Efficient preservation of on - target effect ( On - target effect )

Sheet 40 of 46

( SEQ ID no . 1 ) . 76 3dTATCCGGAAAGUGAUG Z GGAUGS GGCCUUUCACUAC U CCUAC -

S

3

US 10 , 227 , 592 B2


FIG . 11a 5 - Oligo www . mo

o = poo O = P . . . mw Oligo - 3 '

OH C3 Spacer ( 2 )

FIG . 11b

( SEQ ID no . 11 ) 5 mUAAGGCACGCGGUGAAUGCDTDT - 3 ' he 3 ' - dTDTAUUCCGUGCGCCACUUACG - 5 '

( SEQ ID no . 12 ) 5 ' - UAAGGEACGCGGUGAAUGCDTDT - 3 ' mk - 124 - 6CS 3 . dTdTAUUCCGUGCGCCACUUACG - 54

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US 10 , 227 , 592 B2

NUCLEIC ACID INDUCING RNA position 2 from the 5 ' end region , but the silencing of the INTERFERENCE MODIFIED FOR intended target gene is also somewhat reduced . Since then ,

PREVENTING OFF - TARGET , AND USE another kinds of modifications such as LNA modification THEREOF ( Puri et al . , Nucleic Acids Symp . Ser 0 . 2008 ) , UNA modi

5 fication ( Bramsen et al . , Nucleic Acids Res . 2010 ; 38 , CROSS REFERENCE TO RELATED 5761 - 73 ) , and bulge modification introducing a single

APPLICATIONS nucleotide bulge ( Mol Ther . 2011 September ; 19 ( 9 ) : 1676 87 ) also have been developed .

This application is a National Stage of International However , all such chemical modifications were applied to Application No . PCT / KR2014 / 011994 filed Dec . 8 , 2014 , 10 a nucleotide backbone rather than to a base of which claiming priority based on Korean Patent Application Nos . sequence is critical to cause off - targets , unable to affect a 10 - 2013 - 0157498 filed Dec . 17 , 2013 and 10 - 2014 - 0173038 fundamental base - pairing . Because of this reason , they can filed Dec . 4 , 2014 , the contents of all of which are incor not completely block off - target effect albeit somewhat can porated herein by reference in their entirety . be reduced , also having a problem to reduce efficiency of

TECHNICAL FIELD on - target silencing .

DISCLOSURE OF THE INVENTION The present invention relates to an RNA interference inducing nucleic acid and the use thereof , and more particu larly to an RNA interference - inducing nucleic acid compris - 20 Technical Problem ing at least one single strand of double strands , the at least one single strand comprising a modification substituted to a Accordingly , the present inventors have developed modi spacer in the 5 ' end and the 3 ' end region . fied RNA interference - inducing nucleic acids to overcome

the problems of conventional methods , wherein the modified BACKGROUND ART 25 RNA interference - inducing nucleic acids can completely

block the off - target effects while enhancing efficiency of The siRNA ( small interfering RNA ) has been widely used repressing target gene , thereby have completed the present

as a method to repress expression of a desired target gene invention . through RNA interference , but it also causes non - specific An object of the present invention is to provide an RNA repression of other genes , off target effects , as an inevitable 30 interference - inducing nucleic acid comprising at least one disadvantage , raising the serious concern of leading to faulty single strand of double strands , the at least one single strand research results or side effects in therapeutic treatments . The comprising a modification in which at least one of off - target effects are occurred since Argonaute protein , the the sixth nucleotide from the 5 ' end , and core effector in RNA interference , treats siRNA , which is each of the first and second nucleotides from the 3 ' end is artificially introduced in order to induce RNA interference , 35 substituted with a spacer . as a miRNA ( microRNA ) existing in a cell . Therefore , it is Another object of the present invention is to provide a called miRNA - like off - target effect . The miRNA recognizes gene silencing composition comprising the RNA interfer a target gene majorly through base - pairing with a seed ence - inducing nucleic acid . region ( positions 2 - 7 from the 5 ' end ) for suppression , and Another object of the present invention is to provide a the off - targets caused by siRNAs are also induced depending 40 gene silencing kit comprising the RNA interference - induc on sequences of the seed regions as well . The miRNA - like ing nucleic acid . off - target effects in siRNAs have been already reported in Another object of the present invention is to provide a several studies ( Jackson , A . L . , et al . , Nat . Biotechnol . , method for target gene silencing in a cell , the method 21 ( 6 ) : 635 , 2003 ; Jackson , A . L . , et al . , Rna , 12 ( 7 ) : 1179 , comprising a step of introducing or expressing the RNA 2006 ; Birmingham et al . , Nat . Methods , 3 ( 3 ) : 199 , 2006 ; Lin 45 interference - inducing nucleic acid into the cell . et al . , Nucleic Acids Res . , 33 ( 14 ) : 4527 , 2005 ; Anderson et Another object of the present invention is to provide a al . , RNA , 14 ( 5 ) : 853 , 2008 ) , and affect expression of at least method for suppressing off - target effects mediated by a hundreds and at most ~ 1500 of genes depending on guide strand or a passenger strand of the RNA interference sequences of the seed regions and are serious enough to inducing nucleic acid , the method comprising a step of cause up to 30 % of the positive hits in siRNA based 50 introducing or expressing the RNA interference - inducing phenotype screening . Additionally , in the case of miRNAs , nucleic acid into a cell . they are also reported to silence target genes through com - However , the object of the present invention is not limited pensatory pairings within their 3 ' end regions ( 3 ' - compen to the objects discussed above , and other objects which are satory pairing ) when the interactions between seed regions not discussed above may be clearly understood from the and targets become weak ( Cell . 2009 ; 136 : 215 - 233 ) , impli - 55 following disclosures . cating that the miRNA - like off - target effects are likely to be mediated by such mechanism . Technical Solution

In addition , due to such widespread off - target silencing effects mediated by siRNAs , several chemical and structural To achieve the objects above , the present invention pro modifications have been attempted to reduce the off - target 60 vides a RNA interference - inducing nucleic acid comprising silencing while maintaining the efficiency of suppressing an at least one single strand of double strands , the at least one intended target . A modification adding methyl groups to a 2 single strand comprising a modification in which at least one position of a ribosyl ring of the nucleotide ( 2 ' OMe ) was of the sixth nucleotide from the 5 ' end and each of the first studied and used by Dharmacon Research ( Lafayette , Colo . ) and second nucleotides from the 3 ' end is substituted with a to suppress the off - target effects , initially found to be effec - 65 spacer . tive in reducing both the number of off - targets and the extent In an embodiment of the present invention , the at least one of off - target effects especially when the 2 ' OMe is at a single strand may bind to Argonaute protein in the same

US 10 , 227 , 592 B2

manner as ss - siRNA ( single strand siRNA ) which exists as DESCRIPTION OF DRAWINGS a single strand and induces RNA interference .

In another embodiment of the present invention , the FIG . 1 is a diagram showing the activity of silencing spacer may be a compound which affords to maintain the on - target gene expression and suppressing miRNA - like off space of a nucleotide , preferably an organic compound , and 5 target effects by deoxyribonucleotide spacer ( dSpacer ) more preferably a hydrogen carbon chain containing a modification . phosphoryl group or a sulfuryl group , wherein the hydrogen FIGS . 2a , 2b , 2c , 2d , 2e , 2f , and 2g show the effects of carbon may be an alkyl group having 3 carbons ( C3 Spacer ) . modified siRNAs with deoxyribonucleotide spacer

In another embodiment of the present invention , the ( dSpacer ) substitution on gene silencing activity and spacer may be one that cannot base - pair to any other er 10 miRNA - like off - target effects . physiological base , wherein the spacer may be an abasic FIGS . 3a , 3b , 3c , 3d and 3e show the effects of modified

siRNAs with ribonucleotide spacer ( rSpacer ) substitution on deoxyribonucleotide ( dSpacer ) or an abasic ribonucleotide gene silencing activity and miRNA - like off - target effects ( rSpacer ) as a backbone . FIGS . 4a , 45 , 46 , 4d , 4e and 4f show the effects of In another embodiment of the present invention , the RNA 15 modified siRNAs with deoxyribonucleotide spacer interference - inducing nucleic acid may further comprise a t as ( dSpacer ) insertion on gene silencing activity and miRNA mismatch base pairing with an RNA of a target gene by a like off - farget effects substitution or a bulge by an insertion . FIGS . 5a , 56 , 5c , 5d and 5e show the effects of modified In another embodiment of the present invention , the RNA siRNAs with ribonucleotide spacer ( rSpacer ) insertion on interference - inducing nucleic acids may include any nucleo - 20 gene silencing activity and miRNA - like off - target effects . tide inducing RNA interference such as siRNA , miRNA , FIGS . 6a , 6b , 6c , 60 , 6e , 6f and 6g show the effects of a shRNA , DsiRNA , IsiRNA , SS - SiRNA , asiRNA , piRNA , and modified siRNA molecule , which contains deoxyribonucle endo - siRNA . otide spacer ( dSpacer ) substituted for sixth nucleotide from

In another embodiment of the present invention , as in the 5 ' end ( 6 pi ) , on function of miRNA - mediated target miRNA ( microRNA ) , in a case where a complementary 25 gene regulation , compared with the effect from conventional target gene to a whole sequence of the RNA - interference - siRNA modified with 2 ' OMe in position 2 from the 5 ' end . inducing nucleic acid is not existed in vivo , wherein the FIGS . 7a , 76 , 7c , 7d , 7e , 71 , 7g . 7h and 7i show the results RNA - interference - inducing nucleic acid may have no func - evaluating off - target effects in cells with PCSK9 siRNAs tion , and in such a case , the RNA interference - inducing containing the modification of the present invention , 6 pi .

30 FIGS . 8a , 86 , 8c , 8d , 8e and 8f show the results evaluating nucleic acid may be provided as a control . off - target effects in vivo with PCSK9 siRNAs containing the In another embodiment of the present invention , the target gene which the RNA interference - inducing nucleic acid is to modification of the present invention , 6 pi .

FIGS . 9a , 96 , 9c , 90 , 9e , 9f , 9g , 9h , 9i and 9j show the repress can be of any genes , which can be a coding or non - coding gene mediated by RNA , transcribed in an organ - 35 SiRNAs having mismatch base - pairing , 2 ' OMe modification

result comparing the present invention with conventional ism including virus . and UNA modification in position 6 from the 5 ' end and In another embodiment of the present invention , the RNA siRNA duplex with introduction of bulge in position 2 , interference - inducing nucleic acid may be used for silencing examining the effect on gene silencing activity and miRNA target gene expression , and the double strand may be a like off - target effects product from artificial synthesis or a further processed 40 FIG . 10 shows a diagram for the single strand from the product from in vivo modification . RNA interference - inducing nucleic acid , where the position

The present invention provides a compound and / or a kit 6 from the 5 ' end is substituted to a spacer , wherein the containing the RNA interference - inducing nucleic acid for spacer is a hydrogen carbon chain comprising at least 3 silencing gene expression . carbons , alkyl group , affecting activity of repressing gene

Additionally , the present invention provides a method for 45 expression and suppressing miRNA - like off - target effects . silencing target gene expression in a cell , the method com - FIGS . 11a , 11b , 11c , 11d , 11e , 11f , 11g , 112 , 11i and 11 ; prising a step of introducing or expressing the RNA inter show the results for siRNA of which position 6 from the 5 ' ference - inducing nucleic acid into the cell . end is substituted to the spacer with an alkyl group ( C3

Furthermore , the present invention provides a method for spacer ) , wherein activity of repressing gene expression and suppressing off - target effects mediated by the guide strand or 50 miRNA - like off - target effects are evaluated . the passenger strand of the RNA interference - inducing FIG . 122 , 126 , 12c , 120 , 12e , 12f , 12g and 12h represents nucleic acid , the method comprising a step of introducing the results for the modified siRNA of which positions 1 - 2 the RNA interference - inducing nucleic acid . from the 3 ' end are substituted to the spacer , wherein activity Moreover , the present invention provides a method for of repressing gene expression and miRNA - like off - target

suppressing off - target effects mediated by the guide strand or 55 effects mediated by 3 ' - compensatory pairing are evaluated . the passenger strand of the RNA interference - inducing nucleic acid , the method comprising a step of expressing the MODE FOR INVENTION RNA interference - inducing nucleic acids in a cell .

The present invention is to provide an RNA interference Advantageous Effects 60 inducing nucleic acid comprising at least one single strand

of double strands , the at least one single strand comprising The present invention provides a RNA interference - in - a modification in which at least one of the sixth nucleotide

ducing nucleic acid , wherein the RNA interference - inducing from the 5 ' end and each of the first and second nucleotides nucleic acid contains a novel modification to prevent the from the 3 ' end is substituted with a spacer . off - target effects caused by RNA interference for silencing 65 The present inventors have studied the RNA interference target gene expression , thereby offering a method to selec - mediated by small RNAs to find a method to completely tively repress expression of a target gene . prevent the off - target effects while efficiently repress a target

US 10 , 227 , 592 B2

gene of interest , worked based on sequence specificity , In another embodiment of the present invention , the wherein they validated as a result that a modification of the off - target effects of PCSK9 siRNA was found to cause 5 ' end region of single strand from the RNA interference - adverse side effects , in that it induces cell cycle stop in inducing nucleic acid to a spacer , which contains covalent human liver cell , HepG2 , and cell death in mouse liver tissue bonding with no base such as a single abasic nucleotide , 5 caused by the defect in copper metabolism , whereas such showed a specific to block miRNA - like off - target effects and adverse side effects mediated by off - targets was shown to be to efficiently repress the target gene , wherein a modification eliminated in siRNA with the modification of the present of the 3 ' end to contain the spacer , which is also the covalent invention , observed as a result of examining the side and bond with no base such as a single abasic nucleotide , also off - target effects , thereby confirming the elimination of both showed a specificity to block miRNA - like off - targets medi - 10 the adverse side effects and the off - target effects and also the ated by 3 ' - compensatory pairing but to effectively silence conservation of effective silencing activity to an intended target gene , thereby completed the present invention . target , PCSK9 .

In other words , in an embodiment of the present inven - In another embodiment of the present invention , intro tion , the spacer modification in the position 6 from the 5 ' end duction of a mismatch in seed region , a conventional method eliminates off - targets while keeping the best efficiency of 15 proposed to reduce miRNA - like off - targets , was applied to silencing the target gene . The transitional nucleation model position 6 from the 5 ' end of miR - 124 , one of miRNAs ( Nat Struct Mol Biol . 2012 Feb . 12 ; 19 ( 3 ) : 321 - 7 ) , elucidated recognizing and repressing target genes through the mode of by previous research of the present inventors , well accom - seed pairing , showing reduction of off - target effects in the modates these results , demonstrating that the position 6 from validation , whereas the sequence change introduced by the the 5 ' end , called " pivot ” , is a critical element to recognize 20 mismatch responded to new matches to the altered seed , still miRNA - like off - targets . recognizing and silencing new targets showing adverse side

In addition , expanding from using the abasic nucleotide , effects , wherein the same observation of showing the side a nucleotide in position 6 is substituted to a spacer , wherein effects was also confirmed even in the case of introducing the spacer only has a form of covalent bonding between fifth 2 ' OMe modification to position 6 from the 5 ' end , wherein and sixth nucleotide , thereby validated to perform the best 25 the 2 ' OMe modification is a conventional method for reduc efficiency in silencing target gene without causing off - target i ng off - targets . effects . In another embodiment of the present invention , it was

In other words , in examples of the present invention , the validated that all conventional modification methods for spacer modification in the position 6 from the 5 ' end suppressing off - target effects are unable to completely block completely block off - target effects , wherein a deoxyribo - 30 off - target effects , but the present invention can completely nucleotide spacer ( dSpacer ) shows better performance in abrogate off - target effects , wherein the conventional modi on - target activity where the dSpacer is relatively smaller fication methods were 2 ' OMe modification in position 2 than an rSpacer due to the absence of oxygen in 2 ' position , from the 5 ' end , UNA modification in position 7 from the 5 ' thereby the smallest spacer as an extreme case , alkyl spacer end , and introduction of one single nucleotide bulge in ( C3 Spacer ) was validated to perform on - target effect of 35 position 2 from the 5 ' end of double - stranded si RNA . which activity is similar to the activity of the unmodified In another embodiment of the present invention , it is form . Such superior on - target effect , shown by the small validated that spacer substitution of sixth nucleotide from spacer located in position 6 from the 5 ' end , is well matched the 5 ' end , mediating the covalent bonding between fifth and with the previous report of Ago - miRNA complex structure seventh nucleotide , completely abrogates off - target effects ( Schirle et al , Science 2012 , 336 , 1037 - 40 ) and with the 40 while keeping silencing of a target gene . transitional nucleation model , in that , in order to silence I n another embodiment of the present invention , complete targets , miRNA undergoes structural and functional changes abrogation of miRNA - like off - targets mediated by 3 ' end after making base - pairing up to position 6 from the 5 ' end compensatory pairing was observed along with superior region . silencing activity for gene expression , when first and second

In another embodiment of the present invention , the 45 nucleotides from the 3 ' end were substituted to abasic modification of the present invention was practically applied spacers . to miR - 124 , one of miRNAs functioning with seed - mediated As stated above , the present invention may provide the target recognition , showing abrogation of miRNA - depen - RNA interference - inducing nucleic acid to silence target dent target repression and lost in function inducing neuronal gene , wherein at least one strand from the double - stranded differentiation as a result , whereas a conventional 2 ' OMe 50 RNA interference - inducing nucleic acid contains a spacer modification , developed by Dharmacon company for reduc substituted for a nucleotide in position 6 from the 5 ' end , ing the off targets , was unable to block miR - 124 function , where nucleotides in position 5 and 7 have covalent bonding induction of neuronal differentiation , wherein its effect on with the spacer , or contains abasic nucleotides or spacers reducing off - targets is very modest compared with the substituted for nucleotides in positions 1 - 2 from the 3 ' end . present invention , confirmed by genome - wide examination 55 In the disclosure above , the RNA interference - inducing of transcript expression . nucleic acid prefers to form double strand , comprising of

In another embodiment of the present invention , the ~ 18 - 23 nucleotide - long guide strand and the passenger modification of the present invention was applied to siRNA strand complementary to the corresponding guide strand , for suppressing renilla luciferase or PCSK9 as a target gene wherein the most appropriated form is 21 nucleotides , but and on - target and off - target effects were examined , wherein 60 not limited to this . Herein , the double strand can be derived it is validated to show excellent performance in blocking from a stem - loop hairpin structure , of which stem part is off - target effects mediated by the guide strand and increased processed to the double strand by Dicer protein , wherein the selectivity to the target . Especially , in the case of the double strand is siRNA processed from shRNA , varied application for PCSK9 siRNA , which is for reducing plasma depending on strictures of nucleotides , thus not limited to cholesterol level , its therapeutic effect is validated to cause 65 only these cases . In addition , to achieve optimal suppression effective reduction of cholesterol level in mouse animal of off - target effects , it can include two abasic nucleotides in model as much as in the unmodified siRNA . 3 ' end overhang , mismatch base - pairing to RNA of target

US 10 , 227 , 592 B2

gene by the substitution , or bulge formation by the insertion . access to the contents by a syringe needle . The kit may The term “ bulge ' refers to a portion in a double - stranded comprise an exterior package which may include instruc nucleotide , which is not paired and is gaped open due to the tions regarding the use of the components . introduction of one or more nucleotides , and ‘ mismatch Additionally , according to the present invention , the RNA pair ' generally refers to base pair which cannot make 5 interference - inducing nucleic acid effectively represses Watson - Crick base pairing . expression of a target gene , validated by the examples of the

In the present invention , the phrase ' guide strand ' ( anti - present invention , thereby providing a method for silencing sense strand ) refers to the single strand , which is a part of expression of the target gene into a cell , wherein the method said double strand , determined to have sequence for silenc - comprises a step of introducing the RNA interference ing a target , wherein the guide strand majorly loads onto the 10 inducing nucleic acid into the cell , wherein also providing a Argonaute protein , plays role in guiding Argonaute complex method for silencing expression of the target gene into a cell , to recognize target gene , and is a polynucleotide that is wherein the method comprises a step of expressing the RNA substantially or 100 % complementary to mRNA of target interference - inducing nucleic acid into the cell . gene of interest , thereby it is also called “ antisense strand ” , In the present invention , the target gene can be either wherein the guide strand can be the complementary poly - 15 endogeneous gene or transgene , but not limited thereto . nucleotide as a whole or in part to siRNA , miRNA , shRNA , Moreover , according to the present invention , the RNA DsiRNA , IsiRNA . Ss - siRNA , piRNA , endo - siRNA or interference - inducing nucleic acid effectively suppresses asiRNA as examples , whereas the phrase ' passenger strand expression of a target gene while blocking off - target effects , refers to the strand , which forms said doublex structure with validated by the examples , wherein the present invention is the guide strand , wherein it plays role as passenger in 20 a method to inhibit off - target effect mediated either by the helping guide strand to load onto Argonaute protein , wherein guide strand or the passenger strand of the RNA interfer it is a polynucleotide that is substantially or 100 % the same ence - inducing nucleic acids , wherein providing the method sequence with target nucleic acids , thereby it is also called comprising a step of introducing the RNA interference “ sense strand ” , wherein the passenger strand can be the same inducing nucleic acid into the cell . In addition , the present polynucleotide as a whole or in part with siRNA , miRNA , 25 invention can provide a method comprising a step of shRNA , DsiRNA , IsiRNA , Ss - siRNA , piRNA , endo - siRNA expressing the RNA interference - inducing nucleic acid to or asiRNA as examples . abrogate off - target effects mediated by either the guide

In the present invention , the term ' spacer ' refers to the strand or the passenger strand of the RNA interference substituent which can be the replacement of one single inducing nucleic acids . nucleotide and affordable to maintain single nucleotide 30 In the present invention , the phrase " off - target effect space , not limited to the specific one but allowing anything refers to a instance in which the guide strand of siRNA maintaining the space , wherein the spacer prefers an organic causes unexpected degradation of other mRNAs or silencing compound , more appropriately hydrogen carbon chains con - of gene expression of corresponding mRNAs , also including taining ( connected to ) phosphoryl group ( H2PO4 ) or sul - any instance in which the passenger strand of siRNA causes furyl group ( H , PSO2 ) , wherein the most appropriate one 35 degradation of other mRNAs or silencing of gene expression for the spacer is an alkyl group comprising at least three of corresponding mRNAs by pairing to wrong targets , carbons . Additionally , the spacer includes abasic nucleotide despite of the fact that siRNA is originally used only for forms such as dSpacer and rSpacer , representing single degrading mRNA having a sequence complementary to the nucleotide derivatives containing no base at all , wherein the guide strand , thereby inhibiting the gene expression of the spacer also comprehensively refers any compound with 40 corresponding mRNA . modified base which cannot pair to any base including RNA Hereinafter , examples will be provided in order to help to present in vivo . understand the invention . However , such examples are pro

In the statement above , the single strand derived from the vided for illustrative purposes only , and the present inven double strand has ability to bind Argonaute protein inducing tion is not limited thereto . RNA interference . 45

In the statement above , the RNA interference - inducing [ Example 1 ] Comparison of Target Gene Silencing nucleic acid can contain spacer substitution modifications and Off - Target Effect Caused by siRNA Molecules both in sixth nucleotide from the 5 ' end and in first and with Deoxynucleotide Spacer ( dSpacer ) second nucleotides from the 3 ' end . Substitution

In addition , the present invention can provide composi - 50 tion and / or kit for gene silencing , including the RNA inter - As examples illustrated in FIG . 1 , the present inventors ference - inducing nucleic acid . speculated that application of the spacer modification to the

In the present invention , said “ composition ' refers the 5 ' end region of the RNA interference - inducing nucleic acids compound which can be used for silencing expression of a might be able to completely block off - target effects , while target gene while suppressing off - target effects at the same 55 enhancing the repression of a target gene ( on - target effects ) , time . wherein the spacer cannot make base - paring and the RNA

Herein , said “ kit ' consists of the RNA interference - induc - interference - inducing nucleic acids includes all RNAs bind ing nucleic acids for suppressing target gene expression and ing Argonaute protein , wherein the present inventors containers with such nucleotide components inducing RNA focused on the 5 ' end region , especially on the sixth nucleo interference , wherein the container may take the form of 60 tide from the 5 ' end , called ' pivot ' , which is based on the bottles , tubs , sachets , envelops , tubes , ampoules , and the mode of miRNA target recognition , transitional nucleation like , which may be formed in part or in whole from plastic , model ( Nat Struct Mol Biol . 2012 Feb . 12 ; 19 ( 3 ) : 321 - 7 ) , glass , paper , foil , wax , and the like . The container may be thereby have invented the present RNA interference - induc equipped with a fully or partially detachable lid that may ing nucleic acids . initially be part of the container or may be affixed to the 65 Initially , following experiments have been performed to container by mechanical , adhesive , or other means , wherein compare the on - target and the off - target effects between the container may also be equipped with a stopper , allowing modified si RNAs from the present invention and unmodi

US 10 , 227 , 592 B2 10

fied siRNAs that conventionally used . First , a guide strand From the above , the dSpacer substitution between posi RNA was synthesized where a nucleotide in the 5 ' end region tions 2 - 7 from the 5 ' end turned out to eliminate the ( positions 1 - 11 ) including the seed region was substituted to miRNA - like off - target effects while showing the on - target deoxyribonucleotide spacer ( dSpacer ) ( pi ) , and used to pro - activity , wherein the abasic deoxynucleotide is a modifica duce a double strand , which has two dT ( deoxythymine 5 tion that cannot make base - pairing . nucleotide ) overhangs with conventional 19 nucleotides Furthermore , in order to investigate the superior on - target structure via perfect reverse complementary to passenger activity shown by the dSpacer substitution between posi strand , wherein the passenger strand was synthesized with tions 2 - 7 of the siRNA in detail , the on - target silencing out any modification . Such RNA molecules were chemically activity was estimated across various concentrations to synthesized by ST Pharma , Trilink Technologies or Bioneer 10 measure IC50 ( inhibitory concentration 50 ) as a result ,

thereby validated that the substitution in position 5 or 6 is the company , further purified by HPLC , then a duplex between most efficient , as represented in FIGS . 2d and 2e , wherein the guide strand and the passenger strand was produced as the substitution in position 6 especially performed the most illustrated in left panel of FIG . 1 ( e . g . abasic nucleotide excellent maximal inhibition rate ( Imax ) among the modi substitution at position 6 from the 5 ' end , 6 pi ) by following 15 Following 15 fications that showed elimination of the off - target effects . the protocol provided by the companies . While doing this , a The above results well fit into the transitional nucleation control ( NT ; non - targeting , SEQ ID NOS : 3 and 4 ) was model , a new mode of miRNA target recognition , as illus synthesized by using siRNA form of cel - miR - 67 , a micro trated in FIG . 27 , wherein the results also match with the RNA ( miRNA ) expressed only in C . elegans . previous research reporting that a base - pairing in position 6

The modification stated above was applied to siRNA 20 from the 5 ' end , especially called " pivot ” , is critical to ( siRL , SEQ ID NO : 1 ) designed to silence renilla luciferase recognize miRNA targets ( Nat Struct Mol Biol . 2012 Feb . ( renilla luciferase is derived from the insert of psi - check2 12 ; 19 ( 3 ) : 321 - 7 ) . vector , Promega company ) . In detail , 75 nM siRNAs pro - In addition , as shown in FIG . 2g , the guide strand having duced as the duplex above ( SEQ ID NOS : 1 and 2 ) were the single abasic deoxynucleotide substitution in position 3 co - transfected into HeLa cells ( ATCC CCL - 2 ) together with 25 or 6 from the 5 ' end reduced miRNA - like off - target effects psi - check2 vectors , which express renilla luciferase , by mediated by the passenger strand in opposite , wherein the using lipofectamine 2000 reagent ( Invitrogen ) according to complete prevention can be achieved by the same modifi manufacturer ' s protocol , then the effects were examined . In cation on passenger strand , substituting a nucleotide in the case of measuring siRL mediated on - target effects , positions 2 - 7 from the 5 ' end region . psi - check2 vector was used as intact , whereas in the case of 30 Based on the above , it was confirmed that the guide strand measuring the off - target effects , renilla luciferase gene in of the siRNA molecule should be appropriately used by psi - check2 vector was replaced by renilla luciferase in substituting positions 2 - 7 from the 5 ' end to deoxyribonucle PRL - TK ( promega ) that was unable to respond to the otide spacer ( dSpacer ) having defect in base - pairing , on - target effects , then to construct RL - Seed and RL - Nuc , the wherein the most appropriate usage is to substitute position vector included two copies of perfect complement sequences 35 6 to dSpacer ( 6 pi ) , considering the effect on target gene to seed region ( positions 1 - 8 from the 5 ' end ) of siRL ( seed repression and off - target avoidance . sites ; Seed ) and nucleation bulge sites which can bind to miRNA by forming bulge between positions 5 and 6 , syn [ Example 2 ] Comparison of Target Gene thesized as DNA and inserted repetitively into 3 ' UTR ( 3 ' Repression and Off - Target Effect Caused by siRNA untranslated region ) . Herein , HeLa cell was cultured in 40 Molecules with Ribonucleotide Spacer ( rSpacer ) Dulbecco ' s modified Eagle ' s medium ( Invitrogen ) , supple Substitution mented with 10 % FBS ( fetal bovine serum ) , 100 U / ml penicillin , and 100 ug / ml streptomycin , whereas the trans In order to see whether the results in the example 1 above fection was performed in complete medium without antibi - can be reproduced by a similar spacer having ribonucleotide otics . After 24 hours from the transfection , effects of siRNA 45 backbone , a nucleotide in positions 2 - 7 of the siRL ( SEQ were examined by measuring luciferase activity using Dual - NO : 1 ) was substituted to ribonucleotide spacer ( rSpacer ) luciferase reporter assay system from Promega company ( pi - r ) where they showed elimination of off - targets before , according to manufacturer ' s protocol , wherein the renilla wherein the on - target activity of the siRL was examined by luciferase activity was estimated at least three times in calculating IC50 using the same method performed in the replicates , measured by using Glomax Luminometer from 50 Example 1 above . Promega company , then normalized by firefly luciferase As a result , as shown in FIGS . 3a , 3b and 3c , every activity . rSpacer substitution in position 2 - 7 from the 5 ' end showed As a result , as shown in FIG . 2a , siRNA having a dSpacer more than or equal to 54 % of maximal inhibitory rate ( Imax )

substitution between position 1 and 11 from the 5 ' end region relative to the unmodified ( WT ) Among them , every modi shows less than or equal to 60 % of the activity ( relative to 55 fication except 2 pi - r ( applied to position 2 ) has less on the normalized value denominated by firefly luciferase ) , target silencing activity than 6 pi . Exceptionally , the 2 pi - r where the on - target effects were measured at 75 nM . Espe - showed superior on - target repression comparing with 6 pi , cially , although the substitution of position 2 , 4 , 5 , or 6 but still repressed off - targets as shown in FIG . 3d , where reduced less than or equal to 30 % of the activity , where the validated by measuring off - target effects with RL - seed performance is not as much as the unmodified , the effect was 60 according to the method in the example 1 . Furthermore , still significant . However , when the miRNA - like off - target when off - target effects for each modification were measured effects were estimated by using luciferase reporters where at 75 nM siRNA , it was confirmed that every modification previously well - known sites such as seed sites ( RL - Seed ) or except 2 pi - r and 7 pi - r blocks off - target effects ( FIG . 3e ) . nucleation bulge sites ( RL - Nuc ) were inserted , the off - target Based on the above , it was confirmed that the guide strand repression was shown to be eliminated only in the case of the 65 of the siRNA molecule should be appropriately used by dSpacer substitution between positions 2 and 7 , as repre - substituting position 3 or 6 from the 5 ' end to ribonucleotide sented in FIGS . 2b and 2c . spacer ( rSpacer ) , wherein the most appropriate usage is to

US 10 , 227 , 592 B2 11 12

substitute position 6 to dSpacer ( 6 pi ) as in Example 1 above , or 6 from the 5 ' end region , wherein the most appropriate considering the effect on target gene repression and off usage is to substitute position 6 to dSpacer ( 6 pi ) , consid target avoidance . ering the effect on target gene repression and off - target

avoidance . [ Example 3 ] Comparison of Target Gene Silencing 5 and Off - Target Effect Caused by siRNA Molecules [ Example 5 ] Loss of Function in Target Gene

with Deoxyribonucleotide Spacer ( dSpacer ) Regulation by microRNA Molecules Having Insertion Substitution Modification of 6th Nucleotide from

the 5 ' End to Deoxyribonucleotide Spacer ( dSpacer ) As sequence configuration containing deoxyribonucle - 10 and Comparison with the Effect from 2 ' OMe

otide spacer ( dSpacer ) in seed region . a mismatch pairing Modification should be occurred in the part of an abasic in siRNA when it interacts with target gene RNA , wherein the abasic is made As validated by the example 1 above , the siRNA molecule by substitution as in Example 1 and Example 2 . Therefore , with abasic substitution derived from the present invention additionally the abasic can form a bulge when a dSpacer is 15 abrogates miRNA - like off - targets caused by a conventional inserted into the seed region of siRNA , as shown in FIG . 4a . structure of siRNA , thereby to further validated the modi As such , for the case of such dSpacer insertion , there fication by applying to miRNA , miR - 124 ( SEQ ID NO : 5 ) changes in on - target and off - target effect were investigated . was modified to contain deoxynucleotide substitution in Initially , the on - target effects of siRL ( SEQ NO : 1 ) were position 6 from the 5 ' end , then examined by performing examined by measuring IC50 as in the method performed in 20 following experiments below . Initially , the same sequence of example 1 , wherein the siRL contains dSpacer insertion human miR - 124 - 3p ( SEQ ID NO : 5 ) and the sequence between positions 2 - 7 from the 5 ' end , wherein the positions containing 6 pi , dSpacer substitution in position 6 , were 2 - 7 was reported to abolish off - target in the examples above . synthesized , then cotransfected ( SEQ ID NOS : 5 and 6 ) into As a result , as shown in FIG . 4b , every dSpacer insertion HeLa cells with psi - check2 vector containing two miR - 124

between positions 2 - 7 from the 5 ' end showed at least 19 % 25 seed sites ( the perfect complementary sequence to position of maximal inhibitory rate , relative to the unmodified ( WT ) . 1 - 8 from the 5 ' end region ) . Then the luciferase activity was Additionally , as shown in FIGS . 4c and 4d , everything measured at various concentrations of RNA by using the except 2 pi - I and 3 pi - I showed less potent on - target activity method in example 1 above . than 6 pi . Exceptionally , 2 pi - I and 3 pi - I showed better As a result , as shown in FIG . 6a , it was confirmed that on - target activity than 6 pi , but they still silence off - targets 30 miRNA having the 6 pi , the modification of the present ( FIG . 4e ) , confirmed by the results from measuring off - invention ( miR - 124 - 6 pi ) , cannot repress the seed sites target effect using RL - seed as performed in Example 1 . where they are previously known to be recognized . In Furthermore , as shown in FIG . 4f , all modifications ( 4 pi - I , addition to the seed sites , we also validated that 6 pi 5 pi - I , 6 pi - I ) except 2 pi - I , 3 pi - I and 7 pi - I abrogated modification cannot affect nucleation bulge sites ( Nuc ) , the off - target effects , observed at 75 nM siRNA concentration 35 sites known to be recognized by miRNA . by measuring off - target effects for each modification . Furthermore , genome wide validation was conducted to

In view of the above , it was confirmed that the guide confirm whether miR - 124 - 6 pi actually avoids repression of strand of the siRNA molecule should be appropriately used various genes which have been usually affected by the by inserting deoxyribonucleotide spacer ( dSpacer ) into posi - unmodified miR - 124 , wherein miR - 124 , miR - 124 - 6 pi , and tion 4 or 6 from the 5 ' end region , wherein the most 40 the control were transfected into HeLa cells where miR - 124 appropriate usage is to substitute position 6 to dSpacer ( 6 pi ) is not expressed , wherein total RNA was extracted by using as in the example 1 above , considering the effect on target R NAeasy kit ( Qiagen ) after 24 hours from the transfection , gene repression ( on - target ) and off - target avoidance . thereby performing RNA - Seq analysis , a gene expression

experiment based on sequencing analysis , which is provided [ Example 4 ] Comparison of Target Gene Silencing 4 5 by Otogenetics company . As a result from the experiment and Off - Target Effect Caused by siRNA Molecules above , FASTAQ files were generated and further serially with Ribonucleotide Spacer ( rSpacer ) Insertion analyzed by using TopHat , Cufflink and Cuffdiff program ,

wherein values were finally normalized as log 2 ratio by In order to investigate effect of ribonucleotide spacer using the result from original HeLa cells , wherein the values

( rSpacer ) insertion into seed region ( pi - ri ) on the off - target 50 should be reported as significant by statistical analysis effects , IC50 was measured by using the method in Example provided by Cuffdiff , in order to be selected for the analysis . 3 above . As a result , as shown in FIG . 6b , it was observed that the As a result , as shown in FIGS . 5a , 5b and 5c , every control showed both up - and down - regulation in gene

modification in seed region showed superior on - target activ - expression at the same extent , but the miR - 124 increased the ity , more than 20 % of Imax . Especially , 2 pi - r ) and 3 pi - r 55 significant portion of down - regulated genes , wherein , in the showed potent on - target activity better than 6 pi , but they case of the 6 pi modification , such down - regulated genes still silence off - targets as reported in FIG . 5d , wherein the were significantly reduced in their number . Furthermore , as measuring the off - targets was performed by using the same shown in FIG . 6c , heatmap representation significantly method in the example 1 . showed that large number of miR - 124 dependent down

In addition , every modification ( 4 pi - ri , 5 pi - rl , and 6 60 regulated genes were unable to be repressed by mir - 124 - 6 pi , pi - ri ) except 2 pi - ri , 3 pi - rl and 7 pi - rI was observed to wherein the extent of repression mediated by miR - 124 was abrogate off - target effects as shown in FIG . 5e , wherein the sorted to examine total genes for miR - 124 and miR - 124 - 6 off - target effects were estimated for each modification at 75 pi , finally represented as the heatmap . nM siRNA concentration . Furthermore , in order to see whether the 6 pi modification

In view of the above , it was confirmed that the guide 65 abrogates interaction between miRNA and target mRNA , strand of the siRNA molecule should be appropriately used potential cause of the observation above , Ago HITS - CLIP by inserting ribonucleotide spacer ( rSpacer ) into position 4 analyses was performed for miR - 124 transfected HeLa cells ,

14 US 10 , 227 , 592 B2

13 wherein sequences from the Ago HITS - CLIP were analyzed [ Example 6 ] Off - Target Effects of siRNA to generate a map of all binding sites in mRNA , then further Developed for Reducing Plasma Cholesterol and compared with the results from original HeLa cells express Comparison of Improvement by Applying Spacer ing control , thereby precisely identified Argonaute binding Modification sites generated by miR - 124 expression at genome - wide 5 level . Such miR - 124 binding sites were named as de novo In order to evaluate the effect of the present invention , the Ago - miR - 124 clusters , wherein their cumulative fraction modification was applied to siRNA sequences of which was analyzed depending on the extent of repression for the purpose of usage can be expanded from experiments to

comparison , focusing on the target mRNA which contains therapeutics , wherein the siRNA sequences were developed miR - 124 seed sites in these binding , wherein the extent of 10 to lower plasma cholesterol by repressing PCSK9 gene in

liver through inducing RNA interference , wherein the repression was estimated by mRNA profiling results for experiments were performed for PCS - A1 ( A1 , SEQ ID miR - 124 or miR - 124 - 6 pi expressed HeLa cells NOS : 7 and 8 ) and PCS - A2 ( A2 , SEQ ID NOS : 9 and 10 ) As a result , as shown in FIG . 6d , the target mRNAs bound ( Proc Natl Acad Sci USA . 2008 Aug . 19 ; 105 ( 33 ) : 11915 by miR - 124 were significantly repressed relative to total al 15 20 ) , which were developed by Alnylam company . The mRNA expression ( KS test , P < 0 . 01 ) , but the case of miR on - target activity was compared for A1 ( SEQ ID NO : 7 ) 124 - 6 pi was not statistically significant . Based on this , we with different position of abasic modification by measuring confirmed that miR - 124 - 6 pi is unable to repress conven - luciferase activity using the same method in example 1 , tional target mRNAs of miR - 124 in genome - wide level , showing that A1 - 6 pi has the most superior efficiency of wherein , for the miR - 124 - 6 pi , position 6 from the 5 ' end of 20 target gene silencing and , as represented in FIG . 7a , it also miR - 124 was modified to be substituted to abasic single completely abrogates off - target effect mediated by the seed deoxynucleotide . region . A2 ( SEQ ID NO : 9 ) has the same sequence as A1 , but

2 ' OMe modification , developed by Dharmacom company , contains 2 ' OMe modification in several positions to solve has been conventionally used to prevent siRNA off - targets . the problem of innate immune response and also to improve This is an empirical method which applies 2 ' OMe modifi - 25 RNA stability , wherein the 6 pi modification in A2 siRNA cation to position 2 from the 5 ' end of guide strand to prevent molecule also abrogates off - targets as validated in FIG . 7c , the corresponding miRNA - like off - target effects . The pres estimated by the luciferase reporter assay using the same ent inventors applied the 6 pi modification of the present method in Example 1 . Additionally , as represented in FIG .

invention to siRNA for silencing renilla luciferase ( SEQ ID 7d , A2 - 6 pi showed almost the same on - target silencing NO : 1 ) and also applied 2 ' OMe to it , wherein they evaluated 30 ted 30 activity as the unmodified A2 , observed by measuring IC50 the efficiency of on - target effect based on IC50 derived by using the luciferase reporter .

Since it was observed that every siRNA for PCSK9 gene measuring repression in various concentrations using the caused miRNA - like off - target effects in luciferase reporter same method in Example 1 above . assays , additional experiments were performed in human As a result , as shown in FIG . 6e , the 6 pi modification not as calion nol 35 liver cancer cell line , HepG2 ( ATCC HB - 8065 ) , to validate only showed superior efficiency of the repression than the off - target effects actually happening in liver cell . Initially 2 ' OMe , but also showed excellent maximal inhibition rate . we examined whether A2 can efficiently reduce PCSK Furthermore , genome - wide off - target effects were evaluated mRNA as siRNA for PCSK9 gene , wherein the A2 and the by the same RNA - seq method and heatmap analyses above , A2 - 6 pi siRNAs were transfected into HepG2 cells using confirming that 2 ' OMe modification cannot abrogate off - 40 Lipofectamine RNAiMAX ( Invitrogen ) reagent according target silencing in siRNA as much as 6 pi . to the provided protocol , then total RNA was extracted after

Additionally , 6 pi or 2 ' OMe modification was applied to 24 hours by RNeasy kit ( Qiagen ) and reverse transcribed by miR - 124 , of which function is known to induce neuronal Superscript III RT ( Invitrogen ) according to the provided differentiation , to see whether the modification mediated protocol , thereby amount of PCSK9 mRNA was quantitated abrogation of miRNA - like targeting is efficient enough to 45 by performing qPCR with SYBR® Green PCR Master Mix inhibit its biological function , wherein miRNA with these ( Applied Biosystems ) and normalized by GAPDH mRNA modifications were transfected into N2a cell ( Neuro - 2a , level . ATCC CCL - 131 ) , then examined through microscope after As a result , A2 and A2 - 6 pi are validated for on - target 72 hours from the transfection to see whether they can effects that they both efficiently repress PCSK9 , as shown in generate terminal neuron structures . 50 FIG . 7e . As a result , as shown in FIG . 6g , miR - 124 - 2me still After that , NSR RNA - Seq method ( Nat Methods . 2009

September ; 6 ( 9 ) : 647 - 9 ) was used to construct the library possessed the capability to induce the structure of terminal from total RNA , wherein total mRNA expression was inves neuron , but miR - 124 - 6 pi completed lose such ability , tigated by analyzing the library using HiSeq2000 sequence wherein miR - 124 - 2me is the case where 2 ' OMe modifica ned 55 analyzer ( Illumina company ) , thereby genome - wide expres tion was applied . sion was analyzed with heatmap representation by using the Based on the above , considering the effect on off - target same method in Example 2 . silencing , it was confirmed that the 6 pi modification shows As a result , as shown in FIG . 7 , it was observed that excellent avoidance of binding and silencing of miRNA - like mRNA level of off - target genes suppressed by A2 was off - targets , thereby the 6 pi modification completely block 60 recovered by 6 pi modification . the biological function in miRNA , wherein 6 pi modification Furthermore , the off - target genes showing such changes is the substitution of position 6 from the 5 ' end to deoxyri - were analyzed by performing GO ( gene ontology ) analysis bonucleotide spacer ( dSpacer ) , whereas on - target efficiency using DAVID program ( Nature Protoc . 2009 ; 4 ( 1 ) : 44 - 57 ) , of the conventional 2 ' OMe modification is not better than 6 thereby finding that many genes with cell cycle related pi , wherein the 2 ' OMe modification only marginally reduces 65 functions were significantly silenced via off - target effects . off - targets , showing the limitation that the off - target cannot To confirm the putative off - target effects , corresponding be completely blocked siRNAs were transfected into HepG2 , synchronized for cell

US 10 , 227 , 592 B2 15 16

cycle by eliminating 10 % serum ( FBS ) from the media for As a result , as shown in FIG . 8e , it was validated that 24 hours , then analyzed for cell cycle after 48 hours by using cellular copper ion in increased by A2 but not increased by propodium iodide and FACS machine . A2 - 6 pi . Increase of copper ion in liver tissue is known to

As a result , as shown in FIGS . 7g , 7h and 7i , defect in cell induce cell death , thus cell death was quantitated by using cycle was observed as expected when A2 siRNA molecule 5 Annexin V : FITC Apoptosis Detection Kit II ( BD Pharmin was expressed , especially wherein cell cycle arrest in G1 / G2 gen ) , wherein the assay was performed at 72 hours after was increased , but the phenotype was disappeared when 6 pi transfection , wherein the transfection was performed for the was introduced . expression of control ( SEQ ID NOS : 3 and 4 ) , A2 , and A2 - 6

Based on the above , it was confirmed that siRNAs for pi into NCTC clone 1469 , wherein FACS was used after the PCSK9 causes deleterious cell cycle arrest in human liver 10 stain with PI and Annexin V according to the provided cell as unexpected off - target effects , but such adverse off - protocol , wherein A2 induced cell death was observed as the target side effects can be blocked by applying the 6 pi similar extent to the result from 32 um CuSO4 treatment , modification of the present invention , wherein the 6 pi also but in the case of A2 - 6 pi the cell death was disappeared as maintains on - target silencing activity . shown in FIG . 8f .

15 Based on the results above , the siRNAs for PCSK9 is [ Example 7 ] Off - Target Effects Caused by PCSK9 turned out to suppress the copper ion metabolism as unex siRNA Through In Vivo Deliver in Mouse and pected off - target phenotypes when it was injected into mice

Evaluation of the Improvement by Applying Spacer and delivered to liver tissue , thereby increasing copper Modification concentration in liver tissue and inducing cell death as

20 unexpected adverse side effects . Nevertheless , it is turned In order to estimate off - target effects in vivo , siRNA was out that 6 pi modification of the present invention abrogates

delivered to liver tissue by using 7 weeks - old mice ( C57 / adverse off - target effects even in vivo as well , while main BL6 ) for the experiments , wherein the evaluation was per - taining efficiency of on - target silencing against PCSK9 . formed for the off - target effects which was analyzed for PCSK9 siRNAs ( SEQ ID NOS : 9 and 10 ) in Example 6 . 25 [ Example 8 ] Comparison of Target Gene Silencing Each siRNA was delivered to liver tissue by injecting 5 and Off - Target Effect Between Deoxyribonucleotide mg / kg of siRNA via tail veins of 5 mice ( tail - vein injection ) , Spacer ( dSpacer ) Substituent and Conventional then the mice were sacrificed after 48 hours to dissect liver Substituent Modified for Inhibiting Off - Target tissue and extract blood for the experiments . Initially , total Effects RNA including small RNA was extracted from the part of 30 dissected liver tissues using miRNeasy kit ( Qiagen ) , then the Modified by the present invention validated to abrogate amount of each delivered siRNA and the target mRNA , off - target effect and to silence the target gene , a dSpacer PCSK9 , was quantitated by qPCR . For quantification of substituent was compared with conventional modified sub siRNA , we followed the protocol ( Biotechniques . 2005 stituents for the performance in suppressing off - target effects October ; 39 ( 4 ) : 519 - 25 ) using poly ( A ) tailing Kit ( Ambion ) , 35 by measuring IC50 using the same method in Example 1 , which first attached adenosines to the 3 ' end of RNAs , wherein the dSpacer substituent contains a deoxynucleotide wherein reverse transcription was performed by Superscript spacer substituted for a nucleotide in the position 6 . III RT ( Invitrogen ) using oligo - dT containing a specific First , as a conventional substituent , a method of introduc sequence , thereby qPCR was conducted with SYBR® Green ing a mismatch into position 2 - 8 from the 5 ' end was applied , PCR Master Mix ( Applied Biosystems ) and by using the 40 introducing mismatch base - pairing into position 6 from the pair of DNA primers , one recognizes the specific sequence 5 ' end of miR - 124 ( miR - 124 - 6 mm , SEQ ID NOS : 13 and in oligo - dT and the other has the same sequence of siRNA . 14 ) , wherein IC50 was measured . As a result , represented in The Quantitation of PCSK9 mRNA was conducted by the FIGS . 9a , 96 , 9c and 9d , miR - 124 - 6 mm showed superior same method in example 6 . The amount of total plasma silencing activity to perfectly complement target ( perfect cholesterol was measured by ELISA method using the kit 45 match target ) as much as the unmodified miR - 124 ( both from Wako company according to the provided protocol . 1C50 = 0 . 02 nM , SEQ ID NOS : 11 and 12 ) , but it did not As a result , as shown in FIGS . 8a , 86 and 8c . A2 - 6 pi , repress miR - 124 seed targets ( positions 2 - 8 from the 5 ' end ) .

modified to have the spacer , was validated to be delivered to Nevertheless , the introduction of mismatch base - pairing the liver tissue , repress PCS K9 mRNA , and reduce plasma can recognize new sequences as seed targets depending on cholesterol as well as A2 siRNA ( SEQ ID NO : 9 ) . 50 changes in nucleotide base , thereby off - target effects can be

Furthermore , RNA - Seq analyses were conducted by fol - produced by new sequence matches to the seed where the lowing the same method in example 5 above , wherein total mismatch was introduced . Thus , when a mismatch was RNA was obtained from the live tissue where the delivery of introduced to position 6 from the 5 ' end of miR - 124 , each siRNA was confirmed . repression of a new sites which can interact with a new seed By analyzing the results as heatmap representation , as 55 was examined by measuring IC50 , wherein the new site can

shown in FIG . 8d , the 6 pi modification was confirmed to be recognized through consecutive base - pairings from posi reduce genome - wide off - targets . tions 2 - 8 from the 5 ' end containing . As a result , as shown

In addition , functions of off - targets was examined by in FIGS . 9e and 9f , the new seed pairing target , which can using GO analysis , applying to genes repressed by A2 be recognized through consecutive base - pairings from posi siRNA , wherein it was found that off - targets play roles in 60 tions 2 - 8 from the 5 ' end , was silenced as much as observed copper ion metabolism in liver tissue , thereby defect in in the unmodified miR - 124 . copper ion metabolism was expected , examined by using the In addition , as shown in FIGS . 9g and 9h , conventional mouse liver cell , NCTC clone 1469 ( Korean cell line bank ) . 2 ' OMe modification was applied to position 6 from the 5 ' In detail , control , A2 , or A2 - 6 pi siRNA was transfected into end of miR - 124 , examined by IC50 measurement , wherein NCTC clone 1469 using lipofectamine 2000 , and amount of 65 seed sites were still silenced as much as observed in the copper ion was quantified by using QuantiChrom Copper unmodified miR - 124 , wherein the seed sites could work as Assay Kit ( bio systems ) at 72 hours after transfection . off - targets in the case of siRNA .

17 US 10 , 227 , 592 B2

18 Based on the results above , it was confirmed that the ence - inducing nucleic acid has been invented , expecting

method introducing a conventional mismatch to position 6 complete elimination of off - target effect together with con from the 5 ' end can prevent off - targets of initial seed pairing servation of on - target efficiency as much as in abasic nucleo sites , but it has limitation that it causes off - targets of new tide substitution , wherein the modification is applied to seed pairing sites , altered by introducing the mismatch . 5 position 6 from the 5 ' end . Furthermore , the conventional 2 ' OMe modification cannot Initially , as illustrated in FIG . 11a , the C3 spacer was reduce off - targets at all when it is applied to position 6 from adopted and applied to position 6 from the 5 ' end of miR - 124 the 5 ' end . ( SEQ NOS : 11 and 12 ) ( miR - 124 - 6c3 ) , making covalent Next , for the comparison of other modifications , the bonding between 5th and 7th nucleotide as shown in FIG .

2 ' OMe modification in position 2 from the 5 ' end ( 2me ) and 10 11b , wherein IC50 was measured to investigate and compare the UNA modification in position 7 from 5 ' end ( 7UNA ) the effect on on - target and off - target , wherein the C3 spacer were applied to miR - 124 , wherein their effects were mea is the most minimized spacer modification with covalent sured by the same method performed in Example 1 above , bonding and no base . wherein the other modifications were known to applied the As a result , as shown in FIG . 11c , miR - 124 - 6c3 position other than sixth nucleotide and effectively prevent 15 ( IC50 = 0 . 01 nM ) was observed to show better efficiency of off - targets . As a result , represented in FIG . 9i , both 2me repressing the perfect match target than miR - 124 - 6 pi ( IC50 = 0 . 9 nM ) and 7UNA ( IC50 = 7 . 2 nM ) were observed to ( IC50 = 0 . 15 nM ) and its efficiency of silencing the perfect only marginally reduce off - target effect compared with the match was the same as the unmodified miR - 124 ( IC50 = 0 . 01 unmodified miR - 124 ( IC50 = 0 . 7 nM ) , but the abasic deoxy nM ) , wherein the perfect match corresponds to siRNA nucleotide substitution of the present invention ( 6 pi ) com - 20 on - target . pletely eliminated the off - target effect . Furthermore , as shown in FIG . 11d , it was observed that

Furthermore , as shown in FIG . 9j , we observed that silencing activity to seed pairing sites was completed dis 7UNA applied to A2 ( SEQ ID NO : 9 ) can reduce some appeared when C3 was applied to position 6 from the 5 ' end off - target effects , but cannot completely abrogate it , wherein of miR - 124 , wherein seed pairing sites mediate off - target the A2 is the siRNA for silencing PCSK9 gene as on - target . 25 effects in siRNA . Additionally , other method introducing a bulge was applied In addition , the C3 spacer was applied to position 6 from to A2 and examined for its effect , showing reduction of some the 5 ' end of A2 ( SEQ ID NO : 9 ) ; siRNA for silencing off - target effects but not complete abrogation , wherein the PCSK9 gene , siMAPK14 - 1 ( SEQ ID NO : 15 ) ; siRNA for method introducing a bulge was conventionally generated silencing MAPK14 gene ( Jackson , A . L . , et al . , Rna , 12 ( 7 ) : by changing a nucleotide to form single nucleotide bulge in 30 1179 ) , or siRL , showing superior on - target activity together position 2 from the 5 ' end of the guide strand in the siRNA with complete abrogation of off - target effect in every case , duplex structure ( SEQ ID NOS : 9 and 17 ) . wherein the results were validated by measuring IC50 as

Based on the above , it was confirmed that every conven - represented in FIGS . 1le , 11f , 11g , 112 , 11i and 11j . tional method with usage for inhibiting off - target effect can Based on the statement above , we found the C3 spacer reduce some off - target effects , but has limitation that it 35 which is minimal abasic form in position 6 , existing as cannot completely eliminate off - target effect , whereas the covalent bonding between 5th and 7th nucleotide in siRNA , present invention completely abolish the off - target effects . wherein the C3 spacer allows the backbone without nucleo

tide , wherein the spacer can be any covalent bonding only if Example 9 ] Comparison of Target Gene Silencing its size is affordable to be substituted to a single nucleotide and Off - Target Effect for siRNA Molecules with 40 occupancy , thereby completely abrogating off - target repres No Base in Position 6 from the 5 ' End by C3 sion while conserving the better or the same efficiency of

Substitution , Wherein the C3 Substitution Makes on - target silencing from 6 pi . Covalent Bonding Between 5th and 7th Nucleotide

[ Example 10 Evaluation of the Effect from the Based on the observation in example 8 , it is speculated 45 Spacer Substation in Position 1 - 2 from the 3 ' End

that the reason to perform complete elimination of off - target of siRNA Molecules on miRNA - Like Off - Targets effects by the siRNA - 6 pi is that base in position 6 becomes Mediated by 3 ' End Compensatory Pairing and unable to base - pair with off - targets through this region , On - Target Gene Silencing wherein the siRNA - 6 pi has substitution of position 6 from the 5 ' end to dSpacer ( 6 pi ) . Thus , in theory , any spacer 50 The off - target effect caused by siRNA is majorly mediated modification making no base in position 6 from the 5 ' end is by recognizing target genes like miR NA through base expected to eliminate off - target effect , wherein the spacer pairing with the seed region in sequence dependent manner . modification can be anything only if it can afford to be In the case that miRNA recognizes targets in vivo , it is substituted to single nucleotide occupancy , although its reported that miRNAs repress target genes through compen backbone is not nucleotide . 55 satory pairings within the 3 ' end region ( 3 - compensatory

Therefore , as examples illustrated in FIG . 10 , the present pairing ) when the binding between seed region and target inventors expanded the spacer modification from dSpacer becomes weak ( Cell . 2009 ; 136 : 215 - 233 ) . Accordingly , substitution of position 6 from the 5 ' end shown in FIG . 1 to miRNA - like off - target effect could be occurred by the covalent bonding , wherein the covalent bonding can be mechanism of the 3 ' - compensatory pairing , requiring the anything maintaining the abasic skeleton of position 6 from 60 method to prevent it . the 5 ' end , thus designing the most minimized spacer linking Conventional siRNAs generally contain deoxynucleotide between 5th and 7th nucleotide . In other words , as the most thymidine ( dT ) in positions 1 - 2 from the 3 ' end , forming minimized spacer occupying size of a nucleotide in position overhang structure in the double - stranded siRNAs . In the 6 , C3 spacer , which comprises of phosphoryl group and case of dT , since it can base - pair to adenosine ( A ) , it could three carbon molecules , was used for designing the modi - 65 participate in 3 ' - compensatory pairing and induce miRNA fication . Using the C3 modification as the minimum stan - like off - target effect . Therefore , in order to block the off dard of a spacer without nucleotide , a novel RNA interfer - target effect mediated by such 3 ' - compensatory pairing ,

US 10 , 227 , 592 B2 19 20

spacer substitution modification can be applied to positions Additionally , when pi or c3 was applied as a spacer to 1 - 2 from the 3 ' end as illustrated in FIG . 12a . In other words , position 1 - 2 from the 3 ' end of siMAP14 - 1 ( SEQ ID NOS : the present inventors gave attention to the fact that conven 15 and 16 ) or siRL ( SEQ ID NOS : 1 and 2 ) , the same tional double - stranded siRNA structure can be maintained on - target repression activity was observed as in the unmodi even in the case of applying dSpacer of C3 spacer to 5 op to 5 fied , as represented in FIGS . 12f and 12h .

Based on the results above , it was confirmed that the RNA positions 1 - 2 from the 3 ' end , making them as abasic interference - inducing nucleic acids , of which 1st and 2nd nucleotide spacer , wherein efficiency of on - target activity nucleotides from the 3 ' end were substituted to abasic spacer can be maintained as superior , but off - target effects mediated with covalent bonds , have activity to avoid off - target effect by 3 - compensatory pairing can be eliminated , thereby mediated by 3 ' - compensatory pairing while maintaining the invented the present RNA interference - inducing nucleic activity of silencing the target gene . acids . Although the preferred embodiments of the present inven

For this , after substituting positions 1 - 2 from the 3 ' end of tion have been disclosed for illustrative purposes , one miR - 124 ( SEQ ID NOS : 11 and 12 ) to abasic spacer skilled in the art will appreciate that various modifications , modification , the on - target effect and off - target effect medi additions and substitutions are possible , without departing ated by 3 ' - compensatory pairing were investigated by mea 15 from the scope and spirit of the invention . Therefore , it

should be understood that the above - described embodiments suring IC50 using the same method in FIG . 8 above . As a are illustrative but not restrictive in all aspects . result represented in FIG . 12b or FIG . 12c , we observed that the unmodified miR - 124 showed target repression mediated INDUSTRIAL APPLICABILITY by 3 ' - compensatory pairing like siRNA off - target silencing , 20 but such off - target effects were completely disappeared According to the present invention , the RNA interference when 1st and 2nd nucleotides from the 3 ' end were substi inducing nucleic acid provides a novel modified form , tuted to either dSpacer or C3 spacer . showing target selectivity and specificity by abrogating

Like this , when the first and second nucleotides from the off - target effects and also maintaining activity of silencing 3 ' end of double - stranded miR - 124 were substituted to 25 target gene expression , wherein the off - target problem , abasic spacer , such as dSpacer ( pi ) or C3 spacer ( IC50 = 0 . 02 which causes inaccuracy and adverse side effects in the nM ) , the same on - target silencing effect was observed as in usage of conventional RNA interference , can be solved by the unmodified miR - 124 ( IC50 = 0 . 02 nM ) , wherein the on offering such RNA interference - inducing nucleic acids , target silencing effect was examined by using perfect thereby it can be widely used as a method for silencing gene complementary pairing target , on - target in siRNA , as rep - 30 expression for research and for gene therapy without con resented in FIGS . 12d and 12e . cerning .

SEQUENCE LISTING

< 160 > NUMBER OF SEQ ID NOS : 17

< 210 > SEQ ID NO 1 1 > LENGTH : 21

< 212 > TYPE : RNA < 213 > ORGANISM : Artificial Sequence

FEATURE : < 223 > OTHER INFORMATION : siRL Guide 20 > FEATURE : 21 > NAME / KEY : modified _ base

2 > LOCATION : ( 1 ) 3 > OTHER INFORMATION : n = quanosine or abasic deoxyribonucleotide

< 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 2 ) < 223 > OTHER INFORMATION : n = uridine , abasic deoxyribonucleotide ,

abasic ribonucleotide , or 21 - 0 - methyluridine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 3 ) < 223 > OTHER INFORMATION : n = adenosine , abasic deoxyribonucleotide , or

abasic ribonucleotide < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 4 ) < 223 > OTHER INFORMATION : n = guanosine , abasic deoxyribonucleotide , or

abasic ribonucleotide 220 > FEATURE : 221 > NAME / KEY : modified base

< 222 > LOCATION : ( 5 ) < 223 > OTHER INFORMATION : n = guanosine , abasic deoxyribonucleotide , or

abasic ribonucleotide < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 6 )

3 > OTHER INFORMATION : n = adenosine , abasic deoxyribonucleotide , abasic ribonucleotide , or propyl spacer

US 10 , 227 , 592 B2

- continued

?

?

< 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 7 ) < 223 > OTHER INFORMATION : n = guanosine , abasic deoxyribonucleotide , or

abasic ribonucleotide < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 8 ) < 223 > OTHER INFORMATION : n = uridine or abasic deoxyribonucleotide

20 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 9 )

3 > OTHER INFORMATION : n = adenosine or abasic deoxyribonucleotide < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 10 ) 223 > OTHER INFORMATION : n = quanosine or abasic deoxyribonucleotide

< 220 > FEATURE : 1 > NAME / KEY : modified base

< 222 > LOCATION : ( 11 ) < 223 > OTHER INFORMATION : n = uridine or abasic deoxyribonucleotide < 220 > FEATURE : < 221 > NAME / KEY : modified _ base

> LOCATION : ( 20 ) < 223 > OTHER INFORMATION : n = deoxythymidine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 21 ) < 223 > OTHER INFORMATION : n = deoxythymidine

?

< 400 > SEQUENCE : 1

nnnnnnnnnn ngaaaggccn n 21

< 210 > SEQ ID NO 2 < 211 > LENGTH : 21 < 212 > TYPE : RNA < 213 > ORGANISM : Artificial Sequence

FEATURE : < 223 > OTHER INFORMATION : siRL Passenger < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 20 ) < 223 > OTHER INFORMATION : n = deoxythymidine < 220 > FEATURE : 21 > NAME / KEY : modified base 22 > LOCATION : ( 21 )

< 223 > OTHER INFORMATION : n = deoxythymidine


ggccuuucac uacuccuacn n 21

< 210 > SEQ ID NO 3 < 211 > LENGTH : 24 < 212 > TYPE : RNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : NT Guide < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 1 ) < 223 > OTHER INFORMATION : n = 21 - 0 - methyluridine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 2 ) < 223 > OTHER INFORMATION : n = 21 - 0 - methyladenosin 220 > FEATURE : 21 > NAME / KEY : modified base

< 222 > LOCATION : ( 23 ) < 223 > OTHER INFORMATION : n = deoxythymidine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 24 ) < 223 > OTHER INFORMATION : n = deoxythymidine

US 10 , 227 , 592 B2 23 24

- continued < 400 > SEQUENCE : 3

nncucuuucu aggagguugu gann 24

< 210 > SEQ ID NO 4 < 211 > LENGTH : 24 < 212 > TYPE : RNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : NT Passenger < 220 > FEATURE : < 221 > NAME / KEY : modified _ base

2 > LOCATION : ( 1 ) < 223 > OTHER INFORMATION : n = 2 ' - O - methyluridine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 2 ) < 223 > OTHER INFORMATION : n = 2 ' - 0 - methylcytidine < 220 > FEATURE : 21 > NAME / KEY : modified _ base 222 > LOCATION : ( 23 )

< 223 > OTHER INFORMATION : n = deoxythymidine < 220 > FEATURE :

> NAME / KEY : modified base < 222 > LOCATION : ( 24 ) < 223 > OTHER INFORMATION : n = deoxythymidine

Hem Om < 400 > SEQUENCE : 4

nnacaaccuc cuagaaagag uann 24

< 210 > SEQ ID NO 5 < 211 > LENGTH : 20 < 212 > TYPE : RNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : miR - 124 - 3p < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 6 ) < 223 > OTHER INFORMATION : n = cytidine or abasic deoxyribonucleotide NNNNNN < 400 > SEQUENCE : 5

uaaggnacgc ggugaaugcc 20

< 210 > SEQ ID NO 6 < 211 > LENGTH : 22 < 212 > TYPE : RNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : miR - 124 - 5p


cguguucaca gcggaccuug au 22

< 210 > SEQ ID NO 7 < 211 > LENGTH : 21 < 212 > TYPE : RNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE :

3 > OTHER INFORMATION : siRNAs for PCSK9 Al qiude < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 2 ) < 223 > OTHER INFORMATION : n = uridine or abasic deoxyribonucleotide 220 > FEATURE : 21 > NAME / KEY : modified base

< 222 > LOCATION : ( 3 ) < 223 > OTHER INFORMATION : n = cytidine or abasic deoxyribonucleotide < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 4 ) < 223 > OTHER INFORMATION : n = cytidine or abasic deoxyribonucleotide

US 10 , 227 , 592 B2 25 26

- continued < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 5 ) < 223 > OTHER INFORMATION : n = guanosine or abasic deoxyribonucleotide < 220 > FEATURE :

1 > NAME / KEY : modified base < 222 > LOCATION : ( 6 )

> OTHER INFORMATION : n = adenosine or abasic deoxyribonucleotide < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 20 ) < 223 > OTHER INFORMATION : n = deoxythymidine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 21 ) < 223 > OTHER INFORMATION : n = deoxythymidine


unnnnnauaa acuccaggcn n 21

H

H

H

< 210 > SEQ ID NO 8 < 211 > LENGTH : 21 < 212 > TYPE : RNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : siRNAs for PCSK9 Al passenger < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 20 ) < 223 > OTHER INFORMATION : n = deoxythymidine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 21 ) 223 > OTHER INFORMATION : n = deoxythymidine


gccuggaguu uauucggaan n 21

?

< 210 > SEO ID NO 9 < 211 > LENGTH : 21 < 212 > TYPE : RNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE :

23 > OTHER INFORMATION : siRNAs for PCSK9 A2 quide > FEATURE :

< 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 6 ) 223 > OTHER INFORMATION : n = adenosine , abasic deoxyribonucleotide or

propyl spacer < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 7 )

23 > OTHER INFORMATION : n = adenosine or adenine unlocked nucleic acid < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 8 ) < 223 > OTHER INFORMATION : n = 2 ' - o - methyluridine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 15 ) < 223 > OTHER INFORMATION : n = 2 ' - O - methylcytidine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 20 ) < 223 > OTHER INFORMATION : n = deoxythymidine < 220 > FEATURE : 21 > NAME / KEY : modified base

< 222 > LOCATION : ( 21 ) < 223 > OTHER INFORMATION : n = deoxythymidine

.

?

< 400 > SEQUENCE : 9 uuccgnnnaa acucnaqgcn n 21

US 10 , 227 , 592 B2 27

- continued

??? ???

< 210 > SEO ID NO 10 < 211 > LENGTH : 21 < 212 > TYPE : RNA

3 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : siRNAs for PCSK9 A2 passenger < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 2 ) < 223 > OTHER INFORMATION : n = 2 ' - O - methylcytidine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 3 ) < 223 > OTHER INFORMATION : n = 2 ' - o - methylcytidine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 4 ) < 223 > OTHER INFORMATION : n = 2 ' - o - methyluridine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 9 ) < 223 > OTHER INFORMATION : n = 2 ' - o - methyluridine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base

LOCATION : ( 10 ) < 223 > OTHER INFORMATION : n = 2 ' - 0 - methyluridine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base

LOCATION : ( 11 ) < 223 > OTHER INFORMATION : n = 2 ' - o - methyluridine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 13 ) < 223 > OTHER INFORMATION : n = 21 - 0 - methyluridine 20 > FEATURE :

< 221 > NAME / KEY : modified _ base 2 > LOCATION : ( 14 )

23 > OTHER INFORMATION : n = 2 ' - O - methyluridine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 15 ) < 223 > OTHER INFORMATION : n = 2 ' - o - methylcytidine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 20 ) < 223 > OTHER INFORMATION : n = deoxythymidine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base

2 > LOCATION : ( 21 ) < 223 > OTHER INFORMATION : n = deoxythymidine

< 400 > SEQUENCE : 10

gnnnggagnn nannnggaan n 21


> FEATURE : < 223 > OTHER INFORMATION : miR - 124 Guide < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 2 ) < 223 > OTHER INFORMATION : n = adenosine or 2 ' - o - methyadenosine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 6 ) < 223 > OTHER INFORMATION : n = cytidine , 2 ' - o - methycytidine ,

abasic deoxyribonucleotide , or propyl spacer < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 7 )

3 > OTHER INFORMATION : n = adenosine or adenine unlocked nucleic acid < 220 > FEATURE : < 221 > NAME / KEY : modified _ base


? ?

? ?

US 10 , 227 , 592 B2 29 30

- continued < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 21 ) < 223 > OTHER INFORMATION : n = deoxythymidine

< 400 > SEQUENCE : 11

unaggnncgc ggugaaugcn n 21

< 210 > SEQ ID NO 12 < 211 > LENGTH : 21 < 212 > TYPE : RNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : miR - 124 Passenger < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 20 ) < 223 > OTHER INFORMATION : n = deoxythymidine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 21 ) 223 > OTHER INFORMATION : n = deoxythymidine

< 400 > SEQUENCE : 12

gcauucaccg cguqccuuan n 21

E

E

E

N

N

< 210 > SEQ ID NO 13 < 211 > LENGTH : 21 < 212 > TYPE : RNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : miR - 124 6mm Guide < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 20 ) < 223 > OTHER INFORMATION : n = deoxythymidine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 21 ) < 223 > OTHER INFORMATION : n = deoxythymidine

< 400 > SEQUENCE : 13

uaagggacgc ggugaaugcn n 21

V

V

V

V


> FEATURE : < 223 > OTHER INFORMATION : miR - 124 6mm Passenger < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 20 ) < 223 > OTHER INFORMATION : n = deoxythymidine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 21 ) 223 > OTHER INFORMATION : n = deoxythymidine ???

< 400 > SEQUENCE : 14

gcauucaccg cgucccuuan n 21

< 210 > SEO ID NO 15 < 211 > LENGTH : 21 < 212 > TYPE : RNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE :

3 > OTHER INFORMATION : siRNA for MAPK14 - 1 Guide

US 10 , 227 , 592 B2

- continued < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 6 ) < 223 > OTHER INFORMATION : n = cytidine , abasic deoxyribonucleotide , or

propyl spacer < 220 > FEATURE : < 221 > NAME / KEY : modified base < 222 > LOCATION : ( 20 ) < 223 > OTHER INFORMATION : n = deoxythymidine

20 > FEATURE : < 221 > NAME / KEY : modified _ base


< 400 > SEQUENCE : 15

aaccgnaguu cucuguaggn n 21

< 210 > SEQ ID NO 16 < 211 > LENGTH : 21 < 212 > TYPE : RNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : siRNA for MAPK14 - 1 Passenger < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 20 ) < 223 > OTHER INFORMATION : n = deoxythymidine < 220 > FEATURE : 21 > NAME / KEY : modified base

< 222 > LOCATION : ( 21 ) < 223 > OTHER INFORMATION : n = deoxythymidine

?

< 400 > SEQUENCE : 16

ccuacagaga acugcgguun n 21

< 210 > SEQ ID NO 17 < 211 > LENGTH : 20 < 212 > TYPE : RNA < 213 > ORGANISM : Artificial Sequence < 220 > FEATURE : < 223 > OTHER INFORMATION : A2 2bulge < 220 > FEATURE : 21 > NAME / KEY : modified base

< 222 > LOCATION : ( 2 ) < 223 > OTHER INFORMATION : n = 2 ' - o - methylcytidine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 3 ) < 223 > OTHER INFORMATION : n = 2 ' - 0 - methylcytidine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 4 )

23 > OTHER INFORMATION : n = 2 ' - O - methyluridine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 9 ) < 223 > OTHER INFORMATION : n = 2 ' - O - methyluridine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 10 ) < 223 > OTHER INFORMATION : n = 2 ' - O - methyluridine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 11 ) < 223 > OTHER INFORMATION : n = 21 - 0 - methyluridine 220 > FEATURE : 21 > NAME / KEY : modified base

< 222 > LOCATION : ( 13 ) < 223 > OTHER INFORMATION : n = 2 ' - O - methyluridine < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 14 ) < 223 > OTHER INFORMATION : n = 2 ' - o - methyluridine

US 10 , 227 , 592 B2 34

- continued < 220 > FEATURE : < 221 > NAME / KEY : modified _ base < 222 > LOCATION : ( 15 ) < 223 > OTHER INFORMATION : n = 21 - 0 - methylcytidine < 220 > FEATURE :

1 > NAME / KEY : modified base < 222 > LOCATION : ( 19 ) < 223 > OTHER INFORMATION : n = deoxythymidine < 220 > FEATURE : 21 > NAME / KEY : modified _ base 22 > LOCATION : ( 20 )

< 223 > OTHER INFORMATION : n = deoxythymidine

< 400 > SEQUENCE : 17 gnnnggagnn nannnggann 20

What is claim is : molecule of claim 1 , the method comprising a step of 1 . An RNA interference - inducing nucleic acid molecule introducing the RNA interference - inducing nucleic acid

comprising at least one nucleic acid strand , the at least one 20 molecule into a cell , nucleic acid strand comprising a modification at the sixth wherein the at least one nucleic acid strand , which com position from the 5 ' end of the nucleic acid strand , and prises the modification , of claim 1 is the guide strand .

9 . A method for suppressing off - target effects mediated by optional further modifications at each of the first and second a passenger strand of the RNA interference - inducing nucleic position from the 3 ' end of the nucleic acid strand , 25 acid molecule of claim 1 , the method comprising a step of wherein each modification is a replacement of a nucleo introducing the RNA interference - inducing nucleic acid tide residue at the position with a spacer , molecule into a cell ,

wherein the spacer is selected from the group consisting wherein the at least one nucleic acid strand , which com of an abasic deoxyribonucleotide analog which does prises the modification , of claim 1 is the passenger not include a nucleobase , and a C3 alkyl group . 30 strand .

2 . The RNA interference - inducing nucleic acid molecule 10 . The RNA interference - inducing nucleic acid molecule of claim 1 , wherein the at least one nucleic acid strand is of claim 1 further comprising a chemical modification . capable of binding to argonaute protein and inducing RNA 11 . The RNA interference - inducing nucleic acid molecule

of claim 1 , wherein the at least one nucleic acid strand interference . 3 . The RNA interference - inducing nucleic acid molecule 35 CO . scule of comprises the optional further modifications .

of claim 1 , wherein the nucleic acid strand further comprises 12 . The gene silencing composition of claim 5 , wherein a mismatch base pairing with an RNA of a target gene by a the at least one nucleic acid strand comprises the optional

further modifications . substitution or a bulge by an insertion . 4 . The RNA interference - inducing nucleic acid molecule 13 . The gene silencing composition of claim 6 , wherein

of claim 1 , which is selected from the group consisting of 40 the at least one nucleic acid strand comprises the optional further modifications . siRNA , miRNA , shRNA , DsiRNA , siRNA , SS - SiRNA ,

piRNA , endo - siRNA and asiRNA . 14 . The method of claim 7 , wherein the at least one 5 . A gene silencing composition comprising the RNA nucleic acid strand comprises the optional further modifi

cations . interference - inducing nucleic acid molecule of claim 1 . 6 . A gene silencing kit comprising the RNA interference - 45 15 . The method of claim 8 , wherein the at least one

inducing nucleic acid molecule of claim 1 . nucleic acid strand comprises the optional further modifi 7 . A method for silencing a target gene in a cell , the cations .

16 . The method of claim 9 , wherein the at least one method comprising a step of introducing the RNA interfer ence - inducing nucleic acid molecule of claim 1 into the cell . nucleic acid strand comprises the optional further modifi

8 . A method for suppressing off - target effects mediated by cations . a guide strand of the RNA interference - inducing nucleic acid

( 12 ) united states patent ( 10 ) patent no . : us 10 ...clip.korea.ac.kr/uspatent.pdf · ( 12 )...

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