US011279935B2( 12 )

United States Patent( 10) Patent No .: US 11,279,935 B2(45 ) Date of Patent :Mar. 22 , 2022

Akiyama et al.

(54) METHOD FOR SCREENING( 56 )

PROPHYLACTIC OR THERAPEUTIC

U.S. PATENT DOCUMENTS

AGENTS FOR DISEASES CAUSED BY

INTERLEUKIN 6 , INTERLEUKIN 13 , TNF,

G -CSF, CXCL1 , CXCL2 , OR CXCL5 AND
AGENT FOR THE PREVENTION OR

TREATMENT OF DISEASES CAUSED BY
INTERLEUKIN 6 , INTERLEUKIN 13 , TNF,

G -CSF, CXCL1 , CXCL2 , OR CXCL5( 71 ) Applicants : TAK - CIRCULATOR CO . , LTD ,Tokyo ( JP ); UNIVERSITY OF
TOKYO , Tokyo (JP )( 72 ) Inventors: Tetsu Akiyama, Tokyo (JP ) ; Hiroaki
Harada , Tokyo (JP ) ; Yusuke
Yamazumi, Tokyo (JP) ; Takeaki Oda ,Tokyo (JP ) ; Oh Sasaki , Tokyo ( JP ) ;Kazuyoshi Kofu , Tokyo ( JP )( 73 ) Assignees : TAK - CIRCULATOR CO , LTD , Tokyo( JP) ; UNIVERSITY OF TOKYO ,Tokyo (JP )Subject to any disclaimer, the term of this( * ) Notice:

References Cited
8,962,244 B2

2003/0049662 Al
2003/0152956 A1
2005/0261219 A1
2006/0172930 A1
2009/0118213 A1
2011/0200612 Al
2011/0256063 Al

2012/0183538 A1

2/2015 Ryder

3/2003 Monia et al .
8/2003 Ohtani et al.
11/2005 Richards et al .

8/2006 Akiyama et al .

5/2009 Hansen et al .
8/2011 Schuster et al.
10/2011 Lu et al .
7/2012 Trieu et al .

2012/0244170 A1
9/2012 Ciosk et al .
2014/0350083 A1 11/2014 Fire et al .
2021/0079390 A1 * 3/2021 Akiyama

A61P 11/06

FOREIGN PATENT DOCUMENTS
JP
JP

JP
JP
JP
JP
JP

WO
WO
WO
WO
WO

patent is extended or adjusted under 35
U.S.C. 154 ( b ) by 176 days .

2002508944 A
2003520586 A
415 5561
2009507499 A

3/20027/20037/200844-29269 B2

2/200912/2009

2011-526892 A
2012532613 A
1999032619 Al

10/2011
12/2012
7/1999

WO09949065 A

WO2002031136 A1
2003040182 Al
2018 155 710

9/19994/20025/2003

10/2015OTHER PUBLICATIONS

Hagedorn et al . Nucleic Acids Research vol . 45 , pp . 2262-2282( 21 ) Appl . No .:16 /315,513( 22 ) PCT Filed :Jul . 7 , 2017

( Year: 2017 ) . *Fakhr et al. Cancer Gene Therapy 23 , 73-82 ( Year : 2016 ) . *Qiu , et al . , “ Bronchial Mucosal Inflammation and Upregulation of
CXC Chemoattractants and Receptors in Severe Exacerbations of

PCT / JP2017 /025015

Chung , et al . , “ Cytokines in Asthma, ” Thorax, 1999 , 54 : 825-857 .

( 86 )PCT No .:$ 371 ( c ) ( 1 ) ,( 2 ) Date :Corren , et al ., “ Lebrikizumab Treatment in Adults with Asthma , "New England Journal of Medicine, 2011 , 365 : 1088-98 .
Shifren, et al . , “ Mechanisms of Remodeling in Asthmatic Airways, ”
Journal of Allergy, vol . 2012 , Article ID 316049 , 12 pages .
Zhu , et al . , “ Mex3B : A Coreceptor to Present dsRNA to TLR3 , ” Cell

Jan. 4 , 2019(87 ) PCT Pub . No .: WO2018 /008750
PCT Pub . Date : Jan. 11 , 2018
( 65 )

Research , 2016 , 26 : 391-392.

Sadik , et al ., “ Neutrophils Cascading their way to Inflammation ,"Trends Immunol, Oct. 2011 , 32 ( 10 ) : 452-460 .

Prior Publication Data
US 2020/0325479 A1
Oct. 15 , 2020

Foreign Application Priority Data

( 30 )

Jul. 8 , 2016 ( JP )(51 ) Int. Cl.

Asthma," Thorax , 2007 , 62 : 475-482 .

JP2016-136402

C12N 15/113
A61P 11/06( 2010.01 )( 2006.01 )C12Q 1/6883( 2018.01 )GOIN 33/50( 2006.01 )( 52 ) U.S. CI .
CPC
C12N 15/1136 ( 2013.01 ) ; A61P 11/06(2018.01 ) ; C12Q 1/6883 ( 2013.01 ) ; GOIN

33/5094 ( 2013.01 ) ; C12Q 2600/16 (2013.01 )( 58 ) Field of Classification Search
None
See application file for complete search history.

(Continued )Primary Examiner Brian Whiteman(74 ) Attorney, Agent, or Firm — Emerson Thomson

Bennett; Daniel A. Thomson( 57 )

ABSTRACT

Provided are a method for screening agents for the prevention or treatment of diseases caused by interleukin 6 , interleukin 13 , TNF, G -CSF, CXCL1 , CXCL2 , or CXCL5 and an
agent for the prevention or treatment of diseases caused byinterleukin 6 , interleukin 13 , TNF, G -CSF, CXCL1 , CXCL2 ,or CXCL5 . A method for screening agents for the prevention
or treatment of diseases caused by interleukin 6 , interleukin
13 , TNF, G - CSF, CXCL1 , CXCL2 , or CXCL5 having as theindex at least one selected from the group consisting ofchanges in the expression of the MEX3B gene or MEX3B
protein and changes in the function of the MEX3B protein .
4 Claims , 5 Drawing Sheets
Specification includes a Sequence Listing .

US 11,279,935 B2Page 2( 56 )

References Cited

OTHER PUBLICATIONS
Robinson , et al ., Predominant TH2 - Like Bronchoalveolar T-Lym

phocyte Population in Atopic Asthma, New England Journal of
Medicine , Jan. 30 , 1992 , 298-304 .

LeBorgne, et al . , “ RNA - Binding Protein Mex3b Regulates theSpatial Organization of the Rap1 Pathway, ” Company of Biologists

LTD Development, 2014 , 141 , 2096-2017 .

Yamazumi, et al . , “ RNA Binding Protein Mex - 3B is Required for
IL - 33 Induction in the Development of Allergic Airway Inflammation ,” Cell Reports 16 , 2456-2471 , Aug. 30 , 2016 .

Brightling et al . , “ Targeting TNF - a : A Novel Therapeutic Approachfor Asthma,” J Allergy Clin Immunol., Jan. 2008 : 121 ( 1 ) : 5-12 .
Morishima , et al . , “ Th17 - Associated Cytokines as a Therapeutic
Target for Steroid- Insensitive Asthma, " Clinical and Developmental
Immunology, vol . 2013 , Article ID 609395 , 9 pages .
Al- Ramli, et al . , “ TH - 17 Cell -Related Cytokines' Potential Role in
the Pathogenesis of Severe Asthama,” Journal of Asthma, 2008 ,45 : sup1 , 41-44 .

Bradley, J R. “ TNF- Mediated Inflammatory Disease ,” Journal of
Pathology , 2008 , 214 : 149-160 .
WIPO , WO 2018 008750 , “ International Search Report of PCT/
JP2017 /025015 ” , dated Jan. 11 , 2018 .

JP Patent Office, “ Notificaton of Reasons for Refusal,” JP2017
559904 , dated Mar. 13 , 2018 .

Yan Yang et al : “ The RNA -binding protein Mex3b is a coreceptorof Toll -like receptor 3 in innate antiviral response " , Cell Research
Xibao Yanjiu, vol. 26 , No. 3 , Jan. 29 , 2016 , pp . 288-303 , XP055596004 .
S. Kang et al : “ Therapeutic uses of anti- interleukin - 6 receptor
antibody ”, International Immunology, vol . 27 , No. 1 , Aug. 20 , 2014 ,pp . 21-29 , XP055377592 .
Supplementary European Search Report issued in the EP Patent
Application No. EP17824353.1 , dated Jun . 25 , 2019 .
Office Action issued in the EP Patent Application No. EP17824353 .
1 , dated Jul. 16 , 2019 .
Abstract of W02010005527 published Jan. 14 , 2010 .

Abstract of US2006172930 , published Aug. 3 , 2006 .
Buchet -Poyau , et al . , “ Identification and Characterization of Human
Mex - 3 Proteins, a Novel Family of Evolutionarily Conserved RNA
Binding Proteins Differentially Localized to Processing Bodies,”
Nucleic Acids Research , 2007 , vol . 35 , No. 4 , pp . 1289-1300 .
Mandiyan , et al . , “ Molecular and Cellular Characterization ofBaboon C - Raf as a Target for Antiproliferative Effects of Antisense
Oligonucleotides,” Antisense & Nucleic Acid Drug Development,
Mary Ann Liebert, Inc. , 1997 , vol . 7 , pp . 539-548 .
Wolozin , et al . , “ Participation of Presenilin 2 in Apoptosis: Enhanced>

Basal Activity Conferred by an Alzheimer Mutation ,” Science 1996 ,Yoon , et al . , “ Scaffold Function of Long Non - Coding RNA HOTYAIRin Protein Ubiquitination ," Nature Communications, 2013 , vol .
4.2939 , pp . 1-14 .
Yamazumi, et al . , “ The RNA Binding Protein Mex - 3B is Required
for IL - 33 mInduction in the Development of Allergic Airway
Inflammation ,” Cell Reports, 2016 , vol . 16 , pp . 2456-2471 .
International Search Report dated Sep. 27 , 2017 for International
Application Serial No. PCT /JP2017 /025014 .
Translation of International Search Report dated Sep. 27 , 2017 forInternational Application Serial No. PCT/JP2017/025014 .
Draper, et al . , “MEX - 3 is a KH Domain Protein that Regulates
Blastomerre Identity in Early C. Elegans Embryos,” Cell , 1996 , vol .
87, pp . 205-216 .
Supplementary European Search Report dated Apr. 11 , 2019 forApplication Serial No. EP17824352 .
European Office Action dated May 21 , 2019 for Application Serial

vol . 278 , pp . 1710-1713 .

>>No. EP17824352 .

U.S. Office Action dated Apr.30 , 2021 for U.S. Appl. No. 16/315,594 .

Kurreck , J. et al .; “ Design of antisense oligonucleotides stablized bylocked nucleic acids” , Nucleic Acids Research ; 2002 ; vol . 30 ( 9 ) ; p .
1911-1918 .

Seth, P. et al .; “ Structure Activity Relationships of a- L - LNA Modi
mals ”; Molecular Theerapy - Nucleic Acids ; 2012 ; vol . 1 , e47 ; p . 1-8 .
fied Phoshporothioate Gapmer Antisense Oligoncucleotides in Ani* cited by examiner

U.S. Patent

Sheet 1 of 5

Mar. 22 , 2022

US 11,279,935 B2

FIG . 1
Mex3B
2*

EMRXPeLAxST3IVOBENWILD TYPE MOUSEO Mex3b DEFICIENT???T

INITIALPASSAGE PASSAGE* p < 0.05 , n = 6 (WILD TYPE) , n = 7 ( Mex3b DEFICIENT )FIG . 2
3*

2

ELRXPATSOIVENWILD TYPE MOUSEO Mex3b DEFICIENTPASSAGE PASSAGE* p < 0.05 , n = 6 (WILD TYPE ), n = 7 ( Mex3b DEFICIENT)U.S. Patent

US 11,279,935 B2

Sheet 2 of 5

Mar. 22 , 2022

FIG . 3CXCL520*ECRXPLASTIVO5EN

2 WILD TYPE MOUSEO Mex3b DEFICIENT
50

INITIALLATERPASSAGE PASSAGE* p < 0.05 , n = 6 ( WILD TYPE ) , n = 7 ( Mex3b DEFICIENT)

FIG . 440
35

PBS + / +30

NPBS -OVA + / +OVA - /

104)OFCN(UEMLBESR 25

PBS + / + 0 = 3
PBS - / - in 2
OVA +5

OVA Bar SE* p < 0.05

20

5
met

comuning

0

TOTAL NUMBER MACROPHAGES LYMPHOCYTES NEUTROPHILS EOSINOPHILSOF CELLS

6

U.S. Patent

CI)(-GQOVANOaHApLTmReOIrLN

.
FIG
5

US 11,279,935 B2

Sheet 3 of 5

Mar. 22 , 2022

DCGORNTUOPL GINONRHAVLUTAIPONGINRHAOLUTPN GINRHaApOLmUTeIPrN
S-MIOONPEHAVCXLI3TFABOCN

EOSINPHLal)GROUP
GOVA
CI(
7NOaHNApLTAmReIOrLN S-MOVAI:n=7GNPaEHRApCXLOIm3TUFeIBOPrCN=n:

IOVA:n=G7NHRALOTUIOPNal

CG:n4ORNOTRUOPL

t-KTrueaksmetyr
Bar=S.E.

0.05
<
p
*

NLMEYAUCTPRHOIGS???????

TO AL NUMBER COFEL S
16

14

4
10
)cell 105 x ( CELLS OF NUMBER

12

8

6

2

0

U.S. Patent

US 11,279,935 B2

Sheet 4 of 5

Mar. 22 , 2022

Gapmer**:nGI=7NRHAOL UTIPON :n=7GINHRALOTUIOPN

CS(OPENCTIRFOILCPBSOVAMGaEpXm3eBr

IOVA:n=7GNHRALOTUIOPN

CG:n=4ORNOTRUOPL )C(-GOVAINOaHApLTmReIOrLNOMXS-GPVEaACpXImD3FeABCr

30 25 20 15 10 5 0
level mRNA CSF - G Relative

St-testtudent's 0.01**p<*0.05,
S.E.
=
Bar
*

Gapmer***PBSOVAGMaEpXmi3eBr1

3.0 2.5 2.0 1.5 1.0 0.5

6
.
FIG

CPOENCTIRFOILC **S()

CSOPNETCRFOL**MPBSOVAGEaXm3e8r
301level mRNA CXCL1 Relative

level mRNA TNF Relative

Capmer

Gapme
!

**

**

CONTROL(
SPECFC)SC)(POENCTIRFOILCPBSOVAMGaEpXm3e8r

TLFEROAXNIUSYFCTAEIGMSDCamer

PBSOVAGMaEmXe3r8
1

160 140 120 00 80 60 40 20 0
level mRNA 13 - IL Relative

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

0level mRNA CXCL2 Relative

GapmerGarmerCPOENCTIRFOILCS()

CPOENCTIRFOILCS()

MGPBSOVAEaXm3e8r

MPBSOVAGEaXm3eBr

la
5

1

level mRNA 6 -IL Relativewa
0
25 20 15 10
level mRNA CXCL5 Relative

U.S. PatentMar. 22 , 2022

Sheet 5 of 5

US 11,279,935 B2

SBAR=20umCALE

7
.
FIG

TLOAPNIHUSFYGECALGIONHAVLTIONAPDMRINBOSTUP

GICNORHALTUPN

IOS-MNPHEAVLCXIT3AFOBNCGADMRIaNOpSTmUePr

IOVA(C-G)NOaHApLTmReIOrLNGADMRINOSTUP

US 11,279,935 B2
1

2

METHOD FOR SCREENING
PROPHYLACTIC OR THERAPEUTICAGENTS FOR DISEASES CAUSED BY

of IL - 17 is detected from blood serum of a patient having
severe asthma who has resistance to steroids, it is recently
found that severe asthma is caused by an excessive responseby Th17 cells .
5Furthermore, IL - 13 , TNF (in particular, TNF - a) , andG - CSF are also known to be involved in a progress ofasthma ( e.g. , Non -Patent Document 4 ) .

INTERLEUKIN 6 , INTERLEUKIN 13 , TNF,
G -CSF , CXCL1 , CXCL2 , OR CXCL5 AND
AGENT FOR THE PREVENTION OR
TREATMENT OF DISEASES CAUSED BY

INTERLEUKIN 6 , INTERLEUKIN 13 , TNF,
G -CSF, CXCL1 , CXCL2 , OR CXCL5

As an inflammatory cytokine , IL - 13 is known to play arole of enhancing further the allergic inflammation in

10 peripheral tissues , and, in addition to the aspect that it
promotes an allergic response as a main cause of allergic
asthma, it is also known to be involved in intractability of
The present invention relates to a method for screening asthma for which aa steroid agent is ineffective .
prophylactic or therapeutic agents for diseases caused by
Furthermore, IL - 13 is involved in forming of a syndrome
interleukin 6 (IL - 6 ) , interleukin 13 (IL -13),Tumor Necrosis 15 not only in asthmabut also in inflammatory bowel disease
Factor ( TNF ) , Granulocyte - Colony Stimulating Factor or and atopic dermatitis ( e.g. , Non - Patent Documents 5 and 6 ) .
colony -stimulating factor 3 ( CSF 3 ) ( G - CSF ) , CXCL1 ,TNF ( in particular, TNF - a ) is a signal factor whichCXCL2 , or CXCL5 and a prophylactic or therapeutic agent induces an inflammatory response , and even though it is afor diseases caused by IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 , factor that is important in terms of a defense against infec
20 tion , it is also known to be involved simultaneously in a
CXCL2 , or CXCL5 .
TECHNICAL FIELD

disorder that is caused by augmented inflammation. Namely,
TNF is involved in aggravation of a syndrome in many
disorders, and it is known to be involved mainly in a joint
Allergic airway inflammation has been considered as an disorder ( rheumatoid arthritis , psoriatic arthritis, spondy
allergic disorder that is caused by various allergens (e.g. , 25 loarthropathy, and ankylosing spondylitis ), an inflammatory
BACKGROUND ART

bowel disease ( ulcerative colitis and Crohn's disease ), a
However, people ending up dead due to aggravated cancer (ovarian cancer and breast cancer ), a mental disorderasthma are mostly the elderlies, and the airway inflammation (depression, bipolar disorder, epilepsy, Alzheimer's disease,site of those patients having severe asthma are characterized Parkinson's disease, and multiple sclerosis ), a cardiovascu
in that, neutrophils relating to aa defense against infection are 30 lar disorder (heart failure and arteriosclerosis ), a respiratory
seen in large numbers rather than eosinophils which accu- tract disorder (bronchial asthma, chronic bronchitis, chronic
mulate by an allergic response . Thanks to a progress in obtrusive pulmonary disease, and acute lung injury ), type 2
immunology in recent years , it becomes evident that Th17 diabetes , a kidney disorder ( ischemic renal sorder, rejec
type immune cells ( i.e. , cells responsible for an immune tion after transplantation, and glomerulonephritis ), and the
response corresponding to a defense against infection by 35 like (e.g. , Non -Patent Documents 7 and 8 ) .
secreting mainly IL - 17 ) in those patients with severe asthma
Furthermore , G - CSF is known to have an activity ofare responsible for the basic symptom of disease ( e.g. , promoting granulocyte production and enhancing the function of neutrophils.
Non -Patent Document 2 ) .
It has been also reported that a patient with more severe
Furthermore, CXCL1 , CXCL2 , and CXCL5 belong to theNon -Patent Document 1 ) .

case has higher level of interleukin 17 ( IL - 17 ) in blood 40 inflammatory chemokine CXC subfamily. Inflammation sig
serum , which is one type of cytokines. IL - 17 increases nal activates secretion of CXCL1 , CXCL2 , and CXCL5
secretion of chemokines (CXCL1 , CXCL2 , CXCL5 , and thelike) from lung tissues , and those chemokines recruit neu-from various types of blood cells , fibroblast cells , bloodvessel endothelial cells , blood vessel smooth muscle cells ,trophils to an inflammation site . Infiltration of neutrophils alveolar epithelial cells , or the like ( e.g. , Non -Patent Docu
repeatedly induces chronic inflammation , and , when thick- 45 ments 9 and 10 ) .
ening of smooth muscle , fibrosis of airway mucous memWhen CXCL1 , CXCL2 , and CXCL5 are secreted in lungbrane, hyperplasia of airway submucosal gland, or the like tissues due to an augmentation of excessive inflammation inprogresses , irreversible airway remodeling is eventually airway mucous membrane, infiltration of neutrophils, whichcaused . Once having such state, it is easy to have dyspnea , express high -level CXCR2 as a receptor of CXCL1,
making it very difficult to be treated .
50 CXCL2 , and CXCL5 , is promoted . Consequently, as severe
Meanwhile , IL - 6 is an important cytokine that is involved asthma is caused by the infiltration of neutrophils which
in inflammation , hematopoiesis, bone metabolism , tumor have resistance to steroids, chronic inflammation inducing
aggravation, or the like , and the activity of IL - 6 is known to irreversible airway remodeling is caused .
contribute mainly to a transition from acute inflammation to Non -Patent Document 1 : N Engl J Med , 326 ( 1992 ) , pp .
acquired immune response or an onset of a chronic inflam- 55 298-304
Non - Patent Document 2 : Clinical and Developmental
matory disorder (e.g. , Non - Patent Document 3 ) .
It is known that, as IL - 6 binds to a complex of IL - 6
Immunology Volume 2013 (2013 ) , ArticleID609395, 9
receptor subunit and gp130 ( signal transfer subunit)
pages
expressed on a surface of a target cell , IL - 6 intracellular Non - Patent Document 3 : J Asthma. 2008 ; 45 Suppl 1 : 41-4 .

signal is transferred , and a target gene deeply involved in 60 Non - Patent Document 4 : Thorax . 1999 September; 54 ( 9 ) :various types of biological phenomena that are induced by
825-57 .
IL - 6 is activated by the signal.
Non - Patent Document 5 : J Allergy ( Cairo ). 2012 ; 2012 :For the activation of an acquired immune system , IL - 6
316049signal induces Th17 cells , in cooperation with TGF - B signal. Non - Patent Document 6 : N Engl J Med 2011 ; 365: 1088
Severe asthma shows a symptom that is resistant to steroids , 65 1098
and significant infiltration of neutrophils is shown in the Non -Patent Document 7 : J Allergy Clin Immunol. 2008
inflammatory site . However, since significantly high value
January ; 121 ( 1 ) :5-10

US 11,279,935 B2
3

4

Non - Patent Document 8 : J Pathol. 2008 January ; 214 ( 2 ) :149-60 .
Non -Patent Document 9 : Thorax . 2007 June ; 62 ( 6 ) : 475-82 .

expression level of CXCL5 mRNA in embryonic fibroblast
cells of wild type BALB /c mouse and MEX3B deficient

FIG . 3 is a view showing the result of determining theNon - Patent Document 10 : Trends Immunol. 2011 October;32 ( 10 ) : 452-60 .
DISCLOSURE OF THE INVENTION

Problems to be Solved by the Invention

BALB / c mouse .
5

FIG . 4 is a view showing the result of determining an

increase in various immune cells of wild type BALB / cmouse and MEX3B deficient BALB /c mouse in asthma
inducing test .

FIG . 5 is a view showing the result of determining an

10 increase in various immune cells in a test of administering
As described above , in recent years , it became evident gapmer type antisense oligonucleotide in the severe asthmathat IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 , CXCL2 , or CXCL5 model.
FIG . 6 is a view showing the result of a quantitativeis related with severe diseases.
The present invention is achieved in consideration of the 15 RT- PCR test for mRNA expression level of each of theabove circumstances , and an object of the invention is to cytokines and chemokines in each mouse group .

provide a method for screening prophylactic or therapeutic
FIG . 7 is a view showing the pathological tissue image ofagents for diseases caused by IL - 6 , IL - 13 , TNF, G - CSF, lung tissues in each mouse group .
CXCL1 , CXCL2, or CXCL5 and a prophylactic or thera
PREFERRED MODE FOR CARRYING OUT
peutic agent for diseases caused by IL - 6 , IL - 13 , TNF, 20
THE INVENTION
G - CSF, CXCL1 , CXCL2 , or CXCL5 .

Hereinbelow , embodiments of the present invention are
Means for Solving the Problems
described in detail , but, the present invention is not at all
Inventors of the present invention found that biological limited to the following embodiments, and it can be carried
phenomena in broad ranges are ruled by the function of 25 out with suitable modifications within the range of the????? gene and the MEX3B gene is related with an onset purpose of the present invention .
of a disease that is caused by IL - 6 , IL - 13 , TNF, G - CSF, <Method for Screening Prophylactic or Therapeutic AgentsCXCL1 , CXCL2 , or CXCL5 , and completed the present for Diseases Caused by IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 ,invention . Specifically, the present invention is as described 30 CXCL2
or CXCL5With ,regard
to the>screening method according to the first
below.
embodiment of the present invention , by using as an index
The first embodiment of the present invention isat least one selected from the group consisting of changes ina method for screening prophylactic or therapeutic agents expressionof the MEX3B gene or MEX3B protein andfor diseases caused by IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 , changes in functionof the MEX3B protein, it is possible to
CXCL2 , or CXCL5, the method comprising: screening theprophylactic or therapeutic agents using as an index at least 35 screen prophylactic or therapeutic agents for diseases caused
IL - 13 , TNF, G - CSF, CXCL1 , CXCL2, or CXCL5 .
one selected from the group consisting of changes in expres byItILis- 6 , preferable
to screen prophylactic or therapeuticsion of MEX3B gene or MEX3B protein and changes inagents for diseases caused by IL - 6 , IL - 13 , TNF, G - CSF,
function of the MEX3B protein .
The second embodiment of the present invention is
CXCL1 , CXCL2 , or CXCL5 , and it is more preferable toa prophylactic or therapeutic agent for diseases caused by 40 screen prophylactic or therapeutic agents for diseases caused
IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 , CXCL2 , or CXCL5 , by IL - 6 or CXCL5 .
Examples of the function of the MEX3B protein include
comprising a substance for decreasing expression of
MEX3B gene or MEX3B protein or a substance for inhib- a function of controlling the function (i.e. , translation into
protein ) of various mRNAs of IL - 6 , IL - 13 , TNF, G - CSF,
iting the MEX3B protein .
45 CXCL1 , CXCL2, and / or CXCL5 by binding to the mRNAs,
Effects of the Invention
and a function of inducing the expression of IL - 6 , IL - 13 ,TNF, G - CSF, CXCL1 , CXCL2 , and / or CXCL5 . By using as
The method for screening prophylactic or therapeutic an index at least one selected from the group consisting ofagents for diseases caused by IL - 6 , IL - 13 , TNF, G - CSF, the decrease in expression of the MEX3B gene or MEX3B
CXCL1 , CXCL2 , or CXCL5 according to the first embodi- 50 protein and the decrease in function of the MEX3B protein ,ment of the present invention allows screening of prophy- it is possible to screen prophylactic or therapeutic agents forlactic or therapeutic agents for diseases caused by IL - 6 , the diseases caused by an increased expression of IL - 6 ,IL - 13 , TNF, G - CSF, CXCL1, CXCL2 , or CXCL5 ( e.g. ,IL - 13 , TNF, G - CSF, CXCL1 , CXCL2 , or CXCL5 .
According to the present invention , a prophylactic or among severe asthma, rheumatoid arthritis, colitis , Crohn's
therapeutic agent for diseases caused by IL - 6, IL- 13 , TNF, 55 disease , atopic dermatitis, systemic erythematosus, and can
G - CSF, CXCL1 , CXCL2 , or CXCL5 can be provided.
cer, severe asthma, a joint disorder ( rheumatoid arthritis ,psoriatic arthritis, spondyloarthropathy, and ankylosing
BRIEF DESCRIPTION OF THE DRAWINGS
spondylitis ), diabetes, an inflammatory bowel disorder ( ulcerative colitis and Crohn's disease) , atopic dermatitis,.

FIG . 1 is a view showing the result of determining the 60 systemic erythematosus, a cancer ( ovary cancer and breast

expression level of MEX3B mRNA in embryonic fibroblast cancer), a mental disorder ( depression , bipolar disorder,
cells of wild type BALB / c mouse and MEX3B deficient epilepsy, Alzheimer's disease , Parkinson's disease , and mul
BALB / c mouse.
tiple sclerosis ), a cardiovascular disorder (heart failure and
FIG . 2 is aa view showing the result of determining the arteriosclerosis ), a respiratory tract disorder ( bronchial
expression level of IL - 6 mRNA in embryonic fibroblast cells 65 asthma, chronic bronchitis, chronic obtrusive pulmonaryof wild type BALB / c mouse and MEX3B deficient BALB / c disease , and acute lung injury ), type 2 diabetes, a kidneymouse .

disorder ( ischemic renal disorder, rejection after organ transUS 11,279,935 B2
5

6plantation, and glomerulonephritis ), and the like that arecaused by IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 , CXCL2 , or
CXCL5 (e.g. , Int Immunol. 2015 January; 27 ( 1 ) : 21-9 ,Cancer Discov. 2016 January ; 6 ( 1 ) : 80-95 ) ) .

As described above , IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 ,CXCL2 , and CXCL5 may be deeply involved in the symp
2?

toms of severe asthma.

When it the sequence information of the MEX3B gene is

Furthermore, level of the decrease is , although it is not 5 used as a base , expression of the MEX3B gene in various
particularly limited as long as it is a statistically significant human tissues can be detected even in silico . Furthermore,decrease , preferably 112 or less , more preferably 1/4 or less , also in vivo and in vitro , by using a probe or a primer whichand even more preferably 110 or less compared to thea partial or whole sequence of the gene , expression of theexpression or function of the MEX3B gene or MEX3B has
MEX3Bgene in various human tissues can be detected .
protein in the absence of a test substance ( e.g. , system before 10 Detectionof the MEX3B gene expression can be carriedaadministration of a test substance ( e.g. , wild type ) or system out by a common
method such as RT- PCR , Northern blot ,of negative control ( control administered with a substanceSouthern blot . Furthermore, measurement of an expres
not affecting the expression or function of the MEX3B gene andsionamount of the MEX3B gene at mRNA level can be alsoor MEX3B protein) ) , and it is particularly preferable that thecarriedout by a common method such as RT -PCR , Northern
15
expression or function is not observed .
By using as an index at least one selected from the group blot , and Southern blot .
In the case of carrying out PCR, the primer is notconsisting of the increase in expression of the MEX3B geneor MEX3B protein and the increase in function of the particularly limited as long as it can specifically amplify the
MEX3B protein , it is possible to screen prophylactic or MEX3B gene only, and the primer can be suitably set based

therapeutic agents for diseases caused by decreased expres- 20 on the sequence information of the MEX3B gene. For
sion of IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 , CXCL2 , orexample, an oligonucleotide that contains at least 10 con

CXCL5 (e.g. , viral infection , bacterial infection or the like tiguous nucleotides in the sequence of the MEX3B gene or
the expression control region of the gene , and an antisense( Immunity, 2010 Jul. 23 ; 33 ( 1 ) : 106-17 ) ) .
Furthermore, the level of the increase is , although it is not oligonucleotide having a sequence complementary to theparticularly limited as long as it is a statistically significant 25 oligonucleotide can be used as a probe or a primer. Moreincrease , preferably 1.5 times or more , and more preferably specifically, an oligonucleotide which has a sequence of 10
2 times or more compared to the expression or function of to 60 contiguous residues, and preferably 10 to 40 contigu
the MEX3B gene or MEX3B protein in the absence of a test ous residues in the sequence of the MEX3B gene or thesubstance (e.g. , system before administration of a test sub- expression control region of the gene, and an antisense
30 oligonucleotide having a sequence complementary to thestance or system of negative control ).
It is preferable to use as an index at least one selected from
MEX3B gene or MEX3B protein and the decrease infunction of the MEX3B protein, and it is more preferable tooligonucleotide can be used .
The oligonucleotide and antisense oligonucleotide can beproduced by a common method using a DNA synthesizer.
Examples of the oligonucleotide or antisense oligonucle
use as an index the decrease in expression of the MEX3B 35 otide include , in a partial sequence of mRNA aimed to begene .
detected, a sense primer corresponding to the sequence at 5 'As long as the above is taken as an index, the method for terminal side, and an antisense primer corresponding to thescreening can be any screening method such as in vivo , in sequence at 3 ' terminal side . The sense primer and antisense
vitro , and in silico . As a preferred example of the method for primer are oligonucleotides, in which each of them has

the group consisting of the decrease in expression of thescreening , culturing cells expressing the MEX3B gene in the 40 melting temperature ( Tm ) and base number that neverpresence and absence of a test substance and screening change to an extreme extent, and oligonucleotides withprophylactic or therapeutic agents for diseases caused by approximately 10 to 60 bases can be mentioned . Oligonucle
IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 , CXCL2 , or CXCL5 by otides with approximately 10 to 40 bases are preferable .
having as an index the changes in expression of the MEX3B Furthermore, in the present invention , it is also possible togene or MEX3B protein and the changes in the function of 45 use derivatives of the aforementioned oligonucleotide, and a
the MEX3B protein in accordance with the presence or methylated product or a phosphorothioated product of theabsence of the test substance can be mentioned .
oligonucleotide can be also used , for example.
As the cells that are used for the screening method
Furthermore, measurement of an expression amount at theaccording to the first embodiment, fibroblast cells derived MEX3B protein level can be carried out by a common
from mouse embryo (mouse embryonic fibroblasts (MEF ) ) 50 immunoassay such as Western blot or ELISA using an
are preferable .
antibody to be described later. Specifically, the measurement
The fibroblast cells derived from mouse embryo can can be carried out by a common method that is known to a
induce cell senescence by having simple passage .
person skilled in the pertinent art like those described in the
The MEF system is one of the methods that are used for second edition of Molecular Cloning or Current Protocols in
determining a change in various biological phenomena 55 Molecular
Biology, or the like.
accompanied with cell senescence . It is known that inflamFurthermore, analysis of the changes in function of thematory cytokine or chemokine, which has not been signifi- MEX3B protein can be carried out by measurement of thecantly produced at an early passage ( e.g. , passage 3 ) , is presence or absence, or the level of the binding property ofsignificantly induced in the late passage ( e.g. , passage 13 to the MEX3B protein to mRNA , measurement of the presence15 ) . Based on a reconstitution experiment of cell senescence 60 or absence, or the level of the expression of the function ofof airway mucosal membrane, relationship between defi- mRNA to which the MEX3B protein binds , or measurement
ciency of the MEX3B gene and changes in production of of the presence or absence , or the level of the expression ofcytokine and chemokine in MEF cells can be analyzed.
IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 , CXCL2 , and / or CXCL5 .
The inventors of the present invention found that, in cells
Measurement of the presence or absence , or the level ofwith decreased expression of MEX3B , the secretory factors 65 the binding property of the MEX3B protein to mRNA can be( IL - 6 , IL - 13 , TNF, G - CSF, CXCL1, CXCL2 , CXCL5 , andthe like) may show statistically significant decrease .

carried out by any analysis such as competitive inhibition

test .

US 11,279,935 B2
7

8

Protein - level expression amount measurement of the

CDS , the sequence from 1 to 436 positions corresponds topresence or absence, or the degree of the exertion of the 5'UTR , the sequence from 2983 to 4368 positions corre
function of mRNA to which the MEX3B protein binds can sponds to 3'UTR , and the region from 693 to 1528 positions
be carried out by a common immunoassay such as Western corresponds to the intron region of the human MEX3B gene
blot or ELISA .
5 that is represented by SEQ ID NO : 3 .
mRNA - level measurement of the expression amount of
Mouse MEX3B gene encoding the mRNA of mouse
IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 , CXCL2 , and / or CXCL5 MEX3B has a sequence represented by SEQ ID NO : 4 that
expression can be carried out by a common method such as is described later.
Northern blot , Southern blot , and RT -PCR . Specifically, theIn SEQ ID NO : 4 , the sequence from 319 to 2049

measurement can be carried out by a common method that 10 positions corresponds to CDS , the sequence from 1 to 318

is known to a person skilled in the pertinent art such as those positions corresponds to 5'UTR , and the sequence from2050 to 3416 positions corresponds to 3'UTR .
Furthermore, all genes encoding the MEX3B protein
Current Protocols in Molecular Biology.
As the test substance to be provided to the screening (e.g. , protein having an amino acid sequence represented bymethod according to the first embodiment of the present 15 SEQ ID NO : 5 or 6 that is described later) belong to theinvention , any substance can be used . Type of the test MEX3B gene. The MEX3B gene has been originally iden
substance is not particularly limited , and it can be a nucleic tified as a gene activated by TGF - B , and, based on theacid molecule , an antibody, an individual low - molecular analyses thereafter, the MEX3B protein is known as asynthetic compound, a compound present in an extract of a molecule which binds to various types of mRNA andnatural product, or a synthetic peptide. It can be also an 20 controls the function (i.e. , translation into protein ) of thoseartificial nuclease for genome editing to be described later. mRNAs (e.g. , Nucleic Acids Res . 2007 ; 35 ( 4 ) : 1289-300) .
Alternatively, the test compound can be also a compound
Specific examples of the MEX3B gene include a genelibrary, a phage display library, or a combinatorial library. described in any one of the following ( a) and ( b ), and, from
Construction of a compound library is known to a person the viewpoint of screening prophylactic or therapeuticskilled in the pertinent art, and a commercially available 25 agents for human diseases and also from the viewpoint thatcompound library can be also used . The test substance is it is not needed to carry out unnecessary transformation or
preferably a low -molecular compound ( e.g. , compound the like since as a gene derived from human can be directly
library ), a nucleic acid molecule , an artificial nuclease for used , the gene of the following ( a) is preferable.
genome editing , or an antibody, and from the viewpoint of (a ) Gene consisting of the sequence described in SEQ ID
having high specificity to the MEX3B gene or protein , a 30 NO : 1 or 4 of the Sequence Listing ,nucleic acid molecule or an antibody is more preferable, and ( b ) Gene that is consisting of a sequence resulting froma nucleic acid molecule which has a sequence complemen- deletion , substitution , and / or addition of one or several bases
tary to an oligonucleotide contained in the MEX3B gene of the sequence described in SEQ ID NO : 1 or 4 of the( coding region (CDS ) or untranslated region (UTR ) in exon , Sequence Listing , and also encoding a protein which has anor intron ) or in the expression control region of the gene , or 35 activity of inducing expression of a gene activated by TGF - B
an aptamer or an antibody selectively binding to the MEX3B or a gene having an activity of inducing the expression ofprotein is even more preferable.
IL - 6 , IL - 13 , TNF, G -CSF, CXCL1 , CXCL2 , and / or CXCL5 .
The range of “ one or several” in the “ sequence resulting( MEX3B Gene)
The MEX3B gene includes exon 1 , intron , and exon 2 , from deletion, substitution , and / or addition of one or several
and this constitution is highly preserved in human , mouse , 40 bases of the sequence” described in the present specification
described in the second edition of Molecular Cloning or

and other mammals. Furthermore, a CDS and an UTR are

included in exon 1 and exon 2 .

As an untranslated region (UTR) in an exon which does
not encode any amino acid , 5'UTR is present upstream of theis not particularly limited . However, it preferably means
approximately 1 to 20 , more preferably 1 to 10 , and evenmore preferably 1 to 5 .
As the degree of the DNA variation described above ,initiation codon and 3'UTR is present downstream of the 45 those having homology of 80% or more with the sequencetermination codon .
of the MEX3B gene described in SEQ ID NO : 1 or 4 of the
Human MEX3B gene encoding the mRNA of human Sequence Listing can be mentioned, for example, and pref
MEX3B has a sequence represented by SEQ ID NO : 1 that erably a DNA having homology of 85 % or more , moreis described later.
preferably a DNA having homology of 90 % or more , even
In SEQ ID NO : 1 , the sequence from 437 to 2146 50 more preferably a DNA having homology of 95 % or more,positions corresponds to CDS , the sequence from 1 to 436

and particularly preferably a DNA having homology of 98 %positions corresponds to 5'UTR , and the base sequence from2147 to 3532 positions corresponds to 3'UTR .

or more can be mentioned .
(Obtainment of MEX3B Gene)human MEX3B gene . SEQ ID NO : 3 to be described later
represents 836 bases in an intron region of the humansequence that are described in SEQ ID NOs : 1 , 4 , or 15 and5 or 6 of the Sequence Listing of the present specification

Method for obtaining the MEX3B gene is not particularly
SEQ ID NO : 2 to be described later represents a sequenceof about 36 kilo bases including the expression control 55 limited . By preparing a suitable probe or primer based on theregion upstream of the transcription initiation point of the information of the nucleotide sequence and amino acid

MEX3B gene. In the human MEX3B gene , this intron and selecting a desired clone from human cDNA library (i.e. ,base at 695 position in the sequence represented by SEQ ID which the MEX3B gene is expressed) by using them , theNO : 1 .
MEX3B gene can be isolated .
SEQ ID NO : 15 represents the sequence encoding a
The MEX3B gene can be obtained also by a PCR method .
pre -mRNA of the human MEX3B before splicing . In the For example, by using a chromosomal DNA originating
sequence encoding a pre -mRNA of the human MEX3B that 65 from human culture cells or cDNA library as a template andis represented by SEQ ID NO : 15 , the sequences from 437 a pair of primers designed to amplify the sequence describedto 692 positions and 1529 to 2982 positions correspond to in SEQ ID NO : 1 or 4 , PCR is carried out .

region is present between the base at 694 position and the 60 library prepared by a common method from suitable cells inUS 11,279,935 B2
910(c ) Protein consisting of an amino acid sequence which has
The reaction condition for PCR can be suitably set , and a homology of 95 % or more with the amino acid sequence
condition in which a reaction process consisting of 30 described in SEQ ID NO : 5 or 6 of the Sequence Listing andseconds at 94 ° C. ( denaturation ), 30 seconds to 1 minute at also being activated by TGF - B or having an activity of
55 ° C. ( annealing ), and 2 minutes at 72 ° C. ( elongation ) is 5 inducing or controlling the expression of IL - 6 , IL - 13 , TNF,
taken as 1 cycle , for example , and , after performing 30 G - CSF, CXCL1 , CXCL2 , and / or CXCL5 .
cycles , the reaction is allowed to occur for 7 minutes at 72 °From the viewpoint of screening prophylactic or thera
C. , for example , can be mentioned . Subsequently, an ampli- peutic agents for human diseases and also from the viewfied DNA fragment can be cloned in a suitable vector which point that it is not needed to carry out unnecessary transcan be amplified in a host such as E. coli . Operations 10 formation orthe like since a protein originating from humanincluding production of the probe or primer, construction of can be directly used, the protein of the above ( a) is prefera cDNA library, screening of a cDNA library , and cloning of able .
a target gene or the like are known to a person who is skilled
SEQ ID NO : 5 represents the amino acid sequence ofin the pertinent art, and they can be carried out according to 15 human MEX3B protein . SEQ ID NO : 6 represents the aminoa method described in the second edition of Molecular acid sequence of mouse MEX3B protein .
Cloning or Current Protocols in Molecular Biology, or the
The range of one or several ” in the " amino acid sequence
like .
resulting from deletion, substitution , and / or addition of one
The gene (mutated gene ) that is consisting of a sequence or several amino acids of the amino acid sequence ”
resulting from deletion , substitution , and / or addition of one 20 described in the present specification is not particularly
or several bases of the sequence described in SEQ ID NO : limited . However, it preferably means approximately 1 to
1 or 4 of the Sequence Listing in the present specification, 10 , more preferably 1 to 5 , and even more preferably 1 to 3 .
and also encoding a protein which has an activity of induc- The “ amino acid sequence having homology of 95 % or
ing the expression of a gene activated by TGF - B or a gene more ” described in the present specification means that thehaving an activity of inducing and regulating the expression 25 amino acid homology is 95 % or more , and the homology isof IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 , CXCL2 , and /or preferably 96 % or more , and more preferably 97 % or more.
CXCL5 can be also produced by any method that is known
As described in the above, a physiologically active pro
to a person who is skilled in the pertinent art such as tein which has a binding activity for a specific mRNA andchemical synthesis, genetic engineering techniques, or a physiologically active protein having an activity of inducmutagenesis. For example, by using a DNA having the 30 ing or controlling the expression of IL - 6 , IL - 13 , TNF,
sequence described in SEQ ID NO : 1 and introducing a G - CSF, CXCL1 , CXCL2 , and / or CXCL5 , both as a protein
mutation to the DNA, a mutated DNA can be obtained . encoded by a mutant gene which has high homology with
Specifically, it can be carried out by using , for a DNA having the gene having sequence described in SEQ ID NO : 1 or 4
the sequence described in SEQ ID NO : 1 or 4 , a method of of the Sequence Listing, are all within the scope of thebringing the DNA into contact with aa chemical agent as a 35 present invention .
mutagen for chemical action , a method of irradiating UVSide chains of the amino acid as a constitutional element
light, genetic engineering techniques, or the like. Site of a protein may be individually different in terms of thedirected mutagenesis , which is one of the genetic engineer- hydrophobicity, charge, size , or the like. However, from theing techniques, is useful in that it is a method allowing aspect that no substantial influence is exhibited on the
introduction of a specific mutation to a specific site , and it 40 three - dimensional structure of a whole protein ( also referredcan be carried out according to a method described in the to as a stereo structure ), several relationships having highsecond edition of Molecular Cloning or Current Protocols in conservancy are known either by experience or by actual
physical and chemical measurements . For example , for
Molecular Biology , or the like .
As described above , even if the DNA sequence is partially substitution of an amino acid residue , glycine (Gly) andchanged due to various artificial treatments of the sequence 45 proline (Pro ), Gly and alanine (Ala ) or valine ( Val), leucineof the MEX3B gene described in SEQ ID NO : 1 or 4 of the (Leu ) and isoleucine (Ile ) , glutamic acid (Glu) and gluta
Sequence Listing including introduction of site directed mine ( Gln ) , asparaginic acid ( Asp ) and asparagine ( Asn ) ,mutagenesis, random mutation caused by treatment with a cysteine ( Cys ) and threonine ( Thr ), Thr and serine ( Ser) or
mutating agent, and mutation, deletion , ligation or the like of Ala , lysine ( Lys) and arginine (Arg ) or the like can be
a DNA fragment caused by cut of restriction enzyme, the 50 mentioned .
DNA sequence is within the scope of the MEX3B gene
Thus, even mutated proteins resulting from deletion ,regardless of a difference from the DNA sequence described addition, substitution , or the like on the amino acid sequencein SEQ ID NO : 1 or 4 as long as the DNA mutant is a DNA of the MEX3B that is described in SEQ ID NO : 5 or 6 of thewhich encodes a protein activated by TGF - B , or a protein Sequence Listing are all within the scope of the MEX3B
having an activity of inducing the expression of IL- 6 , IL - 13 , 55 when the mutation is aa mutation which is highly conservedin terms of the three - dimensional structure of the MEX3B
TNF, G - CSF, CXCL1 , CXCL2 , and / or CXCL5 .
(Mex3B Protein )and when the mutated protein is a physiologically active
protein having a binding activity for a specific mRNA or a
The MEX3B protein is any one of the followings.
( a) Protein consisting of the amino acid sequence described physiologically active protein having an activity of inducing
in SEQ ID NO : 5 or 6 of the Sequence Listing,
60 or controlling the expression of IL - 6 and / or CXCL5 similar(b ) Protein consisting of an amino acid sequence resulting to the MEX3B .
from deletion, substitution , and / or addition of one or several
Method for obtaining the MEX3B protein is not particu
amino acids of the amino acid sequence described in SEQ ID larly limited , and it may be a protein synthesized by chemi
NO : 5 or 6 of the Sequence Listing , and having a binding cal synthesis , a protein derived from nature which has been
activity for a specific mRNA or having an activity of 65 isolated from a biological sample or cultured cells or the
inducing or controlling the expression of IL - 6 , IL - 13 , TNF, like , or a recombinant protein prepared by genetic engineer
ing techniques.
G - CSF, CXCL1 , CXCL2 , and / or CXCL5 , or<

US 11,279,935 B2
11

12< Prophylactic or Therapeutic Agent for Diseases Caused by
IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 , CXCL2 , or CXCL5 >The prophylactic or therapeutic agent for diseases caused
by IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 , CXCL2 , or CXCL5

antisense oligonucleotide having a sequence complementary
to an oligonucleotide with 17 or less contiguous nucleotides
is most preferable.
The antisense oligonucleotide is preferably an antisense
according to the second embodiment ( hereinbelow , also 5 oligonucleotide which contains at least one nucleotide hav
simply referred to as an “ prophylactic or therapeutic agent ing at least one structure that is selected from the groupaccording to the second embodiment " ) comprises a sub consisting of a phosphorothioate structure, a crosslinked, and an alkoxy structure .
stance for decreasing the expression of the MEX3B gene or structureFor
exampleas the phosphodiester bonding part connect
the MEX3B protein , or a substance for inhibiting the 10 ing nucleotides, hasa phosphorothioate structure, resistance
MEX3B protein.
tonucleasecanbeobtained, and , from the viewpoint that the
The prophylactic or therapeutic agent according to theis enhanced, incorporation to inside of a cellsecond embodiment is preferably a prophylactic or thera hydrophobicity
a nucleus can be also enhanced .
peutic agent for diseases caused by IL - 6 , IL - 13 , TNF, or Furthermore, as the sugar part of a nucleotide has a
G - CSF, CXCL1 , CXCL2 , or CXCL5 , and it is more pref 15 crosslinked structuresuch as 2 ' , 4 ' - BNA ( 2,4 ' - Bridged
erably a prophylactic or therapeutic agent for diseases Nucleic Acid ; other nameLocked
Nucleic Acid (LNA ))caused by IL - 6 or CXCL5 .
and
ENA
(
2-0,4
'
C
Ethylene
bridgedNucleic Acid) , or an( Antisense Oligonucleotide)
alkoxy structure such as 2 - O -methylaion and 2 - O -methoxy
As the substance for decreasing the expression of the ethylation (2 ' - MOE ) , the resistance to nuclease can be
MEX3Bgene or the MEX3B protein, the aforementioned 20 obtained and also the binding property of mRNA can be
antisense oligonucleotide, which has a sequence comple- enhanced .
mentary to an oligonucleotide contained in the MEX3B geneWith regard to the antisense oligonucleotide, it is prefer( CDS or UTR in exon , or intron ) or in a region for control- able that at least one phosphodiester bonding part connectling the expression of the same gene , can be mentioned .
ing nucleotides has a phosphorothioate structure, it is more
Introduction of the antisense oligonucleotide to cells 25 preferable that 50 % or more of the phosphodiester bond ininhibits transcription or translation of the MEX3B gene so the antisense oligonucleotide has a phosphorothioate structhat diseases caused by IL - 6 , IL - 13 , TNF , G -CSF, CXCL1 , ture , it is even more preferable that 70 % or more of thephosphodiester bond in the antisense oligonucleotide has a
CXCL2 , or CXCL5 can be prevented or treated .
For example, as an oligonucleotide contained in the phosphorothioate structure , it is particularly preferable that
MEX3B gene (CDS or UTR in exon , or intron ) or in a region 30 90% or more of the phosphodiester bond in the antisense
for controlling the expression of the same gene and the oligonucleotide has a phosphorothioate structure, and it isantisense oligonucleotide complementary thereto form a most preferable that all of the phosphodiester bonds in the

hybrid after their introduction to a 11, mRNA of theantisense oligonucleotide have a phosphorothioate structure .

MEX3B is decomposed by a nuclease (e.g. , RNase H )With regard to the antisense oligonucleotide, it is prefer
specific to the generated hybrid double strand so that the 35 able that at least any one terminal nucleotide has a crosstranscription or translation of the MEX3B gene can be linked structure or an alkoxy structure, it is more preferable
inhibited .
that the nucleotides at both terminals of the antisense
As the antisense oligonucleotide, an antisense oligonucle- oligonucleotide have a crosslinked structure or an alkoxy
otide having a sequence complementary to an oligonucle- structure ( i.e. , so - called gapmer type antisense oligonucle
otide that contains at least 10 contiguous nucleotides in the 40 otide ) , it is even more preferable that, in both terminals of
sequence ( CDS or UTR in exon , or intron ) of the MEX3B the antisense oligonucleotide, up to 4 bases from the termi
gene or in aa region for controlling the expression of the same nal independently have a crosslinked structure or an alkoxy
gene is preferable, and an antisense oligonucleotide having structure , and it is particularly preferable that 2 or 3 bases
a sequence complementary to an oligonucleotide that con- from the terminal have a crosslinked structure or an alkoxy
tains at least 11 nucleotides is more preferable, an antisense 45 structure .
oligonucleotide having a sequence complementary to an
As one embodiment of the method for introducing theoligonucleotide that contains at least 12 nucleotides is even antisense oligonucleotide to cells , an embodiment in whichmore preferable, an antisense oligonucleotide having a insertion to a suitable vector is made and further introducsequence complementary to an oligonucleotide that contains tion to a suitable host cell is carried out can be mentioned .
at least 13 nucleotides is particularly preferable, and an 50 Type of the suitable vector is not particularly limited, andantisense oligonucleotide having a sequence complementary it can be a self - replicating vector ( e.g. , plasmid or the like) ,to an oligonucleotide that contains at least 14 nucleotides is for example. However, it is preferably a vector that ismost preferable.
incorporated into a genome of a host cell upon introduction
Furthermore, with regard to the upper limit value of the to a host cell and replicated with a chromosome to which it
length of the antisense oligonucleotide, an antisense oligo- 55 has been incorporated.
nucleotide having, in the sequence (CDS or UTR in exon , orAs the suitable vector, a plasmid derived from E. coli
intron ) of the MEX3B gene or in a region for controlling the (e.g. , pBR322, PUC118 , and the like ) , a plasmid derived
expression of the same gene , aa sequence complementary to from Bacillus subtilis ( e.g. , pUB110 , PSH19 , and the like) ,an oligonucleotide with 40 or less contiguous nucleotides is and also bacteriophage or an animal virus such as retrovirus

preferable, an antisense oligonucleotide having a sequence 60 or vaccinia virus can be used . During recombination , it iscomplementary to an oligonucleotide with 30 or less con- also possible to add a translation initiation codon or a
tiguous nucleotides is more preferable, an antisense oligo- translation termination codon by using a suitable synthetic
nucleotide having a sequence complementary to an oligo- DNA adaptor.
nucleotide with 25 or less contiguous nucleotides is even
Furthermore, if necessary, the antisense oligonucleotide
more preferable, an antisense oligonucleotide having a 65 can be also functionally bonded to a suitable terminator suchsequence complementary to an oligonucleotide with 20 or as a human growth hormone terminator, or, for a fungal host ,less contiguous nucleotides is particularly preferable, and an

a TPI1 terminator or an ADH3 terminator, for example. The

US 11,279,935 B2
13

14
the MEX3B gene , or a DNA encoding that double -stranded
RNA is preferable, and a double - stranded RNA containing
25 or less contiguous nucleotides in a CDS or an UTR of therecombination vector may also have an element such aspolyadenylation signal ( e.g. , those derived from SV40 or
adenovirus 5Elb region) , a transcription enhancer sequence( e.g. , SV40 enhancer ), and a translation enhancer sequence sequence of an RNA to be transcribed from the sequence of( e.g. , those encoding adenovirus VARNA ). The recombina- 5 the MEX3B gene, or a DNA encoding the double -strandedtion vector may also be provided with a DNA sequence RNA is more preferable.
which enables replication of the vector in a host cell , andRNA interference (RNAi) indicates a phenomenon show
examples thereof include SV40 replication origin (when the ing inhibited expression of a target gene when an RNA
host cell is a mammalian cell ) . The recombination vector ( double - stranded RNA : dsRNA ) in which part of mRNA
may also include a selection marker. Examples of the 10 encoding a part of a certain target gene is prepared as aselection marker include a gene of which complement is double strand is introduced to a cell . Examples of the DNA
deficient in a host cell such as dihydrofolate reductase encoding a double - stranded RNA include a DNA having a( DHFR) or Schizosaccharomyces pombe TPI gene, or a gene reverse - direction repeating a sequence of the MEX3B or a
resistant to pharmaceuticals such as ampicillin , kanamycin , partial sequence thereof. By introducing a DNA having atetracycline, chloramphenicol, neomycin , or hygromycin , 15 reverse - direction repeating sequence to cells of mammals,
for example.
the reverse - direction repeating sequence of a target gene canExamples of a host cell to which the antisense oligonucle- be expressed in a cell , and, accordingly , it becomes possible
otide or a vector containing it include a higher eukaryotic to inhibit the expression of the target gene ( MEX3B ) based
cell , a bacterium , a yeast , and a fungus, but it is preferably on the RNAi effect. The reverse -direction repeatinga mammalian cell .
20 sequence indicates a sequence in which a target gene and a
Examples of the mammalian cell include HEK293 cell , sequence in the reverse reaction thereof are present in
HeLa cell , COS cell ( e.g. , COS - 7 cell and the like ), BHK parallel via a suitable sequence . Specifically, for a case incell , CHL cell or CHO cell , BALB /c mouse cell ( e.g. , which a target gene has a double - strand consisting of n
BALB /c mouse embryonic fibroblast cell ) , and the like . A nucreotide sequences shown below,
method of transforming a mammalian cell and expressing a 25
gene introduced to the cell is also known, and a lipofection
Xn - 1Xn - 3
5 ' - X1X2 ·method , an electroporation method , a calcium phosphate
method, and the like can be used , for example.
Yn - 1Yn - 5
3 ' - Y1Y2 .
The prophylactic or therapeutic agents according to thesecond embodiment may additionally contain a carrier for 30 the reverse - direction sequence thereof has a sequence shown
lipofection from the viewpoint of enhancing the incorpora- below .
tion to a cell , but it is also possible not to containany carrier.
Examples of the carrier for lipofection include a carrier
5 ' - Y,Yn- 1
Y2Y1-3 'which has high affinity to cell membrane (e.g. , liposome orcholesterol), and it is preferably lipofectamine or lipofectin, 35
3 ' - X,Xn- 1
X2X1-5 'and more preferably lipofectamine.
For example, as the expression of the MEX3B gene is ( herein , with regard to the bases represented by X and theinhibited by introducting the antisense oligonucleotide bases represented by Y , those having the same subscript are
together with aa carrier for lipofection to cells of a patient by the bases that are complementary to each other ).
administering, via injection or the like, to a lesion or whole 40 The reverse -direction repeating sequence is a sequence inbody of a patient, diseases caused by IL - 6 , IL - 13 , TNF, which the above two types of sequence are present via a
G - CSF, CXCL1 , CXCL2 , or CXCL5 can be either prevented suitable sequence . As the reverse - direction repeating
sequence , a sequence having a target gene upstream of theor treated .
Furthermore, as the antisense oligonucleotide has at least reverse -direction sequence and a sequence having a reverse
one structure that is selected from the group consisting of a 45 direction sequence upstream of a target gene sequence arephosphorothioate structure, a crosslinked structure, and an considered . The reverse - direction repeating sequence usedalkoxy structure and it is used in combination with a carrier in the present invention can be any one of them , but it isfor lipofection, incorporation to a cell or a nucleus of a preferable that the reverse - direction sequence is present
upstream of a target gene sequence. The sequence present
patient can be further enhanced .
The administration amount of the antisense oligonucle- 50 between a target gene sequence and a reverse - direction
otide as an effective component is , for single administration , sequence thereof is a region in which a hairpin loop is
generally within a range approximately 0.1 ug to 100 mg per formed when transcription into an RNA is made ( shRNA :kg of bodyweight.
small hairpin RNA ). Length of this region is not particularly( siRNA )limited as long as a hairpin loop can be formed , but it is
As a substance for decreasing the expression of the 55 preferable to be approximately 0 to 300 bp , and more
MEX3B gene or MEX3B protein, a double - stranded RNA preferable to be approximately 0 to 100 bp . It is also possible( small interfering RNA ( siRNA ) containing at least 20 that aa restriction enzyme site is present in that sequence .
contiguous nucleotides in a CDS or an UTR of the sequence
According to the present invention , by incorporating aof an RNA to be transcribed from the sequence of the reverse -direction repeating sequence of a target gene to a
MEX3B gene , or a DNA encoding the double - stranded RNA 60 downstream of a sequence of a promoter which is operable
can be also mentioned . A double - stranded RNA containing in mammals, the reverse -direction repeating sequence of aat least 21 contiguous nucleotides in a CDS or an UTR of the target gene can be expressed in cells of mammals. A
sequence of an RNA to be transcribed from the sequence of sequence of a promoter used in the present invention is not
the MEX3B gene , or a DNA encoding that double - stranded particularly limited as long as it is operable in mammals.
RNA is preferable . A double -stranded RNA containing 30 or 65 For example, when the double - stranded RNA or DNA areless contiguous nucleotides in a CDS or an UTR of the administered via injection or the like , together with a carrier
sequence of an RNA to be transcribed from the sequence of for lipofection used for facilitating the incorporation to cells ,US 11,279,935 B2
15

to a lesion or whole body of a patient followed by incorpo-

ration to cells of a patient, severe asthma can be inhibited .

The administration amount of the double - stranded RNA or

16

individually . Cas nuclease fused to a protein transduction

domain such as Tat can be also delivered to the inside ofcells . Preferably, a eukaryotic cell or a eukaryotic organism

DNA as an effective component is , for single administration , is simultaneously transfected or contiguously transfected
generally within a range approximately 0.1 ug to 10 mg per 5 with Cas9 nuclease and a guide RNA . Contiguous transfec
kg of bodyweight.
tion can be carried out by a first transfection using a nucleic
acid encoding Cas nuclease and subsequently a second
MEX3B gene or MEX3B protein may be an artificial second transfection is 3 , 6 , 12 , 18 , 24 hours later, but it is notnuclease for genome editing such as Clusterd Regularly 10 limited thereto . Expression of a guide RNA can be also
( Artificial Nuclease)

The substance for decreasing the expression of thetransfection using a naked guide RNA . Preferably, the>Interspaced Short Palindromic Repeats (CRISPR) /Cas carried out by using a unit for expressing guide RNA . The
nuclease , and an artificial restriction enzyme ( artificial unit for expressing guide RNA is preferably a CRISPR - Cas9
nuclease ) using Transcription Activator -Like Effector based transcription unit which includes a target sequence
Nuclease ( TALEN ) and zinc finger nuclease (ZFN) . TALEN (i.e. , a partial sequence of the MEX3B gene) and a guideis an artificial nuclease including TALES, i.e. , domain 15 RNA, and those having a promoter region for expressing theformed by polymerization of four types of units which guide RNA ( promoter of RNA polymerase III ( e.g. , pro
recognize and bind any one of four types of bases (A , T, G , moter selected from U6 promoter and H1 promoter) ) , target
and C ) , and TALEs recognizes at least a partial sequence of sequence (i.e. , the MEX3B gene) , and a guide RNA are
the MEX3B gene and binds thereto .
preferable, and those in which the promoter, a sequence
ZFN is an artificial nuclease in the form of a chimeric 20 complementary to a target sequence (i.e. , at least a partial
protein which includes a zinc finger domain and a DNase sequence of the MEX3B gene) , and a guide RNA are
domain . The zinc finger domain has a structure in which connected in aa seamless manner are more preferable. As theplural units of a zinc finger, which recognizes specific CRISPR /Cas nuclease, a Cas9 mutant which cuts off, as a
3 -nucleotides, are polymerized , and it is a domain recog- nickase , only one strand of a double - stranded DNA in ordernizing and binding a DNA sequence with a multiple of three 25 to prevent the off -target may be used . Examples of afor binding, and the zinc finger domain recognizes at least a single - stranded restriction type Cas9 mutant include Cas9
partial sequence of the MEX3B gene and binds thereto .
(D10A) . When a guide RNA which has a target sequence
CRISPR /Cas nuclease includes a guide RNA and Cas complementary to one strand of aa target DNA and a guideRNA which has a target sequence complementary to the
nuclease ( preferably , Cas9 ) .
The guide RNA means an RNA which binds to Cas 30 other strand extremely close to that one strand are used innuclease as a DNA digesting enzyme and has ?a function of combination , for example, the single - stranded restriction
guiding Cas nuclease to a target DNA ( at least a partial type Cas9 mutant can cut off that one strand with specificity
sequence of the MEX3B gene ). The guide RNA on its of 20 bases while further cutting off the other strand with
5 ' terminal, a sequence complementary to a target DNA (at specificity of 20 bases so as to cut off a DNAwith specificity

least a partial sequence of the MEX3B gene),and when the 35 of 40 bases . Accordingly, it becomes possible to enhance

guide RNA binds to a target DNA via the complementary

remarkably the target specificity.

sequence, the guide RNA guides Cas nuclease to a target
The administration amount of the artificial nuclease or the
DNA . Cas nuclease functions as a DNA endonuclease, cuts nucleic acid encoding the artificial nuclease as an effective
off a DNA at aa site in which a target DNA is present, and can component is , for single administration , generally within a
specifically reduce the expression of the MEX3B gene, for 40 range approximately 0.1 ug to 10 mg per kg of bodyweight.
example .
The prophylactic or therapeutic agent for diseases caused

At least a partial sequence of the MEX3B gene as a target by IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 , CXCL2, or CXCL5
preferably has 15 to 25 bases , more preferably 17 to 22 according to the second embodiment can be administeredbases , even more preferably 18 to 21 bases , and particularly systemically or topically, either orally or parenterally .
preferably 20 bases.
45 Examples of a method for parenteral administration include
As a eukaryotic cell or a eukaryotic organism carrying the intravenous injection such as dropping addition , intramus
MEX3B gene is transfected with a composition which cular injection, intraperitoneal injection, and subcutaneouscontains a guide RNA specific to the MEX3B gene or a injection. The administration method can be suitably
DNA encoding the guide RNA , and a nucleic acid encoding selected depending on age and symptom of a patient. The
Cas nuclease or Cas nuclease , the expression of the MEX3B 50 administration amount varies depending on age , administra
gene can be reduced .
tion route , and the number of administrations, and it can be
The nucleic acid encoding Cas nuclease or Cas nuclease, suitably selected by a person who is skilled in the pertinentand the guide RNA or DNA encoding the guide RNA can be art . Examples of the preparation form suitable for parenteral
introduced to inside of cells by various methods that are administration include those containing additives such as
known in the field of the pertinent art, for example, micro- 55 stabilizing agent, buffering agent, preservative, isotonic
injection , electroporation, DEAE -dextran treatment, lipofec- acid , or the like, and those containing a pharmaceuticallytion, nano particle -mediated transfection , protein transduc- acceptable carrier or an additional product are also accept
tion domain -mediated transduction , virus -mediated gene able . Examples of those carrier and additional producttransfer, PEG -mediated transfection of protoplast, or the include water, an organic solvent, a polymer compound
like , but it is not limited thereto . Furthermore , the nucleic 60 ( collagen , polyvinyl alcohol , or the like) , stearic acid, humanacid encoding Cas nuclease or Cas nuclease , and the guide blood serum albumin ( HSA) , mannitol, sorbitol, lactose , andRNA can be incorporated to the inside of a biological a surface active agent, but they are not limited thereto .
organism by various methods for administering a gene or a ( Aptamer or Antibody Selectively Binding to MEX3B Pro
protein that are known in the field of the pertinent art, for tein)example, injection or the like . The nucleic acid encoding Cas 65 Substance for inhibiting the MEX3B protein can be any
nuclease or Cas protein can be incorporated to inside of substance such as high molecular compound ( nucleic acid orcells , either in the form of a complex with guide RNA or

the like ) , antibody, and low molecular compound as long as

US 11,279,935 B2
17

18

it can inhibit the function of the MEX3B protein . As one iting a sufficient antibody titer can be used as a source forpreferred embodiment of the substance for inhibiting the supplying blood serum or cells for producing the antibody.
MEX3B protein , a prophylactic or therapeutic agent for The polyclonal antibody can be produced by separating anddiseases caused by IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 , purifying the blood serum .
CXCL2 , or CXCL5 , in which an aptamer selectively binding 5 The monoclonal antibody can be produced by preparing ato the MEX3B protein is used, can be mentioned . An hybridoma according to fusion between the antibody -gen
aptamer indicates aa nucleic acid pharmaceutical which con- erating cells and myeloma cells derived from non - humansists of a single - stranded RNA or DNA and inhibits the mammal, culturing the hybridoma or causing an animal tofunction of a target protein as it binds to the protein based have ascites cancer by administering the hybridoma to an
on its steric structure . The aptamer has a high binding 10 animal, and separating and purifying the culture liquid orproperty and specificity for a target protein and low immu- ascites . As the antibody -generating cells , antibody - generat
nogenicity , can be produced by chemical synthesis, and has ing cells in spleen cells , lymph nodes , or peripheral bloodhigh storage stability. Nucleotide length of an aptamer can be used , and , particularly preferably, spleen cells can beselectively binding to the MEX3B protein is not particularly used .
limited as long as it can specifically bind to the MEX3B 15 As the myeloma cells , established cell lines derived fromprotein . However, it is preferably 15 to 60 bases , more a mouse such as P3 -X63Ag8 - U1 ( P3 - U1 ) strain [ Current
preferably 20 to 50 bases , even more preferably 25 to 47 Topics in Microbiology and Immunology, 18 , 1-7 ( 1978 ) ] ,bases , and particularly preferably 26 to 45 bases . The P3 - NS1/ 1 -Ag41 ( NS - 1 ) strain [ European J. Immunology , 6 ,aptamer selectively binding to the MEX3B protein can be 511-519 ( 1976 ) ] , SP2 /0 -Ag14 ( SP - 2 ) strain [Nature, 276 ,obtained by Systematic Evolution of Ligands by EXponen- 20 269-270 ( 1978 ) ] , P3 - X63 - Ag8653 ( 653 ) strain [ J. Immunology, 123 , 1548-1550 ( 1979 ) ] , and P3 -X63 -Ag8 (X63 ) strain
tial enrichment ( SELEX ) method .
As another preferred embodiment of the substance for [Nature, 256 , 495-497 ( 1975 ) ] , which are myeloma cell lines
inhibiting the MEX3B protein , a prophylactic or therapeutic of 8 -azaguanine resistant mouse ( derived from ( BALB / c ) ,agent for diseases caused by IL - 6 , IL - 13 , TNF, G - CSF, can be used . The hybridoma cells can be produced by the
CXCL1 , CXCL2 , or CXCL5 , in which an antibody selec- 25 following method . First , antibody - generating cells andtively binding to the MEX3B protein is used , can be myeloma cells are admixed with each other, and, after being
mentioned . Either a polyclonal antibody or a monoclonal suspended in HAT medium [medium obtained by adding
antibody can be used as long as it can specifically bind to the hypoxanthine , thymidine, and aminopterin to normal
MEX3B protein . The polyclonal antibody can be produced medium ], they are cultured for 7 to 14 days . After culture ,by separating and purifying blood serum that is obtained 30 part of the culture supernatant is collected and reacted withfrom an animal immunized with an antigen . The monoclonal an antigen according to an enzyme immunoassay or the like ,antibody can be produced by preparing a hybridoma anti- and those not reacting with a protein not including the
body - generating cells obtained from an animal immunized antigen are selected . Subsequently, according to limiting
with an antigen and myeloma cells that are fused to each dilution, cloning is carried out and those recognized withother, and culturing the hybridoma or causing an animal to 35 high and stable antibody titer by an enzyme immunoassay
have ascites cancer by administering the hybridoma to the are selected as hybridoma cells which produce a monoclonalanimal and separating and purifying the culture medium or antibody. The monoclonal antibody can be produced bymice ascites . The antigen can be produced by purifying the separation or purification from culture obtained by culturing
MEX3B protein from various cultured human cells , or by the hybridoma cells or from ascites obtained by intraperi
introducing a recombination vector which contains a DNA 40 toneal administration of hybridoma cells to an animal toencoding the amino acid sequence of the MEX3B protein or cause the animal to have ascites cancer .
a mutant sequence thereof or a protein having part of them
As a method for separating and purifying the polyclonal
to a host such as E. coli , yeast , animal cells , or insect cells antibody or monoclonal antibody, a method such as centri
and separating and purifying the protein that is resulting fuge , ammonium sulfate precipitation, caprylic acid precipi
from expression of the DNA . The antigen can be also 45 tation , or a method based on chromatography using DEAE
produced by synthesizing, by using a peptide synthesizer, a sepharose column , anion exchange column, Protein A or
peptide having a partial sequence of the amino acid G -column , or gel filtration column or the like may be used,sequence of the MEX3B protein .
either singly or in combination thereof, can be mentioned .
With regard to a method for immunization , it is possible
When an antibody is referred in the present specification,
to have direct subcutaneous , intravenous, or intraperitoneal 50 not only a whole - length antibody but also a fragment of an
administration of an antigen to a non - human mammal such antibody may be referred . The fragment of an antibody is
as rabbit, goat, rat, mouse , or hamster, but it is also prefer- preferably a functional fragment, and examples thereofable that the antigen is administered while it is bound to a include F ( ab ' ) 2 and Fab ' . F (ab ' ) 2 and Fab ' are produced bycarrier protein having high antigenicity such as sukashigai treating immunoglobulin with a proteinase (e.g. , pepsin or
hemocyanin , keyhole limpet hemocyanin , bovine serum 55 papain ), and they are an antibody fragment produced byalbumin , or bovine thyroglobulin , or administered with a digestion either before and after the disulfide bond present
suitable adjuvant such as Complete Freund's Adjuvant, between 2H chains in a hinge region .
aluminum hydroxide gel , or pertussis vaccine .
When the antibody is used for the purpose of adminis
Administration of an antigen can be carried out, after the tration to a human , it is preferable to use a humanized typefirst administration , 3 to 10 times with an interval of 1 to 2 60 antibody or a humanized antibody in order to reduce theweeks . Blood is taken from postorbital venous plexus on immunogenicity. Those humanized type antibody andDay 3 to Day 7 after the each administration , and investi- humanized antibody can be produced by using mammals
gation is made to see whether or not the blood serum reacts such as transgenic mice . The humanized type antibody iswith the antigen used for immunization , according to mea- described in Morrison, S. L. et al . [ Proc. Natl . Acad . Sci .
surement of an antibody titer by enzyme immunoassay or 65 USA , 81 : 6851-6855 ( 1984 ) ] and Hiroshi Noguchi [ Journal
the like . With regard to the antigen that is used for immu- of Clinical and Experimental Medicine, 167 : 457-462
nization , a non -human mammal having blood serum exhib- ( 1993 ) ] , for example. A humanized chimeric antibody can beUS 11,279,935 B2
19

20

produced by linking the V region of a mouse antibody to the PCR , and , as a result of cross -breeding the mice each other,
C region of aa human antibody by genetic recombination . The a homozygote F2 was obtained . Genotype of the F2 mousehumanized antibody can be produced by replacing a region was confirmed by PCR .
of a mouse monoclonal antibody other than the complemen- ( Isolation and Subculture of Embryonic Fibroblast Cells )tarity determining region (CDR) with a sequence derived 5 Trypsin powder (manufactured by GIBCO ) was dissolved
from aa human antibody.
in PBS ( phosphate buffered physiological saline) so as toFurthermore, the antibody can be also used as an immo
bilized antibody which is immobilized onto an insoluble

carrier such as solid phase carrier, or as a labeled antibody

have a concentration of 0.25 % ( W / V ), and then subjected tofiltering sterilization by passing it through a 0.45 micrometer

which is labeled with a labeling material . Those immobi- 10 filter (manufactured by Advantec ) ( prepared at time of use) .

lized antibodies and labeled antibody are also within the
13.5 Days after starting the cross -breeding of mouse , thescope of the present invention .
whole body of the female C57BL/ 6 mouse was subjected toAmong the antibodies that are described abo the anti
alcohol sterilization using 70 % ethanol, and , after opening
body which specifically binds to the MEX3B protein and 15 the abdomen , the uterus containing embryonic mice wascan inhibit the function of the protein can be used as a collected . After cutting off the portion connected withprophylactic or therapeutic agent for diseases that are caused umbilicalcord with scissors , the embryonic animals wereby IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 , CXCL2 , or CXCL5 .
When the antibody is used in the form of a pharmaceutical taken out one by one from the uterus, and then immersed incomposition as a prophylactic or therapeutic agent for PBS . After removing the head , intestines, paws , and taildiseases that are caused by IL - 6 , IL - 13 , TNF, G - CSF, 20 from the embryonic animal , it was transferred to 1 mL
CXCL1, CXCL2 , or CXCL5 , a pharmaceutical composition trypsin solution which has been kept on ice , and minced tocan be produced by using the antibody as an effective a size of 2 to 3 mm by using sharp scissors . After transfer tocomponent and also using a pharmaceutically acceptable a 15 mL tube, the liquid volume was adjusted to 5 mL per
carrier, a diluent ( e.g. , immunogenic adjuvant or the like), a embryonic animal by using the trypsin solution, and thenstabilizer, a vehicle , or the like . The prophylactic or thera- 25
peutic agent containing an antibody for diseases that are shaken for 10 minute at 37 ° C. , 60 to 100 cycles /minutecaused by IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 , CXCL2 , or while monitoring the digestion state . To terminate the
CXCL5 can be formulated, after filtering sterilization and trypsin reaction, 1 mL of bovine albumin serum (FBS ) was
freeze -drying, into administration form in an administration 30 added and suspended well . Further, to remove the cellvial or stabilized aqueous preparation. Administration to a lumps, the resultant was filtered using a mesh of 100 mm cellpatient can be carried out by a method that is known to a strainer (manufactured by Falcon ) . After centrifuge ( 280xg,
person who is skilled in the pertinent art, for example,
intraarterial injection, intravenous injection, or subcutane for 5 minutes, 4 ° C. ) of the filtered cell suspension , theous injection . The administration amount may vary depend- supernatant was removed and the obtained precipitates wereing on bodyweight or age of a patient and administration 35 suspended in a basic medium (DMEM (High Glucose)
method, but aa suitable administration amount can be suitably (Dulbecco's modified Eagle medium : manufactured by NIS
selected by a person who is skilled in the pertinent art . The SUI PHARMACEUTICAL CO . , LTD .), 10% FBS , penicil
administration amount of the antibody as an effective com lin streptomycin ) and the resultant was sown on a 100 mm
ponent is , for single administration , generally within a range 40 culture dish , at a ratio of 1 well per embryonic animal . On
approximately 0.1 ug to 100 mg per kg of bodyweight.
the next day, the medium exchange was replaced, andEXAMPLES
embryonic fibroblast cells were used for experiments after acouple of passages using trypsinization .
Hereinbelow , the present invention is described in greater
Each of the embryonic fibroblast cells of the wild typedetail by showing examples, but the scope of the present 45 BALB/c mouse and embryonic fibroblast cells of theinvention is not limited to those examples.
MEX3B deficient BALB /c mouse was cultured in 35 mm
Example 1
cell culture dish (BD Falcon : 353001 ) having 5 % FBS

containing DMEM (Dulbecco's modified Eagle medium :(Production of MEX3B Deficient (Knock - Out) BALB / c 50 manufactured by NISSUI PHARMACEUTICAL CO . ,Mouse )
LTD . ), in which 2.5 ug /mL of Fungizone is contained , in a
As a genomic DNA of the MEX3B gene, the genomic carbonate
gas incubator (37 ° C. , 5 % CO2 in air ), and, after
DNA introduced to BAC clone (RP23-272F2 ) was used . The the cells becamein confluent state , subculture was carriedtargeting vector was constructed in a manner such that a out.
gene cassette in the BAC clone resistant to neomycin is 55

substituted with the DNA region of exon 1 and exon 2 of the
MEX3B gene . The construct was introduced by electroporation to BALB / c -derived ES cells ( supplied from Dr. Yasui

at Osaka University ) and selected using an antibiotic( Quantitative RT -PCR Test)
For each of the wild type mouse and MEX3B deficientmouse , by using a dissolution buffer TRlsure (manufactured
by BIOLINE ) for the cells at the early passage (passage 3 )(G418analyzed). Presenceor absence
homologous
cells at the late passage (passage 13 to 15 ), total RNA
wasby PCRmethodof, FISH
method, recombinationand Southern 60 andwas. By using Primescript (manufactured byblot , and 4 ES clones were identified . The selected ES cells Takararecovered
Bio Inc. ), a reverse transcription reaction was carried
were injected to blastocyst of a C57BL/ 6 mouse , and thentransplanted in a surrogate parent in order to obtain a out to obtain cDNA . After that, by using Light Cycler 480
chimeric mouse . By cross -breeding the obtained male chi- 65 (manufactured by ROCHE ) , quantitative RT -PCR was car
meric mouse with a female BALB / c mouse , F? mice each ried out . The quantitative statistical analysis was carried out
having a MEX3B heterogenous mutation were identified by by more than 3 independent experiments.
1

US 11,279,935 B2
21

22

A sequence of the primers used for the quantitative

Mex3B certainly regulates IL - 6 and CXCL5 , and also , when
it is seen the other way around, it is demonstrated that an
onset or a progress of disorders caused by IL - 6 or CXCL5(e.g. , severe asthma) can be inhibited by inhibiting the
5 function of the MEX3B gene product.
(Severe Asthma Inducing Test)( SEQ ID NO : 7 )RT - PCR test is as follows.
?????

primer Fw1 :5 ' - CGTCGTCCTCTGTGGTCTTTCCCGGGGGTG - 3 !

MEX3B primer Rvl :( SEQ ID NO : 8 )5 ' - TCAGGAAAAAATGCGGATGGCCTGAGTGAC - 3 !

Mouse GAPDH primer Fwl :( SEQ ID NO : 9 )5 ' - AGAGACAGCCGCATCTTCTT - 3 'Mouse GAPDH primer Rvl :5 ' - GACAAGCTTCCCATTCTCGG - 3

On Day 0 , each of the females of 8 -week old wild type

BALB /c mouse and MEX3B deficient BALB /c mouse wassubjected to subcutaneous sensitization with 50 ul of Com
and 20 ug of egg white albumin (OVA : Sigma- AldrichCompany) emulsified in 50 ul of PBS . On Day 21 and Day

10 plete Freund's Adjuvant (CFA : Sigma -Aldrich Company )22 , all mice were forced to inhale an aerosol which consistsof 0.1 % OVA in PBS ( 6 or more mice/group ) . Furthermore,

15 as a control, inhalation of an aerosol, which consists of PBS ,( SEQ ID NO : 10 )was carried out . On Day 23 , the numbers of the various typesof immune cells in bronchoalveolar lavage were measured .

FIG . 4 is a view showing the result of determining an

Mouse IL - 6 primer Fwl :( SEQ ID NO : 11 )increase in various immune cells (macrophage , lymphocyte,

20 neutrophil, and eosinophil) of the wild type BALB / c mouseand MEX3B deficient BALB / c mouse in bronchoalveolar
lavage fluid of the severe asthma model. In FIG . 4 , PBS +/ +( SEQ ID NO : 12 )5 ' - GCTACCAAACTGGATATAATCAGGA - 3 'Mouse IL - 6 primer Rvl :5 ' - CCAGGTAGCTATGGTACTCCAGAA - 3 !

Mouse CXCL5 primer Fwl :indicates the number of cells in the wild type BALB /c mousewhich has inhaled PBS as a control, PBS - / - indicates the25
numberof cells in the MEX3B deficient BALB / c mouse( SEQ ID NO : 13 )5 ' - CAGAAGGAGGTCTGTCTGGA - 3 !

Mouse CXCL5 primer Rvl :( SEQ ID NO : 14 )5 ' - TGCATTCCGCTTAGCTTTCT - 3 'which has inhaled PBS as a control, OVA + / + indicates thenumber of cells in the wild type BALB / c mouse which has
inhaled OVA , and OVA - / - indicates the number of cells in

the MEX3B deficient BALB /c mouse which has inhaled

30 OVA . As it is evident from the results shown in FIG . 4 , theFIG . 1 is a view showing the result of determining, by
quantitative RT -PCR , the expression level of MEX3B

wild type BALB /c mouse and MEX3B deficient BALB /cpassage 3 as early passage of each wild type mouse , a

onstrated that severe asthma has been induced . On themouse , which have inhaled OVA , all exhibited increased

total cell number and increased macrophage number com
mRNA in embryonic fibroblast cells of the wild type pared
to control, and they were sensitized with OVA . Fur
BALB /c mouse and MEX3B deficient BALB / c mouse . FIG .
1 shows a mean value of 6 wild type mice and a mean value 35 thermore, as the increased number of neutrophils and theincreased number of lymphocytes are shown from the wildof 7 MEX3B deficient mice with p value at significance level type
BALB/ c mouse which have inhaled OVA , it is dem( <0.05 ) . Y axis indicates, with regard to the actual data at

contrary, as the number of neutrophils and lymphocytes inrelativenumerical
value
when
the
gene
amountofaquan40the
MEX3B deficient BALB/c mouse which have inhaledtification subject divided by GAPDH amount is set at “ 1 ” . OVA are decreased and this showed statistical difference, it
The same shall apply for FIGS . 2 and 3 that are described
below. As it is evident from FIG . 1 , as a result of analyzing is demonstrated that symptoms of severe asthma have been
the cells of the embryonic fibroblast of the wild type mouseameliorated . From the results that are described above,

searching a substance that can inhibit the function of theand MEX3B deficient mouse at early passage ( passage 3 ) 45 MEX3B protein ( e.g. , low molecular compound , protein ,and late passage (passage 13 to 15 ) , it was shown that the all nucleic acid or the like ) is useful as a method for screening
cells derived from MEX3B deficient mouse exhibited no prophylactic or therapeutic agents for diseases that are
expression of Mex3B , both at early passage and late pas- caused by IL - 6 , IL - 13 , TNF, G - CSF, CXCL1 , CXCL2 , orsage .
CXCL5 .
Furthermore, FIG . 2 is a view showing the result of 50
Example 2
determining, by quantitative RT - PCR , the expression levelof IL - 6 mRNA in embryonic fibroblast cells of the wild type
BALB /c mouse and MEX3B deficient BALB / c mouse . FIG .

( Test of Administering Gapmer Type Antisense Oligonucle

3 is a view showing the esu of determining, by quantitative otide in aa Model for Severe Asthma)
RT -PCR , the expression level of CXCL5 mRNA in embry- 55 On Day 0 , 8 -week old female wild type BALB / c mice
onic fibroblast cells of the wild type BALB /c mouse and were subjected to subcutaneous sensitization with 50 ul of
MEX3B deficient BALB /c mouse . As itis evident from CFA (manufactured by Sigma- Aldrich Company ) and 20 ug
FIGS . 2 and 3 , as a result of analyzing the cells of the of OVA (manufactured by Sigma -Aldrich Company ) emul
embryonic fibroblast cells of the wild type mouse and sified in 50 ul of PBS .
MEX3B deficient mouse at early passage and late passage , 60 For 5 consecutive days starting from Day 16 , inhalation
it is shown that, in the case of the cells derived from MEX3B administration of a gapmer type antisense oligonucleotide
deficient ouse , the expression of IL - 6 and CXCL5 is sig- (i.e. , 5 ml of 10uM solution was subjected to prepare aerosol
nificantlylowered in the cells at late passage.
by using nebulizer, and , after filling it in a container, in
From the above results in FIGS . 2 and 3 showing that, in which mice were exposed to it for 20 minutes) was carried
the cells that are deficient of Mex3B gene , the expression of 65 out. As gapmer type antisense oligonucleotides, a control
IL - 6 and CXCL5 is lowered in fibroblast cells that are in the gapmer and a mouse MEX3B specific gapmer were admincourse of aging at late passage , it is demonstrated that the istered by inhalation. Hereinbelow , they are also referred toUS 11,279,935 B2
24

23

as “ OVA inhale -gapmer ( control) inhalation group ” and" OVA inhale -MEX3B specific gapmer inhalation group ” ,respectively. As a mouse MEX3B specific gapmer, aa gapmer
type antisense oligonucleotide ( 5 '- ACATAAACGAGTGGT PCR primer
3 ' : SEQ ID NO : 16 ; total length : 15 bases ) that is comple- 5
mentary to the sequence from 3135 to 3149 sites included in MouseTNF
3'UTR of SEQ ID NO : 4 , which represents the mouse Fw
Mex3B gene , was used . Furthermore, at both ends of eachgapmer type antisense oligonucleotide, 2 bases of LNA MouseTNF( 2 ', 4 ' -BNA ) were added and natural DNA was employed as 10 Rv
bases filling between gaps while the phosphodiester bond MouseG - CSF
connecting each nucleotide was phosphorothioated.
Fw
On Day 21 , Day 22 , and Day 23 , all mice were forced to MouseG - CSF
inhale an aerosol which consists of 0.1 % OVA in PBS ( 7mice /group ). Furthermore, as a control group (i.e. , control ), 15 Ry
inhalation of an aerosol, which consists of PBS , was carried MouseIL - 6
Fwout ( 4 mice / group ).
On Day 24 , samples were collected and the numbers of MouseIL - 6
the various types of immune cells in bronchoalveolar lavage Rv
20
fluid was measured . The results are shown in FIG . 5 .
- 13
In FIG . 5 , the control group represents the cell numbers in MouseIL
Fw

TABLE 1 - continued

Sequence ofSEQ

PCR primer

ID
NO

TCTTCTCATTCCTGCTTGTGG

19

GAGGCCATTTGGGAACTTCT20CCTGGAGCAAGTGAGGAAGA21GGGGTGACACAGCTTGTAGG

22GCTACCAAACTGGATATAATCAGGA11

CCAGGTAGCTATG
GTACTCCAGAA

12CCTCTGACCCTTAAGGAGCTTAT23

CGTTGCACAGGGGAGTCT24

CAGAAGGAGGTCTGTCTGGA

13TGCATTCCGCTTAGCTTTCT

14

AAAATCATCCAAAAGATACTGAACAA

25

CTTTGGTTCTTCCGTTGAGG

26

the mice which have inhaled PBS as a control, OVA

inhalation group represents the cell numbers in the mice MouseIL - 13
which have inhaled OVA , OVA inhale - gapmer ( control) Rv
inhalation group represents the cell numbers in the mice 25 MouseCXCL5

which have inhaled OVA after inhaling in advance thegapmer ( control ), and OVA inhale -MEX3B specific gapmer
inhalation group represents the cell numbers in the mice
which have inhaled OVA after inhaling in advance theFw

MouseCXCL5
Rv30
MEX3B specific gapmer.
MouseCXCL2
As it is evident from the results shown in FIG . 5 , in the Fw
severe asthma model , the mouse group which has inhaled
OVA showed both increased total cell number and increased
MouseCXCL2
macrophage number compared to control, and sensitized Rv

with OVA . Furthermore, as the neutrophil number has 35 MouseCXCL1
27AGACTCCAGCCACACTCCAA
increased in the mouse group which has inhaled the gapmer Fw( control) in advance, it is demonstrated that severe asthma is MouseCXCL128TGACAGCGCAGCTCATTG
induced .
Rv
On the contrary, as the neutrophil number has decreasedin the mouse group which has inhaled the MEX3B specific 40
gapmer and there was significant difference, it is demonAs it is evident from the results shown in FIG . 6 , because
strated that the symptoms of severe asthma are ameliorated . the expression of all of IL - 6 , IL - 13 , TNF, G - CSF, CXCL5 ,Accordingly, it is demonstrated that a MEX3B specific CXCL1 , and C L2 has significantly increased the OVA
gapmer functions as a prophylactic or therapeutic agent for inhalation group compared to the PBS inhalation group , itdiseases that are caused by IL - 6 , IL - 13 , TNF, G - CSF, 45 was confirmed that the severe asthma model was induced as
expected . Furthermore , compared to the gapmer ( control)
CXCL1 , CXCL2 , or CXCL5 .
administration group , expression level of IL - 6 , IL - 13 , TNF,
( Quantitative RT- PCR Test)
G - CSF, CXCL5 , CXCL1 , and CXCL2 tends to decrease inLung tissues were removed from the mice of each group the
MEX3B specific gapmer administration group and there
and they were disrupted by a polytron homogenizer in a
TRlsure (manufactured by Bioline ) solution . According to 50 were significant differences between control and MEX3B

specific gapmer administration groups, and it was shown
the product protocol , total RNA was collected and an that, by inhibiting the expression of the MEX3B , those
amplification reaction was carried out by using PCR primers inflammatory
factors can be inhibited .
that are shown in the following Table 1. Then, expression (Pathological Tissue
Analysis of Lung Tissues)level of mRNA of each cytokine ( IL - 6 , IL - 13 , TNF, andCollected mouse lung tissues were fixed with 10% for
G- CSF ) and each chemokine (CXCL1 , CXCL2 , and 55 malin solution, and then embedded in paraffin ( Tissue- Tek ).
CXCL5 ) was measured . The results are shown in FIG . 6 .
Thin slicing was carried out by using a microtome (manu
TABLE 1

Sequence ofSEQ

PCR primer

ID
NO

MouseGAPDH
Fw2TGTGTCCGTCGTGGATCTGA

17

MouseGAPDHTTGCTGTTGAAGTCGCAGGAG

18

PCR primer

Rv2

factured by LEICA) , and the thin slice was adhered onto aslide glass having APS ( aminosilane ) coating (manufactured
by Matsunami) and subjected to H & E staining (hematoxylin
60 and eosin staining) according to a standard protocol (reference document: Cell Rep . 2016 Aug. 30 ; 16 ( 9 ) : 2456-71 ) .
The pathological tissue images were photographed by using
an Olympus microscope system . The results are shown in
FIG . 7 .
65
FIG . 7 is a view showing the pathological tissue image ofthe lung tissues in each mouse group , in which degree of theinflammation response in lung tissues is shown .

US 11,279,935 B2
25
As it is evident from the results shown in FIG . 7 ,according to the outer appearance, an inflammation response
has not occurred in the control inhalation group ( i.e. , PBS
aerosol inhalation ), which is the same as the mouse not26
showed the almost same results as the control inhalationgroup , and it exhibited no inflammation . Those results
suggest that, according to administration of an MEX3B
specific gapmer, onset of asthma that is resistant to steroids
received
any treatment, and significant infiltration of 5 is dramatically inhibited . As such, it is demonstrated that theimmune cells was not shown . From the OVA inhale - PBS
MEX3B specific gapmer can function as a prophylactic or
administration group and OVA inhale - control gapmer therapeutic agent for diseases that are caused by IL - 6 , IL - 13 ,administration group , thickening of bronchial epithelial cells TNF, G -CSF, CXCL1 , CXCL2 , or CXCL5.
and significant infiltration of immune cells were observed ,clearly indicating that an inflammation was caused .
10
SEQUENCE LISTING
On the other hand, the OVA inhale -MEX3B specificgapmer administration group, which is a group administeredwith a gapmer exhibiting the effect of inhibiting Mex3b , ATF - 207_ST25.txt
9

SEQUENCE LISTING< 160 > NUMBER OF SEQ ID NOS : 28
< 210 > SEQ ID NO 1
< 211 > LENGTH : 3532
< 212 > TYPE : DNA< 213 > ORGANISM : Human< 400 > SI

CE : 1

tcaagaggcg gggaggagag gaggaaaaag cgctgagtga gggcgggcgg gcgggcggga

60

gggagggagt ggaggagccg gggaggggct ccttaaagaa acgcttctgt cgctcgcctg

120

ctcgcttttc gctcggcatt gcggccagcc agccgggcac tcggggggca cgcgcggcca

180

ccgctagagc tctgccccca ccccacccgc cagcaggtct ggggtgggga cccaggtggg
ggctcctgca gccactgccc ggtgcggacc gcacggagcg acccactcct cccagcaccg

240

aggaagaagc aacggagccc tcagcaggcg accggcctcc ccgcccctga ccacccgott

360

cccggctgcc tttgtggccg cagcttctcg ccgccgagcc gagggccggc gggggcgcgg

420

cgcgcacggc cgagcgatgc ccagctcgct gttcgcagac ctggagcgca acggcagcgg

480

cggcggcggc ggcggcagca gcggaggggg agagaccctg gatgaccaaa gagccctgca

540

gctcgcgctc gaccagctct ccctgctggg gctggacagt gacgagggcg cctctctgta

600

cgacagcgag ccgcgcaaga agagcgtgaa catgaccgag tgcgtgccag tacccagttc
tgagcatgtc gccgagatog tggggcggca aggttgtaaa atcaaagcgc tgcgggcgaa
gaccaatact tacatcaaga ccccagttcg cggggaggag cctgtctttg ttgtgacggg
caggaaggag gatgtggcca tggctcggag ggagatcatc tctgctgccg agcacttctc

660

840

catgatccgc gcctcccgga ataagaacac ggcactcaac ggcgcggtgc ctgggccgcc

900

caacctgccc gggcagacca ccatccaagt gcgggtaccc taccgcgtgg tggggctcgt

960

ggtggggccc aaaggcgcca caatcaagcgcatccagcag cagacgcaca cgtacatcgt

1020

gacgcccagc cgggataagg agccggtgtt cgaggtgacc ggcatgccag agaacgtgga

1080

tcgcgctcga gaggagattg aggcgcacat tgctctgcgt accggcggca tcattgagct1140

cacagacgag aacgacttcc acgccaacgg caccgatgtg ggcttcgatc tgcatcatgg

1200

gtccggcggg tccggcccag gcagcctctg gagcaagccc acccccagca tcacgcccac

1260

ccccggccgc aagcctttct ctagctaccg caacgacagc tccagctcgc ttggcagtgc

1320

ttccacagac tcttatttcg goggcgggac cagcagcago gcagcggcta cccagcgcct1380

ggcggactac agccccccta gccccgccct gagctttgcg cacaacggaa acaataacaa

1440

taacggcaat gggtacacct acacagcggg gggagaagcc tcagtgccat cccccgacgg

1500

ctgccccgag ctgcagcoca cttttgaccc ggctcccgct cccccacctg gggcaccact

1560

300

720
780

US 11,279,935 B2
28

27- continued
tatctgggcc cagttcgagc ggtccccggg aggcggacct gcagctccgg tatcttcttc
ctgctcttct tctgcatctt cgtctgcttc ttcctcctcc gtggtcttcc ccgggggtgg

1620

cgccagtgcg ccctccaacg ccaacctggg gctattggtg caccgccggc tgcaccctgg

1740

caccagctgc ccgcgcctgt ctccaccctt gcacatggcc ccgggggcgg gagagcacca
cctggctcgc cgggtgcgca gcgacccggg tggaggaggc ctggcctacg ccgcttatge
caacgggctg ggggcacagc tgcctggctt gcagccgtcg gacacgtcgg gctcctcctc

1800

ttcgtccagc tcctcctcca gctcttcatc ctcttcctcc gggcttcggc gtaaaggcag

1980

ccgcgactgc tccgtgtgct tcgagagcga agtgattgcc gcgctggtgc cctgtggcca

2040

caacctcttc tocatggagt gcgccaatcg catctgtgag aagagcgagc ccgagtgccc
ggtctgccac accgcggtca ctcaggccat ccgcatcttt tcttaaaggc agcgggcgct

2100

gctagtgcgc accgtgctgg gggaaggggg aacccctccc catcctcttt ccccagcgct

2220

cgcctgcctc cctgggtgcc ccccctctcc cttctccttc ccggccccac caacactctg

2280

agatccgaga ggagcttgga aagctgtagt atccgctcat ttttaaaatt taatttttaa

2340

gtaaaggaat ttgccaggat atctgcatca agagtactgt agcctgggaa acctgaacac
ctgaaatgca tgctctataa ataataggaa cggcgacatt ctagtaatga tagtttttac

2400

2460

actgtactta ataggaagct tccaaaagaa gaaaacccca caagttttcc attttcttaa

2520

agtaggaaaa aatgaacagt aataattatg atgaagatga tagtagtgct atgggatgtg

2580

tggactgttt agtgtgttcc cctttgtggg tgggttccta tgatacttat tatagaacac
agtggatcct ttttgaatgt tcgtggaagggccaggagtt cctgtgaaac caggatactg

2640

1680

1860

1920

2160

2700

cagctttatt aaagttaaag aaactgtaac atatctctta tatattaaaa acgtttaaaa
gttttaaaga gaaattgcat taatacagat tgaagtattt tattcttttt tgacttgaaa

2760

aattatattt catattgcaa agatgtttac aagtatttta atttaagttc agtgaacttt

2880

tttgtagctg ggttaaatct ttttatttta gtatggcctt atggcaaaga acactgtatt

2940

attttaataa tcacacaatt gtgacggaat tacaaccata aaatgtgtaa tgttttgaac
agtattctgt tgggatggag attttatagg ttcagacaaa tcttctagat ctgcttcacc

3000

cagcatattt tctattcagt gatataaagc atattttatt ctatattatt acaaaaacgg

3120

aaatgtataa acatgtcaaa aagaactgtt gatgctttct aacatttgta taaatagaat

3180

tcagtgcaag ttacaaaaat tctgttgcac cactctagtt ttagtatttc tattttaata
catttgttta ccacttgttt atgtatatgt aggtgatgtt acttgagctt aaatgtactt

3240

tactgagcaa agtttaaaaa acaaagtata ttttatttta tgataaaggg cctttaacct3360

catggtcaaa tactaatatt atatttgctg agacaagatt tgaaattgta tcaagagttt
tatttttctg acatttaaag ttctacataa taaaggtaaa acttaagtaa tggtgctact

3420
3480

tcatttttta agtatttcta tataaataaa atattgaaga aaatcttaaa aa

3532

2820

3060

3300

< 210 > SEQ ID NO 2
< 211 > LENGTH : 35919< 212 > TYPE : DNA< 213 > ORGANISM : Human< 400 > SEQUENCE : 2

ctccaggtgt gttaacaacaaca ataatggaat agtttgaaat gccaggcaca60

gtctgaagtg tgttatagac ttgtattatc tcactcaagc ctcacagcat ccctgcgtga

120

aggtggaatc attatcccaa atttgcagac aagactgagg ctcagagaga tcagtaagta

180

US 11,279,935 B2

29

30- continued

gtagagctga gattcaagcc tggattcgct tgactccgta gtccatgtac taactaacca

240

ctaggttaaa ctcccttgtt gggcctgacccatgaattgg aaaaccagaa cctggcccca
tcttcgtcag tacttggctg ggtcaccttg agaaagtcac tttccctctc ctgtcctctg

300

atttattatg agtcaaatac aggtagtacc tatcagggtt tectgaagag gtacctatga

420

aaatgatttg tttccctgct gtaagtgata agaaaacctc caaagataat gcccccctgt

480

ctttttaagg cttctcattc tttcttatgg cactttctgc tcatgagtgg aaacttatgt

540

gtatttactg cagggacaac cacattttgt ccgcagagct atggggtgca gcaaaggaag

600

gtaggatttg gtacatttcc ccaactctct cctgaccaga tccatatatt ctcagctggt

660

tggaggcgtg actccaatct aactgaccca caaaacctct gcctctctcc cttctctgct
tgactcatcc attcttcaga acccagtttg ggagatctct tccagaaagg cttctgtgac

720

catcagtctg agtgtggggc ttctccttga gctcccacca agctctctct cttttttttt
tcttttttcc acacaggatt gtgctctgtc actcaggctg gagtccagtg gtgcgatcat
agctcactgc agccttgatc tcctgggctg aagtgatctt tccacctcag cctcccaagc
agttgggacc acaggcatgc atcatcacgc ctggctaatt aaaaattttt tttttcagag
aaggggtctt gctatgttgt ccaggctggt cttaaactcc tgggctcaag cgatcctccc

360

780
840
900
960

1020
1080

gccttggcct cccctagtac tggggttaca ggcatgaggg actgagctgg gcaccacgct

1140

ctccttgtaa cttcttcctc tctccacaaa ggcagggact ttgattgtct aatgcctgat

1200

ctgctgccca gcaggaacgc tctcagtaaa agatgaatga atgaaaaatg ggtcagatgc

1260

acctgcaatg atgtgtgatt cattctcaaa gttgtgtggg tgtctggttg gactctgctc

1320

atgttcctcc tgtgtcccca ggtcccactt ctcactgatg cgccccaggc agtctgtggt

1380

gctctactct ttctccaggg ccacctatca ctgaacctac aggatggccc tgatgaatca

1440

ggggcaggtg gcacaggtgg atggcagggg tacaatgtgc cagaagtgag gtgttcaccc

1500

cctgttcagg tggtttgggt tggagtctaa gctggtttcc tctattcctg tcttactcaa

1560

gggaaattcc tacccctggc caagacagaa ataggagaaa ccagcttaaa atccatccct

1620

ttcctctgag acagaaaacc atcaaagtgt aacacattca ccttatctaa ggaaacctca

1680

aatttactca aaatggcaga gagagaaaaa tgcgtctctt gtgtgtgtgg agttggagcc

1740

acataatgca cattcagagg gtttacaccc ctcatggagg attcttctcc ttataaagtt

1800

gccacaaaag aagagacaca gccatggatg ctcacaacct aactaagatt aggataagcc

1860

tatggggcgg ggcggtgggg cagacagaca ggaatacaat gccttctgcc tctgcatgcc

1920

atttgtcttt cagggaaatc tggctgagaa ctgctgtaga tacttgctat cagcaagaca

1980

ctctcccatt tcctacccca gagttatgag gagtgtacgt gctgctggat gggagcagcg
cctgggcaga ttccatagac agaaatgtga ttctgatgga aataaaacaa acaaacaaag

2040

2100

caaactcagc aaaagtctca gcagtcatga accttctggg aaagagctgc actaaattac

2160

cgagtctctt catttagatg aaagtgcgtg tcttgagaca tcattgttat tttaaccato2220

tgggaggaaa gtattctccc taaagcctga gctccttgaa ggcagagctg tgtcttacat

2280

aactctacat catccatggc gcctggttgt agtagttgcc ttcccaatac ctcttctttt

2340

cttctttacc aacagaacct ctgtattgtt ggggtttgaa gtaataagct acatttccca

2400

ttcttcttgc acagagagat ggcaaatgag atgtaagcaa aattgattaa agcgctgtta

2460

gagctgtttg aaagggattg agaagaatgc ctttttgctg tttccctttc ctcctgcctg2520

US 11,279,935 B2
32

31- continued
ctgactggaa tttacatgtg atgacttgta tttcagcago cattttgtga ttatgagttg

2580

actttgaaga tgaaagccac atgctataag aatggtgaag caaaaagata ggtttctaaa

2640

gcttctgaac agcaaaggaa acaatcaaat gtaccccata aatatataca cctattatat

2700

gcccataata attaaaaaat taaaaagggg ctgggagcag tggctcagac ctataatccc
agcactttgg gaggccaagg caggtggact gctgagctcg ggagtttgag atcagcctgg

2760

gcaacacggt gaaaccctgt ctctaaaaaa aaaaaaaaaa aaaaaaatta gctgggagtg
gtgtgtgcct gtgttoccag ctactcagga ggctgaggtg ggaggattgc ttgagcccag
gaggttgagg ctgcagtgag ccatgataac gttactgcac tccagcctgg gcctgttatg

2880

2820

2940

3000

caaaagtgca tgggctctgg gagagaggtt gggaggctcg cctttgaata tgggtattaa
gcttcagtgg atgaattata tatcatttat taatatcttg gctgctcaga aaccttttta
ctcacttgca gotttggcca catcttaaac agctgcatag ctagctctaa acaggtgaga

3120

attctgaaca ctacatctca aatttccttt tttttttttt ttgagacaga ttcagattct

3240

tgctctgtca cccaggctgg ggtgcagtgg cacggtcttg gctccctgca acctccgcct
cccgggttca agcgattctc ctgcctcagc ctccccagta gctaggatta caggcgcatg
ccaccacgct cggctacttt tggtattttt agcagagacg gtgtttcact atgttggcca

3300

ggctggtctg gaactcctgg ccttgtgate tgcccacctc ggcctcccaa agtgctggga

3480

ttacaggtgt gacccaccac gcccagccac atttcaaatt totaagagct caggcacagg

3540

gaggtgagca tgacatcagg taagggaagt aagggttcgg tctgtcgttc ctgctggaca
ctgaaggtgg acgtgtattt gatgaccaga tagaagatgg gtccttgtgt agtcttttca
cttcagagga gttcctttgc tctcttgggg cttcgggttc cctttccatc cagatgccca

3600

3660

gatgctccta gactggttct tccctttctt ccctggcttt ctgttttctg tgottgctca

3780

ggggaatagt gtgggaagac agtttccctc acaggttcaa ggcggtagag cgttgttgag

3840

ggtctcctgc tggagaagtg gaactttgcg actgtcactc aggagtgcaa tgcagtttct

3900

gagatgaaaatttgcgaacc aatttcacgt ggtcaatgtt gtgaatgaat ttatagctag

3960

tgaggctctg agtgctgatc agctgggatg actttggagc tagatctgaa tactaatggg

4020

actgggtgac tttagttggt gagaattttg agaccctctt tgttttgttt aaaatggtct

4080

gaggcttttt tttttttttt tttttttttg agatggagtc ttgctctgtc goccaggcta

4140

gagtcagtgg tgtgatctcg gottaccgca acctccgcct cccaggttca agcgattctc

4200

ctgcctcago ctgctgagta gctgggacta caggcgcctg ccaccacgcc caggcaattt

4260

ttggattttt agcagagacg gtgtttcacc atgatggcca ggctggtctg gaactcctga
ccttgtgatc tgcccacctc ggcctcccaa agtgctgaga ttacagccat gtgccaccac

4320

acccaggcgt attatatcaa taacaatttc ttagttttga caaatgtacc atgattgtgt

4440

aagatgttac tagtaggcaa tgctggataa agggtataca agaatcatat atgccatctt

4500

tgtaactctt ctgtaaatct aaaattattc caaaattaaa agtttaaaat aatttttaaa

4560

aagtcaatca ccaaaaagta agtacattta ggacatagat acatgcaaga catgtgtaac

4620

ataactgaaa atcacagatt ttggagtcgg acgtacctgg tttctcatcc tggctgcccc

4680

aattgctaac tgcaatgttg ggcaaatttc tttattcctt caagctctgt gttccaatct

4740

ctaaaatagg aatcctaatt atatatactt caaggggttc ctgggaagag taaatgag

4800

aatactggca aagtgcctta tcatagtgcc tgattgcctg atacatagta agagctcaat

4860

aaaagtagct tttattattc ttgcaggtga tcaaagaaat gcagagtcaa acaatgttgg

4920

3060

3180

3360
3420

3720

4380

US 11,279,935 B2
34

33- continued

caaatgattt cacacctact gaatcggcaa caactgtgat ccccagtgcc tgacagagtg

4980

tgctgaaacc ggtacattca taaactgtaa accagtacag ctatttggaa aagcaacgta

5040

gcaatataga cacagcaaga gactgcagaa atggtcacaa cctttcgccc agacctcctc

5100

ttaatgattt gcagggttcc aggcaagggt ataaatggag cctcacatac catatgtata
agtagtttaa agttagaaat caggttacaa actgttaaat gaaaatatgt tctagcctcc

5160

tgccttggca aatatactgt caccaccacc tggaagggct caagttaaaa attctcagat

5280

gcctctgcat cctgtgttgg aacatgacgg catggggaga gccagttgct agcccccaga

5340

cccactgtct ttccaattcc taagtctgtc cagcactgag ggggcctcat gtgaatgtga
gtggacagcc tagcccatgc gtccaaactc tgttcactac cccctgcctg tggccactcc

5400

tcagatccgg gtgtgtctac tggtggtgtg attggatctc aggaggagag actcagggac

5520

cagggctggg caggccctgg agatgagcat ggggccagtg ggcagggact tctgggagtc

5580

ctaggcacct ggagaatggt ctaaaaggga ggacgtggat ttgcaatggg catgttccct

5640

tggcctgcag actcctcacc aggtgggagg gacgcagccc aaggagggcc agggggacac

5700

cccctgcctg atctcactgc agtactcctt agagtccatc caaaggaaag aaatagttca
gcagaataaa aaggctacgg gcacaaagat agtatcactg tgttgcattt gacactaaaa

5760

aaacccaatc taatgttcaa cagtagggaa atgttaagac aacaatgata aagctattca
gagaagtagt atgctaccac caacactggc acatgtgaca accatgtcat aacatgggat

5880

aatgtttatg tgtagatata atagtaaatg aactctggca actgtgcaaa catctgtgcg

6000

ccctaggtga gatgcggaag tcaacctgca gacatgagaa cagctgcctc aaagagggtt

6060

gaactctttc aggcagctga aagattttcc tcccaaaatt gtctctttcc tgtggctggg

6120

gagaagcagg gacttttcag gaactaatgg gtaagagctg gctggttaga ctggagtttt
ttgtgcaact ctactcagga gttggagata agaacctgct acctgctgca tggaagccag

6180
6240

aaaagcactt tagctaatca cactccctct ctgcgtgggc tgaagatatc cttgagtcct

6300

cccagccctg agcccttctc caggggccag cacacccaga aaccacgcgt gcagtgtttg
ctctggatgc ccaggatagt ctctgctgcgcgtgtgaagc tctgccatgg gctgggtatg

6360
6420

gaacgggcca tgtcacactg agtcctcgca acaacctggt gagatgggag cctcagcatg

6480

gctgacttgg acactgagat gctggtccag caccacgcag ttggcaagca gagaagccag

6540

cctttgaaac tctgtgcttc catttgggca cgtacactgg gaaatcaaaa ggcagtgttc

6600

cctttctccc cacaaactgc ttctagtgtc ccatggggcc agaggaactc aatcaaaaca
atattccata gatgtgggat agtggtagca caaataaatt aacaattgca cttctgggca

6660

5220

5460

5820

5940

6720

ctcgactgtc cccacggttc taaaaatgaa aagactctgg tatttaattt atattttata
agagtgaaaa aggccaggca cagtagctca cacctttaat cccatcattt tggaaggcca

6780

aggtgggagg attgcttgaa ctcaagagtt caagaccagc ctgggcaata tggcaaaact

6900

ctacaaaaaa attagccggg tgtggtggtg catgcctata gttccagcta ctcagagact

6960

gaggtgggag gatcacttga gcctgggagg tcgagactgc agtgcgccaa gatagcacca

7020

ctgtacttca gcctgggaaa cagtgagatt ctgtctcaaa aaaaaaaaaa aaaaaaaaaa

7080

aatttgataa ttgcctttgg taaggaagag ctgattttag catggaacag agggtggcaa

7140

acaacagcct tcaagccaaa tctattccac tgcttgtttt tatatgtccc atgatctaaa

7200

taatgggttt tacattttta aatggttggt ggggggaaat gtgaaactta catgaaatto

7260

6840

US 11,279,935 B2
36

35- continued

cagtctccag atctataaat aaagttttat tggaacacag ccacattcag ttgtgcatat
gtattgtcta cggcagcttc tgcactatac cagcagagtt gaggagttgt aacagggaga

7320

tcatatggcc tgaaaagctg aaaaatttta ctatctggtc ttttacagaa agactttgct

7440

gacccctgaa atggaagaag actacaggcc tagttattta gtgataattg aacccagctt

7500

tcattgtttg agtagcttct gcatgtcgat tgttaagctt tacttaccta gattacttca

7560

aatcctacaa agtacatatt atcgatcccc tgagcaaagc taggatatta gctgaagcct

7620

taaattcaaa ttcttacttt ttaaatttaa tttaatttaa ttaaaatttt ttttttagag
acagggtctc gagctctgtt gcccaggctg gagtcagtgg catgatcata gctcactgca

7680

7740

gcctcaaatt cctgggctca tgtgatcctc ctgccttggc ctcccgaagc actgggatta

7800

cagtgcctgg tctcaaattc ttacttccat accatggcag aggagcagag caagacctct

7860

tcagaaacat gagctctaac caaggcaggg aaaatatatt tagaggtcct gaatactgaa

7920

aacattacag tgtaccttcc ccagccaccc ctctgtataa tccaaaatat taaacattgt

7980

tgaactcaaa actgagtgaa agcaagtcca acagagttct aattccctcc atgataacta
catgtttttc taaaatagta tccgactatt ttatgagaat tttttggtgt ccctaggtat
atacttgctt catttgttgg gtgattaaac cccaaaactg accaaaatct gttggccttg

8040

ctaagtctga gctcagggca cagaagaaga gtctcacatt gtgacagcca cctggcgtgg

8220

cgatagagtc ctgttctttc caccaagaga gaagccgtcc ctagagaagt ctctcattta

8280

agaaagcaga acatatctac tcaaaaagcc atggggccac ctgaatcaaa tgcctgtcag
accttctccc tcacctcagg tctgtgtctc cctaagccca tgtcatgatc agagatgtto

8340

8400

cttaaagctg ggctccccag cccccaggcc atgcaccagt acaggtctgt ggcctgttac

8460

gaacaggagg tgagtggcag gtaagcaagc attactgatg gtaatggcag gtcatctgga

8520

gtgtctgctg ccatcatact ggctgcagca ggaatgcgca agtggggctg tgtgctccat

8580

gaagccagcg ggaaccaggg acaagtggga gctccacctc ttccgagttg gggctggagc

8640

tccctgggtg ccacttcagc tgcccaaatg gcagctgcag acccaggcct cttgcagatg
ggagccccgc cctcccaggc atagcaggca gacaagttcc tgggcagaag gaggtgggtc

8700

ctggtgaggc cccaccttca ggccagggag ggtctgaagt ctgtgggccc agctgccagt
ctggtgaacc agagtgggga cttgtgctgc cttttttggg ctcatccatg gccactcatt
ggaccaatca gtacatactt cctcccctct gaggcccata aaagccctgg gctcagccag

8820

8940

agcagaggaa aggacggaga gacactggga tagccagctg cagagagaag ataacctctc

9000

atcagggaac tacagaggac aggacaacca gcctgcagag aggagctacc ctctctgcta

9060

ggagctaaac acttgtcagg acaccctagc tacagaggag ctgcccactg caagtcttct

9120

ttgagctgtt ctattgctca gtaaagctcc tcttcatctt gctcaccctc cacttgtcca

9180

cataccttat tcttccatgt tgcaggacaa gaactcggga cocactgaat ggtgaggcta

9240

aaagagctgc tgtaacacaa acagggctga aacatgcccc ttgcttgcca cattgtgggt

9300

gaagagaagg agagaagagc tgcagccctt cagggatccc agacctggga gctccctgag

9360

ccagagccgt gactccctct ttggggttct gtggttcctg gcatctgcaa gcttctgggt

9420

gccactgcat tccccagtgc cagccaggga agtggcttgc agtgcacctg gtccagccac

9480

gcag agagcgctgc tggcacctgt agctgcctgc cccacagcag ccaccagtat

9540

gtctgactga gcagtggccg gaccccatgc tcgctcacac acccctcact gttccacacc

9600

cgactcaccc ttggcaggcg tgggacccag gccagcagtg tgaactgagt acagcctgcc

9660

aacc

7380

8100
8160

8760

8880

US 11,279,935 B2
38

37- continued

aggcccagca ggcctgagca aaacttgggt aaaaacatca ctggccacag aggtttctgg

9720

ctaggaaaac aacaccccaa ggatcctgta acattcatcg gaacgccacc tcccgttaga
tcagcggtgg catgagattg tcataggagc acaagcccta ttgtgaactg tgcgtctaag

9780

ggatctaggt tgtgtgttcc ttataagaat staagatcgc tgagattgat gagatcactt

9900

gagcccagga gtttgagacc agccttogta acacggtgaa accccatctt taccacaaaa

9960

acaaaaatta gctggatgtg gtggcgtgtg cctgcagtcc cagctacagg gactgaggtg

10020

ggaggatcac ttgagcccag ggaggttgag gctgcagtga gctgagattg caccactgca

10080

ctccagcctg ggtggcagag taagacctta tctcaaaaaa caaaaccaaa atggtttatt

10140

gtaaaaaaaa tgagtatggt caatattttt ttctcctcag ttctacttta atttcaagca

10200

ctcaagtgag gatcaagcat ctgatgagaa tgggagagaa aggcagagag agctgtgtct

10260

gttcaggaaa ccgcatcctt tcacaattag ggaaacaaca attttttttt gaggcagggt

10320

cttgctctgt tgcccagggt gaagggcagt ggcacaaatc atagctcact gcagcctcga

10380

cctcccgggt tcaagcaatc cccttacctc agtctctcaa gtagctggga ctacacacat

10440

gcaccatcac gtctggctaa tttattttca tttttaattt tttagtagaa acagggtctc
tccagaaaaa ctacagatgg atcagacaat taaatactga accctgtgga acaaatagaa

10500

10560

gagaaagtga aggcttgtct atccctagac acactctttc tggaacatcc gtcttcctca

10620

cactgtcccc tcttgacttc catgataccc taggctgctg gtttttctcc tacctctctg

10680

gccagtcttt ctcagtcttc ttggccggct ccccctgttt ctgagcccca atccctatat
gttggagtcc ttttgggtct caattttttt ttactccttc tcttttaggg ttttcttcct

10740

atgagatctt tcagtctcta gcctttaatg ccactgagtg ttgccatttc tactcccatg

10860

ttcataattc cagccctgat gtctggcctg acacttagag actgtccacg gagggataca

10920

ggaacaaagg aagtgacagg agtgacaaag gaaaagatgc gtttgtgtgt ttctgcacgt

10980

ttctgtgtct gtttgtctgt gtctctgtag gttctgcatg tccaaaaaag aatacagacg

11040

aagaggccaa caacaagggt ggacatattg cagcaaacat gaaaaacaaa gaattgctac
cttatacata aaatgttcat aatattagaa aacaaacaca ttaaggctcc aagaataaaa
caggcaagaa gcagggtcag aaagttctca gaagagaaga tacatttagt atacaagcat

11100

11160

agaaaacgtc cagtcttaga acttatatca atgcaaatga aagcaagtag atacatagtt

11280

atataagaag aacaaaggtt gagaggttgt gaatgctgtt gttgttgaag cgagtgaaac

11340

aggtgctctc ccatagcccg caaactcgta tagtcctgtt ggaaagcatt ttgctgcatt

11400

tgacccagta attatacttg tggcataaat cctaaagaaa tcattccata agctaaaaag

11460

9840

10800

11220

caaactttta agaagcatct atacagaagt agattaagta agccttgata aatcactaga 11520
ttttggaata tctcagattt tectgacaat gggaatcacc caaagtactt accatgtatg

11580

caagttcctc tccctgggaa attctgattc tgtacctctg ggatggtgcc caggaatcta

11640

tattttaaaa aggcacttta ggctattott atcttcagac acatttggaa aatactgata

11700

gaggaaatat aatgaaatgc catcatgagg tcattaaagc taatgtgtgc aaagatcgta

11760

taattctttt tttttttttt ttttttttca gacaaagtct cactgttgtc ccccaggctg

11820

gagtgcaatg gcacgatctc tgctcactgc aacttccacc ttccgggttc aagcaatttt

11880

cctgcctcag ccttcccagt agctgggatt acaggtgccc gctgccatgc caggctactt

11940

tttgtatttt aagtagagac agggtttcac catgttggcc aggctggtgt cgaactcctg

12000

US 11,279,935 B2
40

39- continued
acctcaggtg atccgcccgc cttggcctcccaaagtgctg ggattacagg cgtgagcaac
catgcctggc tgcaaagatc atataattct atggaaaaat acttaatgac attttgagtt
cctagaatgt caaatgaaat agcagaatat aaaagtctgt gaagtattgg tctattcaac
aaataaatat ttactgagca cctactaagg gccagccagg tcgtggggag acagaagagaacaaaacagc tgccatttat cctcatggag cttcgagtct aatgggaaga taaagagtaa
agttaagaaa acacacacac ?????????? acacacacac acaaaattcc atacttacct12180

aactgcatgg ccatgatctc tttacaaaca cattgtctccttccatgggc ggagagctgc

12420

tgcagaacca actgcttttg gtgggattct agctccatta cttagcaagt gottgcaact

12480

ctgtagcago ttaggaaaga actgtggttg gaaggaaaga gtgtggtgca tggatttggt
aaaagagtaa aaagagtggc tgggcacggt ggctcatgcc tgtaatccca gcactttggg

12540

12060

12120

12240
12300

12360

12600

aggccgaggc gggcagatca cgaggtcagc agtttgagac cagcctaacc aacatggtga 12660

aaccccgtct cttctaaaaa tacaaaaatt agccaggcat ggtggcacac gcctgtagtc
ccagctactt gggaggctga ggcgggagaa ttgcttgaac ctgggaggtg aaggttgcag

tgagcagaga ttgcaccact gcattccagc ctgggcgaca gagtgagact ccatctagaa
aaaataaaaa taataaaaga aaaaagtaag aagagtagga gcagaaagaa agtgagttaa

12720
12780
12840
12900

taaatgacca gggctgtttc tgaattctgt atggtggggc agaaaagatt ttctttaagt 12960

agacttctgg gtacctgtag acagacgtgc tgaaaaccca ctggcagaac agcagccctg

13020

gtctggaggg accatgttgg tctccagaag ctaataccac aggtcagagt tcagatcacc
caccccaaat ctgggatctg ggatcagtat gagtctagga ctggggcaaa agcccggggt

13080

13140

ggggatagag ggctgtgtga attgacctgc cattcagaaa cagcctcaag tttaggottg

13200

gagttatctc caaatgagat tatgaatact gtcacagaga gccacccgta agatcgaaaa

13260

aaaaaaaaat taaccaggac ttttacactt gtgtgttatg actgagctgt attgactctg

13320

atgcagtctg aatattttaa aaataaatca tacgcttatc tgggaaccaa aaaaatatgt
tgcattatag ttggttgaag taaatattta atatcctgga tatgatagtt tttataatto
tgggttggca tttcttcctt gtatcaaaaa ctatactaag gccagttgca gtggcttgtg
cctgtaatcc cagcactttg ggagcctgag gtgggcggat cacctaaggt caggagttca
agacaagcct ggccaacatg gtgaaacccc gactccacta aaagtacaaa attagccagg

13380

catggtcgcg tatacctgta atcccagcta cacgggaggc tgaagcagga gaatcgcttg

13 680aacctgggag gcggaggttg cagtgagcgg agatcatgtc attgcactcc agcctgggca

13740

aaaagagcga aactccgtct caaaaaacaa acaagtaact atattagaat cctcaaacgc

13800

tggaagtggt ttaagcctgc cctgactctg agaggctatt ccagggtaag aatgctgccc

13860

agtactctca aaccctgtag aaaaggggct ggtgcttatt tccttagagg catttctgtg

13920

tagtttatct ttggtgctaa tgcggtcctt ttcaacttac cgtgttaaac aggacattag

13980

ggttctgtta agaagagatg cactttggga ggccgaggcg ggcggatcac ggggtcagga

14040

gattgagacc atggtgaaac cccgtctcta ctaaaaaaaa atacaaaaaa attggtcggg

14100

catggtggcg ggtgcctgta gtcccagcta ctccggaggc tgagacagga gaatggcgtg

14160

aacccaggag gcggagcttg cagtgagccg atatcacgcc actgcactcc agcctgggca

14220

ggagaacctc

14280

tcctgggata tgtcctacca ggaagcatat ctgcaggato agattacaca gattacagga

14340

ggtaacagag ctggggactt agaaacagca gctgtctagt ttcctattta tttactcatt

14400

acagagcaaa aaaaaaaaaa aaaaaaaaaa aaaagatgtc gctgag

13440

13500
13560
13620

US 11,279,935 B2
42

41- continued
ctgagcactg tccattggaa tttttttagg aaggcaggtt tggtgtgggc taggtagttg

14460

agatcctgag acctgctggt cttgatctgt agggagctga ctggatgacc ttggccccag

14520

gcctgccata gatgtaacag ggaagatgca cttggccatc tccaagggcc actgcagccc

14580

agaacattct acgttaataa gactcaagag tgtttgccca atggcaaaaa caaaagtcac

14640

accttccaac tcagaccatc caaacggttt atgaacttcc aagttttatt tgttgaatcc

14700

cacaggtcta tcatcattgt aaactcttat tccccccagt aatatatctg taataatcct14760

gaagcatgaa tatcttgaaa atagctgcag atgcctatat tttcccagca gagccgataa

14820

aagagacttt tgtttacata ttcatcattc agagtgcttt gagaaattag gaagtaaatc
gagttgcaat tctgcaagtg acttggccct taggactgtc ttacagtgtg tggtgtgtac

14880

14940

cttgtgccta ttcaggacca ttcctatcaa aatgaatttg taatacaaac atgtcttcag

15000

ccaatgggac cacagaatct caccttgtca taagagatgc aatcagaagt attgtgcaaa 15060

cacctttccc tgacagaata agttcctggc aacccagtag ccccctgcat aatcaaacag
taagccagat cctaaatgtt ctatataaat gtagaatatg gaaactgtag tcaaacaaaa

15180

atagcaatag tggggtagaa accaggactc tgtacttctg tatttcagtc cttgtgtcct

15240

tcagtccttc tcaggccacg tttagtttgt tttaacaacc tcagcctaga actgccctta

15300

attctggcaacaagggccct cctctggccc catgotttca aggatctgct ctcctgcagg

15360

tgtggctcct ttcttagggg gaaatgccgg gctaaggaat gtatccctca aggcctgggc

15420

ctccctattt tagaacattc tctggatacc caggattccc cgctcaaatg ttctgggcct

15480

atttctagga cctgcccagg ttctttgctt ggtccatcct cctgagggca gactatactg

15540

aagccttagg ctaaagaagg ggcagctttt ttgtggggag acggccagca gaacttggac

15600

ctgtgggccg agatgtgcac ccaggtgcat gtgaggactt tctcagtgca agatggagct15660

ggagggagtg ggatgggaaa ggaggtggta tcatgtgctg gcagctggct ctccccatgo15720

caacgtgttc cgggaaaaaa ttccaaggag cctgagaact cttaacttgg atctagtctt

15780

ctaggtcata aatgaaggtg tgcatatata tcaaggtaga aggatatgtt atctttaaaa

15840

gttttgttag tttcagccag gcatggtggc tcacgcctgt aattccagca cttcgggagg
ccaaggtggg cagattacct gaggtcagga gttcgagacc agcctggcca aaatggtaaa

15900

accccatctc tactaaaaaa aaatacaaaa taattagctg ggtgtggtga cgggcacctg
taattccagc tacttgggag gctgaggcag gagaatctct tgagcccggg aggcagaggt

16020

16080

tgcagtgagc tgagatcacg tcactgcact ccagcctggg tgatagagca agactccatt

16140

tcaaaaaaaa aaaaaaaggt ttgttagttt gattggtaat gtttaaatat ttagacatgg
agtaggtggg tctcccactt gtgatcttgt tctgagtgaa aatgttaggg gctgagctgc
cttaagcaaa tctcttcacc tctgaggctt ggagctcctc tgtaccagga agccttgggc

16200

16320

tccttgaggg ctactggccg gtccagctct ttctttcttt gaatgttggc tgatgaggct

16380

gaaactgatg aagaaaaatg taggcaactt ctttccatgg ctgctttagg tgggattctt

16440

ttcagtgcaa gggacaacaa tctaactcaa atcaccttaa gcaaaagaga gattctttct

16500

gttcacttgc ttcagacatg ttcatattta gttactcaaa caccatgttg tccataattt

16560

attcctattt cttcttctgt tttcgttttt tgagatggag tcttgctctg tcacccaggc

16620

tggagtgcag tggcaggatc tcggctcaga gcaacctctg cctccagggt tcaagcaatt

16680

ctcctgcctc agcctcctcc tgagtagctg ggactacagg catgcaccac caagcccagc

16740

15120

15960

16260

US 11,279,935 B2

44

43- continued
taatttttgt atcttcagta gagatagggt ttcaccatgt tggctaggct ggtcttgaac
tcctaacctc aagggatcca cccacctcag cctcccaaag tgctgtgatt acaggcgtga

16860

gccaccatgc ccggccccta tttcttgact cagctttttt tctctattgg cttcattttg

16920

gggcaagcca agatggccac aagcacctcc aagcttgaat ttggcaactt cagcaaaaga

16980

gaacattcct ttcctaatag gttcagcaaa aatcctaact aagcctctca ttggccctgg

17040

ctgggttaca tgctcatcct ctaaacaaat cactgtgact caactgccag gaagctgggg

17100

gagtggaaga cggcggactc gcctgaacca cagggcagtg tggggcaaag ggtatttcca

17160

acaggaaaag aggggttctg atgctggaag aaggatgcta tgtaggcaaa atcaacagat

17220

gtttcaccag caatgottac cacatgaact atgaggacaa aggaaacaga aacatacaaa
tatttatgaa agacagcgat gaaatggtgc ggtggaaaat tgtgctagag aagccaatcc

17280

tgccccctca acgcagecac actgagcgag ctgtgggtca ggggtaccca ccctaccctc

17400

tccttggctc caaagcagat gttcactaca gtcaacttag acattgtgac tcagagactg

17460

caagttctgt cagacacttg gactgaggat ctacatggag ataagcagag aaaggtggtc

17520

tgtgagagaa taaagcggaa atgcaaagaa caccagagac caggcagccc cagatagacc

17580

aagagctatg actccttccc tccagccagt ggctccagtg ccttgcaagg ctcagctgta

17640

16800

17340

tttcctgccc ttggtagaga ctcctgggtc cctccaacat gttttcgcgc gcgtgtgtgt 17700

gtgtgtgtgt gtgtgtgtgt gttacagctg gatttaacag tttgctgttc cacaaaccac

17760

aaataaaaca aaacacaata aaaacataaa ggtggggaaa tgctgttttt tcttctaaca
tgtaagaatt gagtcaggtg tttctcaaga ctgttgtgat ttttatctta gtttttcaca

17820

17880

aacatccaga gaagggatag ggaagcgtgc acgtggaacg acaaggcggc tgagttcaag

17940

gtctggagca agaagctctg catccctgca gctctgatgg ggacggagac cctctgggca

18000

gagatatgca gggaaatgct cattactctg taccccctac cccaacaccg cttttgccaa

18060

gaggtggact gtgctagcac caatgtggag agttttggac atccacgcca tatctgtagc

18120

agctgcggct atctcaatgc ataagctaca ttctcaaaga atttcacgtc aaaccagtgt

18180

ttataaacgg agcccgattt tcagtgottg gagagaacct acacgcttcc gttaaaatot

18240

ctcttctaga aaagtgaaac acaacctgtt aatcagcatt tatataaggc aatattttaa
aaatcagaaa aaagcttttt ttaaaaaagg caaagttgac aagacaaata agctggatga

18300

atcaaatttc tgotttgttc tgctttcact aattgctgac agagccgagt tgcggcctga

18420

ttataaagct gtgcttcago caccttagca aatctgttgc tggaagacaa ggagcgagca

18480

cttacttttt gagagtaaac tgtattttat tttttattat actttaagtt ttagggtaca
tgtgcacaac gtgcaggtta gttacatatg tatacgtgtg ccattttaaa agcaccgggg
gactggtcta tttaaaagaa caatggggtg agtggcgctc taatgacgga ctccagcttg
ggggctttga ctatcctagt tcttcttggg ccccttttgg ctgagcgtga agcaaataaa

18540

18720

caaacacccc gatggagagg tccttaacca cggtgtgcag cagcatcacc tggggagctg

18780

tagcccagta cacacgccca gccccaccca ccagccttac tgagagggga agggatcctg

18840

cctgtgtgag ctggaaaagc tctccaggtg attccgcttc aagcttccgg ttaagcacga

18900

aggctaatga gccttattta aaagctcagg tttttactca gcaaaatctg ttgcttttaa

18960

tgggact ctctccccgg

19020

aaatgaaata agcaacccct gagaaacggc gctgggacaa tagggcagca gtttctcctc

19080

acgtcctagg agggaaagtc gactgcaggg agttatatga cttaatatgc agctaaccag

19140

aat gaccgc ctt

aa accago

tg ttgca

cca

18360

18 60018 660US 11,279,935 B2
46

45- continued
ccttttaatt aacttgacat gccagataac aaagctatta ctaagtgaga gtttcttaat
tgaaaggggg aagagagatt tgttttatat tttcaaaata atctataatg agcgttacta

19260

atttgacaat cagaatttgt gtgtgtgtga aaaatagcta ttttgagcta tgaaaaggga

19320

ggatgagcag acctgcacac ggcagcttag cttgctgtgg ttttgcctta gctggtagac

19380

ggtgcctggt agtgcatgac tgaaagatgg tgtggttgat tctattagtg tgcaattgtt

19440

cgcaggcatt ttacctagaa atcacttgtg ccatcgtggc catagtattt ttccagcctc

19500

atctcttact gaactgtacc tctagctctg agtccagtca caatgaacta atttgtagtc
tctcagtcat ccatgctttg cacatgcagt ttcctctcta tggaatatga aatacctccc
cttccctcct cttccttccc ttcagcttcc agactcctgc cagcaagctt ataagtcacc

19560
19620

tttcaaatct cagttcaaaa gcctcctctg agaccatctc tocacttgca agactcgggg

19740

gctccatcta tctcctggga tccctcgacc aggcactcta gactgtcatt atagcaactc

19800

attgagtgta ttacgatggt gtgcttttgt gcctgtctcc ccaccattca tatagtcaac

19860

acgcacttgg tagatgtcat catttgatca ctgcagcaat cctacagggt accttttact

19920

gtgactccca tttcccagtg gaagaaactg aggctcaaac atagtagcct gcccagctct
cactacaagt agatggttta cgtgtgatac aatccaggtt catcttgctt ctgagtttgt

19980

19200

19680

20040

gctcttaact gccatgtgag caaacttcaa tcagaaagtg ttttttcttt tttcttttct
tttttttttt ttttttttgt gacagagttg ctctgttgcc caggctggag tgcaatggca
caatctctgc tcactgcacc tccacctccc aggttcaaac gattctcctg cctcagcctc

20100

ccgagtagct gggactacag gcgcccacca ccacacccag ctaatttttg tatttttagt

20280

agaggtgggg tttcaccatg ttggccaggc tggtctcgaa ctcttgacct caaatgatcc

20340

accttggcct cccaaagtgc tggaattaca ggcgtgagcc accgcaccct gcctgagaga

20400

gactgtcttt tcgaatttgt atcttcaatg cccagcctac aggagatgct cagtggataa

20460

tgatggataa ttatttagca gttatccatt gccaaaagaa tggataacat cactgctacc

20520

ggctttggca gacctcactc caggtaaatc attccctggg ggcattaatt acctgctggt
gttccctggt taaaacacct gtgacaccag cctacacata cgcacagggc tctgaggtga
tttttttttt aatgacaata aagacataaa aagatataag gtaaacctac gtgggctgct

20580

20640

tatagcactg t?agttatgt gtgtgttttc atttacttca aaaactgagg atattaaaat

20760

aaatatgact ttattccatt tagttgagat tgtcattttt ttttaatttg gaagagaatt

20820

taagaaatga tgactttatt aacaacattg gcgtaggaca gtagcagact gggttaaaat
actgtatcaa tgttgaattt actaaagttc atcactgtag tacagttttg gaagagaata20880

tctccattct cagaaaatgc acactgaaga atttaggggt aaagggctat aatgcacaca

21000

actgactcgg atgtctcaga aaataagtgt gtgtgcatgt gtgtggtttg tatgtacgtc

21060

tgtgtatgag gaagtgaaag ggactaatgt taacaataga tgaatctgtg tgaaaaataa

21120

atgcatatto tggctgggca cagtagctca cacctgtaat cccagcactt tgggaggttg

21180

20160
20220

20700

20940

aggcaggcgg atcacttgag gtcaggcgtt tgggaccagc ctggccaaca cagtgaaacc 21240

ctgtctctac taaaaaaaaa aaaaaaaggc tgggtgtggt gacatgcgcc tgtagtocca

21300

gctacttggg aggctgaggc aggagaatca cttgaaccca ggaagtggag gttgcagtga

21360

gccgaggtcg ctccactgca ctccagcctg ggcaacagag aaagactcca cctcaaaaac

21420

aaaacgaaca aagaatggat gcgtattctt catattattc ttatttttgc aaattttctg

21480

US 11,279,935 B2
48

47- continued
caagtttgaa attatgtcca aacgaccttg gaagaaatga acatccctga gaggaaggtg

21540

taatgagagt gtttctttga aatacagtga gcaagagctg gcagggccgg t?agtgtctg

21600

acagtggcat gaggtttctt tcaggctgac ttgcaatggc tggttttctt tacctattga

21660

tgtgtcttga atatgtttac agttcagctg gcccagttct ctgagaggca attgggtttc

21720

cttgcagatg tgccgcggga aggacagtgg tgcctgtgct tggagaagcc cctggccacg

21780

tgctaggaca gagggacata tggtggcacg actccagcca cagatgcaac tccctcagga

21840

acagtggggc ctgcagttgg aaggggcccc ctctggctca cagtgctgtg tggacagtag

21900

catctgtgtg actgcaaact cgcccactgt gagtgggaac agtcgaggct atctgtggcc

21960

ttgcatctgc tggtcccatt caccagggca acactgtgca tggccaattc tagacctccc

22020

ggacgcagtg catgcaactc agcgttagaa tgtcaatttc gaagtactag aatgtcattt22080

tgctgttaca gtcaccccac aagcattttc tgagcatctt ctacttggga ttgcatcctt

22140

tttgctaggg actgtgtcat gtgcgtacac acagcatctt atttttagct ttcaggctga

22200

cttgattgaa catggtgcag ttaatgtgcc ccccattttc caaaagtaga aaactgtgac

22260

acagaggtta agtggtttct gaggtaacat agaaaaaata tgtagcagag atttcctaaa
caaacttgca gcctggcagg aggggtaaga agatccacga ggaacattgg atcaggttgg

22320

22380

aggaaagagg tgccaagaga gacccaagat cttggctggg aagtgaaaag gagtgtggcc

22440

acttttgatt ctggggtagg aggctttatt ggggaggtgg catttgcgtg gagactaaca
ggatgataag aattttgaaa ggaaaatcat aaagagcttt ccaatggagt cgagaaaaca

22500
22560

tggtaaaaaa acaaaatagg agttgagato ataacaactg tcatttattg agtttttcta 22620

tatgctagga actgtgagag gcgctttaga taaatcaact cattgaaaca ggagttttct

22680

taagtgagcc ctattagcat cctcatttta gggtaaatgg aggcatccat ggaaggttac

22740

atgcatctag tgtggtaaca aagcccacaa gottagaaag tgcttcatga taatagtgaa

22800

atgatggtaa taataggaaa aaggaggggg aggaggaggg agaggggaag aggaagagga

22860

agaggagtgg ggaggaggag aggaaaagga gaaggagagg aggggagggg gaggaggaag

22920

aagcagcagg aggaagagtg gaaggagaag gggaagaaga aggaggggca aggggggagg

22980

aggatgagaa gaagggagag gagaaggagg gggaggagaa gggggaaggg ggagaaaaca
agagggagga gggggaggag gagaaggagg gaggtaggag gaggagtggg ggacaaggag

23040

23100

ggaaaggaga aggggaagag ggagaggatg aggaggggga ggtggaggaa tgaggaggga

23160

gaggaagaga gaggagagga aggagaaggg ggaagggaga gtagagaaag gaagaggagg

23220

gaggagaggg aagaggaggg aggagaggga agaggaggaa acgagaagag gggagaagag 23280

gagggaggag ttggggagga ggaaggggag aagggagagg agagggagga ggaggaatga 23340

aaataggcag ggggaggagg agaagaggga ggatgggaga agaggtagga gggggaggag

23400

agggtggagt ggggttaggg gttagtcctg gaggcatcag aggcttctct agggagcgtg

23460

gtttctatcc tactctacct tagccttgac caagcaggca gatagcatta gtattcacto
cttgcttggg agcacttgaa aagtttataa aaaggcgtag gctgtttgga taaaagatge

23520

tctggagaca agacagtgca tcttcctcag tcacttcctc actcctttct cggctctgat

23640

tctggtgttt gatgtcttcc cacaactcgg caaagaaaga agaaaatccc tctactcact

23700

gtgcagtttg taggttattt aaagg

gtg gttccttacc cggcagggtg

23760

cagggatgga gtgggataca accatacaac caacccctca gagggcaagc gcagctcatt

23820

cacagtggcc aggatcccta gggcagcctc gggcgtctgt cccgcagcag aggacactct23880

tg catt23580

US 11,279,935 B2
49

50- continued

catgaggcgc tggaaactca gctcagggcg toccaggtcc cacgggcgcc ttccagcctg
ggacacctgt gccaaacctg aacagggaag gtgcagaaaa gcgtgtgtgt gcgcatgcac

23940

gtgtgtgtgc taaaatataa atgaacgatt aagggtttcc tctgctggat attctctgca

24060

gtggggagaa gaggtgggag aaagtagctg tttttgccgc ttggggaatg tttgcccaac
tggtaaaggt agaattgttc ttttccaaag gaaagttgtg tgggagttgc ttccttaccg

24120

24180

aaaacaagga aaagcattag tgaaaagatg taagatcaac aggcaatcaa aaatgcacac

24240

toccagtgtt aagggcacag gtgaattttc cttctaggat tctcaataca aacctatttt

24300

gaaataaaat gcctctaggt cttccctgcc taaacccacg tggggagcac taatagctgc

24360

gtacaaactt cattaaactg gccacagctg aaaggagctg gtgccaaccc actgcttttt

24420

gctgaccgaa gtatccggct gggtttttgc cctcattctg aagtgactgc tgcccatccc

24480

ccacaccttg agcattgttt tactagacat cttctagtgt agggtaactg gaagaaaagt

24540

aatggaatac caagtgccat tcatcatatc cctgcctctt ctattttgtg gtgcagggat

24600

gctgagggga aaatgagtct cctgtaagtg cttggagatc ctcccccact ccacctcctg

24660

catttccata agcatttggc atcagagtct tctggtaaac cccccagctt ctacaattgg
acaattggct ttgtatcacg gcaggaattc gtgcctctga agtttctagg gaagctctac

24720

agaagcaata aagtggtaga gttgtttgaa taaaattagc ttctatgctt gtaggaagca

24840

tacccaattc cagttagctg tatgaaatge aaccattttg gaggtttgtg aaaatatgca

24900

aggctcccgg cttcaggaga aaaacagatt tacattcctc cctgtctcag cagggagtct

24960

tcaatcaaag gcaaccaaca aatcaaacat gattatctgt cagcacattg gccctggttt

25020

gtaattatac atttatttgt gtgtgatcaa tgctagtctc ctcactggat totaaactcc
ctgagagctt ttctgtctta attaccctga actccagtgc ccacacagta cctggcacat

25080

25140

agctcatgct gtaaatatct gtggaaagaa ggaagaaatg gagaaaagga agggaggaag

25200

gaacagaagg gagaaaggaa aacggaaaaa aagaaatgtt ccaggtggaa ggattttttt

25260

ttttttttac aatcatcctg cccaacagta tataggtttg cctgtgatct ttttctcata

25320

cataagatgg agataatagt ccttgtctca atagttattt tacgttattt tctaactcaa
atagttaaaa gtcatttgta ggcccctgct ctgggcccat gotttgtagg atctcactgt

25380

25440

cgctgtcctc aggctgtgcc ccttcctaga gtatgaagtc ccggtgggac tttaagccta

25500

tgctcccctt ccccttcctc cgcatctgtg gggacttctc cccgacagtt cacgcaggtc
tccaccacaa gtgtgcatgc cctaggccca aggggtgtcc aaggagtgac tatttgtagg

25560

ggatgagcca tggatggaac tgtggtgtct gcacacatgt gcctaagacc ctctcccatt

25680

gtgggatgga gcctgcagtt cagtaggggg aatgaaggga ggtgtgaatc atggctcttg

25740

acccaggccc gcctctctct gtgtggccag ggttcagggc agaacctcag gaggctggaa

25800

gttgtagctt ctaacctggc ctttcaggtc atgatgaagg tctatttgtc agggtaagag

25860

gttcaaacct cctgcaatag tcctattcag cagctgatag cttggtttat aacttttgaa

25920

tatttagatg tattccacgt gggcaaatag gaacacacac acatatatat gtatatttgt

25980

gcatttatat gtgtgggcat gtgtgtgcac gtgtgtgtac atatataatt ctatagttca

26040

cctgttagtg taccttgatg atgaaaagtg agggaggacc ctgactttgg acagcatgga

26100

tctgcctgcc acttaccagc taaactgttt ccttagggca aaaatagaga caacaatagt

26160

24000

24780

25620

acctatttag ttggattatt gacattatta aaagagagca actaacatac tcctagaata 26220

US 11,279,935 B2
51

52- continued

taagtgctcc atgcatatta taaactaaca attgtttcct gctcctttca cttcatgcat

26280

gaagacatat tcactcactc ataaatactt gcattatatc tatctatcta tctatctatc

26340

tatctatcta tctatctatc atctatcaac aaagtcttgt tctgttgccc aggctagagt
gcagtggtgt gatcctagct cactgtagcc tcgaactcct gtgctcaagc aattctccca

26400

cttcagcctc ctgagtagct gggactacag atgtgcacca ccatgcctgg ctaatttttg

26520

catttatttg tagagatgag ctctcactat gtcgcccagg ctggtctcga actcctgggc

26580

tcaagtgatc cttctgcctc ggcctcccaa agtgctggga ttaggagtgt gagccaccat

26640

actcggccat tatgtatatt tcttattcaa tctgagaact aactataata ttattcattt

26700

actattttag ttcaagttga tttcctttta aaacatacac accaagttta tgtgtattga

26760

taccagtggt ttcccaggtg tttttttgtt gttgttttgt tttttcagat tctcattctg

26820

ttgctggagt gcagtggcgc tatcttggct cactgcaacc tctgcctccc gggttcaagc

26880

gattctcctg cctcagcctc ccgagtagct gggactacag acacgtgcca ccacgcccag

26940

ctaatttttg tatctgtagt agagatgggg tttcaccatg ttggccagga tggtctcgat

27000

ctcttgacct catgatccac ccgccttggc ctcccaaagt gctgggatta cagacgtgag

27060

ccaccatgcc tggccttata taggtatttc acaaaatgat ttcctctatt aggaccatat

27120

tcaaaaagcc cttcccctac tccacaatta cagaagcagt cacatacatt ccatttgatt

27180

cttctctatg atttcatttt tgaaacttaa ttccttaacc cacctggaat ttattgcggt

27240

gttgttgtga ggttaagaaa aaaaaaaatc aatctaatat aatgtttcta aaaagccagt

27300

26460

tgtctcggcatcatttttgg aataatcttt cccctcatca ttcatttaca tttctacctt 27360

tgtcacatag ttaatacttt ttttcaattt gggtttcttt ctaaacctgt aagtcaattt

27420

taatgatcta gttaactttt gatgccagta tataccgttt taattcttgc aggtgcataa

27480

cagttaatac agcagagcgc aagggctgct tagttttctt gtttttccct tttattttgg

27540

gtgggttggg gaagggaaca aggtcttgct ctgttgccca ggctggagtg cagcagcgtg

27600

atcacagctc accacagcct caacctcttg ggctcaagcc atcttcccac ctcagcctcc

27660

tgagtagctg tgattacagg ggtgcgccac catgaccagc taattttttt tttttttttt

27720

tgtaaagaca gtggtcttgc tttgttgcct aggctggtct cgaactcctg ggctcaaacc

27780

cacctcagcc toccagagtg ctagaattac aaacatgage cactgtgcct ggccattttt

27840

cttctttttc aaagttgatc aattatgtga gcatcaccac tagaatctgc aaatgtctcc

27900

tgtatcggca acgagggagg taccataagc acccttcttt tacagatgag gaaactgagc

27960

acagaaagaa gagacacttg ctcaaggtca caaaaacaag gcaggtccag gaacagtgaa

28020

acacaggcag totagctgca gggaccacga tcataacctc tgcaaagtgg ggcagatcca

28080

acctgagaat caatctcctt caagtcggag cattttctcc tcatatctat tattactgac

28140

ttcttagtat ttgttttatt tatttggttg tttaatgatt tcatatgttt ccctgtgttt

28200

cgttatatac tttctattta taatgcttct taaagttttc tggttacaga ctattttctt

28260

tctttttttc tgagatgtgg aacttaccat ggttcatatt ttaaactctt tggctaggtt

28320

tactaataat tgtgcttacc cgaaagcatt ctttaactgc tatttctttg gttttcaagg

28380

tcagtgactc tttatatggc tgcatacaca ttttagtaca atgataactg gataatttta

28440

gaac ataaaaatga ttctattatt agctaaaatt

28500

tcagcagcac tgttgacata atttttattg ttttaattaa attgcatgga aaagaaataa

28560

ttttatcata agaataaaga ggatgatagt tcaaagttat ttgtgagaat atcgtggctg

28620

ataaagttgt ata atata gt

US 11,279,935 B2
54

53- continued
tattgcatta gcatcttgac ttttaaggtg atgtttaata gagacacaac aattctttag

28680

gtcgtaaagg caattcttgc tgttttaaaa aaatgttatg acaacaggtg tgatcacata

28740

aagtattagt aaagatgaaa caattttaaa tattttccac tctatattct actgctttat

28800

aagaaggctg acaagaaaac cctcatttca ctagatcata tctacatctc actggcagga
aagaagggcc catatgagtt acttacctaa gtaggaacta ttattcctgt tttacagatg

28860
28920

agaaaaccaa agctcagagg ctaagtgact agttgaatgt cagcagacca attgaagaaa

28980

actcaaaatg tcaccactca ccaaattcac ctgtctatcc agccagtgca cacatccttg

29040

gcagatgtct cctaattaag tggagtgtgt gtgtgtttgt gtgtgtgtgt gtgtgcgcgc

29100

gcgcgcgcgt gcgcgcgcag tgaggtgggc caccccgcct gcaggcagct ctaagtcata

29160

gttccgttaa ttatttattt tatatgatga ttttgagtoc ttttgtagtc ctggctgtct
ttttatgagc acatttctat ttgagaaagt cagcaggaat ctggattctg gaatcactga
attccaatge tagctccaat tcttcctaga aacgtgatct tagaaacgtt aatttatata
acagaaatga taatacatat cttacaggta tggtgtgaga gttaaatgag aagttgtcca

29220

tgtatttagc attgtgcctg actggtatca tgtgctaatg catactgctc attttatttt

29460

29280
29340

29400

atttccatgg tatggacgtc cttttaagaa tattcgcoca ctgtagctgg gcgcggtggc 29520

tcacgcctgt aatcccagca ctttgggagg ccgaggcagg cggatcacga ggtcaggaga

29580

tggagaccat cctggctaac acagtgaaac cccatctcta ctaaaaaaaa aaaattacaa
aaacttagct gggcatggtg gcgggcacct gtagtcccag ctactcggga ggctgaggca

29640
29700

ggagaatggc gtgaacccgg gaggcggagc ttgcagtgag acgagatcgc gocactgcac

29760

tccagcctgg gcgacagagg gagactgtct caaaaaaaaa aaaaaaaaaa aggatattcg

29820

cccagtggaa caactttggc tgctggtccc tcctctgtct cccccaagca gggttatata

29880

gttogttgag acattccctt caccaaaagg cattcctcag ctcagggaaa ggaaagtgga

29940

gaggaatggt gagtgagacc gtctttaaat atcctgaact actgccccct tccttattgt

30000

ctactatcta catttgggga gtgattctct tagcaacctc tcaggtttga tatcagcaat
actgactcat gatactaccc caaagccaaa gcatgaaaca tggaaggtgt tcactaggga

30060

30120

caataccaga tttccttaaa tcacaaagca acagagcaca tcacactgga tgtctcacto

30180

tggcttcctg cccggctcca tatcaacggg caattgacgt tggggggagc catttcctac

30240

tctggccttc agcttgcggt cagctgagtg tgttggacca cacactctct agcatcctgc

30300

gattctaacc ttctatggat ttcatcagtt ttatagatat gagttcagcc tacgactttg
ggaaacaaag gattcaactg taaaaaacat ttgaattcca ctgatctatt ttatctctgc

30360

30420

tcaggaagaa aaaatggcac cttcttcctg aagtttgtgt ttccggtctc cttgatttct30480

gagagatttt aaaattactt tctatttttc ttactctgtt ctgatattgc aaaatggtta

30540

ttaccaatta ttaactaagt gatgtttaat tttagaagtc aagctacttg taagggccca

30600

tataaatgtg tgctaggatt agctttagat ttgttttctg cttctattta cagcaaggac

30660

aggactgggg agtggtttta ttttattaag ctttttagca tgatttaatt tttaaaacto

30720

aggacgtatt actttaaaaa ttaatcgaat aaagatgtat gttcataact tgaaccacac

30780

agaaaagtaa aatacaaata aaattgtgat ctcattactt aaagaataac cattgttggc

30840

catgtgcggt ggctcacgcc tgtaatctca gcactttggg aggccaaggc gggcgcatca

30900

cttgaggtca ggagttcaag accagcctgg ccaacgtggt gcaacctcat ctctactaaa

30960

US 11,279,935 B2
56

55- continued
atacaaaaaa aattagctgg gcatggtggc gtgtgcctgc aatoccagct acttgggagg
ctgagccagg agaattgott aaacccggga ggccgaggtt gcagtgagca gagattgcgc
cactgcactc cagcctgggc gacagagcaa gactccatca caaggaaaag aaaacaagca

31140

ttgttaaaat tttgatgcaa ttccttgtag cctactcatt cactgtttct aatgcttact

31200

aagtgtttag taacacatac atacacactg cacacacaca cattgtctcc ttcttcacto
aacataagga tagcatttcc tatattatta aaatattttt aaaaatgtgt gtgtgttaca

31260

tgtgttatat gtcacacaca cttttaacca cacaaagaca caggagaaca tcaaaaatca

31380

agatgotttc acgtcccctc acacatgtga tccttataat cctgtgtgag aagcaggact

31440

ggaaatattt ccaacttcca gctgaaaatg catagctcag agacaggaag gaacttaacg

31500

ttggttaaga tcacaggctc ggggtaccac ataccagctg tgtgaccctg ggcacaataa

31560

tggatctaag atttgtgggg cgcgaagcct gtatcatttt aggggttctc cttaaagaaa

31620

ataataaaac atttacctgt gaaaatttta gtgatttcat gtgatcttgt aagtatattg
ctaggacctc ttccagggcc ttggaagggt ctgtgttaag tgaggagccc tgtagcttca

31680

tttcatctgc ttggaggcaa atctgccttt atttgggtaa gtgacttatt ttcatctgta
aagtggggat aataaaagtg cttgcctctg agtagttaga aagattaaac aagatactgt

31800

31020
31080

31320

31740

31860

attgtaaagc attttgtaaa cttcctggca cattggcaca taataagtgc ttaataaggg 31920

tcacctgtga ttactaattt tattcaaaat catgtcattg tggctggtgt catttgagct
gcttcccaga gactgagcga ttcctgtaca tacctaaagt tacaccttca agatgctgca

31980

gottggctca ctcactcatt taataagcat ttaggagttc ctgctagatg ctatgcactg

32100

32040

tgctaggctc cagtctcaat tcattctcac accacattca ctggaagcac aactcaggta 32160
tcccctcctc tgggaagccc ttctaggacc ctaagccgac tcactactat ctgggcactt

32220

atctctctgt gtgactgggt ttggacatca ctagtattca aggaaaagac attatcctgc

32280

ccctctgcca cctttgttca tttcagttca gtataatcga tacttctctc tgtgcctata

32340

atacagtatg gaggtgctca ataatcttaa atgagttgac tgagcatgtg tttgttttca

32400

caggaagatt tccgtttctt ttgcaagcta tttaatttgc aaaagtaagc aagatgtgca

32460

caaaatagtt ttgagacatc tggaaacaaa acaaggttgc ctcatcatta atcttgtttt

32520

tgtgtgataa ctttactttt tctcatttta tgacaactga aattctctat tatactgcca
gacctcattc tgcaagcttc tctcctgtgc taataaaaaa tattatggcc acatcgagaa

32580
32640

gtccttaggg aatgtgttct gcatcaggaa aagtaattaa gotctggggt cctaaaggag

32700

cctgggaaag tctatccaga aactatgggg ttaggatgta ttatctattg aaaaacagaa
coggactttg caagttttac aatatcttgg cttattaaag ccactaactg agaattaaaa

32760

tttctggatt gttactttta caacagcagt caagtgtcgg aaaatatcct gaccagtcag

32880

taaggtatac aaatagagaa ggcaaggagt agctgtgagt aaagcgccaa ctacatttgo

32940

tgagtacctg ggaatttacg agttagcttt tgctatcatt atccgtctcg ctaaagcaac

33000

cctatgaggt aggtgggaag ctggatgccg tcctcttttg acagccaaca ctgaagaaca

33060

gctggggctg gaaactagat ctgtcagaca tagtaactgc ttctatcact ctcaccaccc

33120

tcccatgctc ttgagagtgc cttcgctttt gtgggtgtct tcagaaaagc atgcggatot

33180

ctgtatagg tttactcaag tgttcctctc tgt agggt gacctgggct acatgaacat

33240

gggcacctca ggatcggcag ctttccatga attcctatgg taaggggttc agcttatcct

33300

tcatgtgggt gaagatgtat gatttcattg aacattggca gttacagcca agtacaagtc

33360

32820

US 11,279,935 B2
58

57- continued
ctgccttcag gatccagcag ggaagaatga cattaaacat gtcaccttgg agaaatgttg

33420

cattcacctt ccgggtaatg acccagccac ccagggactc ctctgaaata tocaatcctc

33480

taagtttaaa cacgtttcag aattaatcca tctgaattct tatcgctgta gatgcaagga

33540

gaaggagtta tggcaggtct gaaactatta gcggccaagc tccctagcag aagcaacctc

33600

tccctacacc ccattatccg tgcacagctc cttatcagcg gaggaacaac tttactgtca

33 660ccatattttc tacttggaaa aaaaaaagaa acctcagcag gcccaatccg aaggctatga

33720

aatttcaaag aaacagagct aagaggcgag aaactagccc tttgtgttaa aaagatggaa

33780

atgctccaag cgcctttcca gtctgctttc tgtttacagg ccttgcgtga gcgcgggagg

33840

ctgggcctca tctctggctc tccccagcca ggacccctta ctcaaaaccc tccatctcgg

33900

caagaaccct cattcccaag ttagttggat tccaagtcca taaaccgggt caaaagaata

33960

cagtaatttc ctactggagc totaagtggt ggttttcacg gaggactcgg caccatcctc

34020

ccacctgcct cctcccatcg aacattcctt tttaagaaaa acacacacca ggctccggga

34080

agctgacgta aacatcctga ggaaataacc actgtaagtg gottctaaac ccatttcccc

34140

aaggaaataa tcagcacctc tttcccaaga cagctccagg taagcaagcg gtgaagggag
ctgcaccacc gcgttctcaa aagtaaaggc gottgcggaa ctgtctactg atttgtaagc

34200

gcgcgcccat ccactgtcgc atttaatctt ccgaacgacc cacgaggtgg taaaggcggg

34320

gattattttc ttcattttac gggtgagaaa ccgaggccca cagcgctcgt tcctgtagcc

34380

ggggtcaggc agggcacacc taggggagcc tctgccttcg agccccggga ccgccccgcg
gcaccacgag atctccccag tcagcccgtg tgcggctgcg gccgcatggc ctcattgttt

34440

34260

34500

tgggaagacg tccctctgtg aggatcttgg gcgactctat gaaccgcggg cccagccgct 34560

ctgcggaacg atccgcagcg ggacgcaaag ggcgcggcga goccggcggc tcccggaaca

34620

aaggacagca gagcgcgcgg ggcccgaagg ggcgtccgcc ctagtgggcg goggaggggg

34680

atgcgcgcgg agaggaccag cgcgcaccgc cccggcccgc ccctcaaatc ctccttcctt

34740

ctctctcgca ctcggagacg gtccctggga gatccgggag ggggcgcagc tcgccgtcct

34800

cgccggcaag tgccgtcgca acctccctct gccgccacccccaccccgag aagcgcacaa
agagcggccg ggcagggagc gctcacctcc cgtcggggcg gggccgcggg cggggccggc

34860

34920

gcgcgctccg cagcgcccgc cttaaagggc ccgcccgctc ctcgcgcgca ctgcgcatgo

34980

tcggccgcgg ggcacggagg ggagggatgg gaacagcggg cgcgcacgcc cgcgccgccc

35040

gctggcgctg tggcctgcgt ccccgcggtg goggcgcggc gctcgtgcgc gtgtcagaac

35100

ccgcagggtc tcggcccggg cgccgggcgt gtgctggaca ctggtgccat gcattccaga

35160

ggtcgccagc ggccgggcgg gccgagctcg cgcggcagcc agcttttagc tttcccatcc

35220

tcagtttcag ggccagttgg gcgaagtgct gcccaaggag acagaaatge aggcgcgggc

35280

gcgcgcgcgc gcggttgcca gtgtgggcgg gcgtgtgctt ttgtgtctcg tgcgctcccg

35340

cgcaggtgga caacccgcgc cgggagtcac tctgcccgtg cgccccaaga cctggagtcc

35400

cgttttccgt gtcggagggc tcgggtcgcc cttttgggcg gactacagac tcttctcaca

35460

gaggaagctt tggctccacg gatggtttat ttggagccac taacgggact tectgaggac 35520

cgctctttta gagcccggct ccaggcatca gagttgggcg cgttttcttt tctcagtcag

35580

gcgagggagg agacttaggg acaatgattg tcctcggctg ataggtacat ccaagtgtcc

35640

cgagcctgcc agtcccgcat tagacaacaa agagaggcgt tagcggcggc gggggagggg

35700

US 11,279,935 B2

59

60- continued
cgcggccggg aggaggacgg ggcgggctgc ggcgagcgcg ggggtggggg gtggcgctct
ctctccgctc gctcaacccg cggcctcctc ccgcccccgg cctggcgcgc gccaatcgcc
tcccccagcg atagtgtcag tagcattgtg gtgtcagctc ccgcctgtga cgttgcgcct
ccctcgtccc cctccggagt cgtctggctg cagaggctc

35760
35820

35880
35919

< 210 > SEQ ID NO 3
< 211 > LENGTH : 836< 212 > TYPE : DNA< 213 > ORGANISM : Human< 400 > SEQUENCE : 3

gtaggtcgag gagggatggg gaggtgggtg ggggtggtgt agggtctcag gccactgcct
cgaggggaag gcgtgttcag tatcgggatc cgcggcgtgg ggtgccttgg ggagagagga

120

agagccgcgc gatgggccgg cgccccagac aaagaaagtg caggcaggct gtctcccgga

180

gccgcgccgc tgccctggcg ggatgcactt tcttctgcta aaatcactgc cttctcccct

240

aacgcccccc aaccagcoca cctccagaaa gacaatttaa atgtaagatg cttgggggag
ggggcctttg atcagtcctt tgggaggagg aaggaggagg agtgagcata ggatgggagg
aggattctgg atttctgcaa agcggaatgg agcccagagg aggaacaatg ggtcccggga

300

60

360

420

ctcacacccc accccctacc cccagtcgac cagcgctgag gcatcgcgac ttcagctgcc
ttccccgagc cccttccccg tgtcttcaga gotgacggcg cgcccagctg gatcccagcg
gcatctcccc agatgacttt tctgggattc tcgggtttgg ttcgggacga ctgcagtcac

480

tgggggaggg ccaggcagcc aatgggctgt tcctcgagcg cctcgcgggt gggatccgct
gcccagcccg tggcgcggcc cgaggtcagt gagggagacg ccccctttcc gcccatctct

660
720

tgtcctgccg tctcgctgtc ctggacgcgg gctgtccccg gtccccgcgg ttaccccagg

780

ataatgggcg tgtctgtctc tctctcccac tccctcctcc gcaccctggt tcgtag

836

540
600

< 210 > SEQ ID NO 4
< 211 > LENGTH : 3416
< 212 > TYPE : DNA< 213 > ORGANISM : Mouse< 400 > SEQUENCE : 4

tgttcgcttt gcgctcggca ttgtggccag ccagccgggc actcgggggg cacgcgcggc

60

cgccgctoga gctctgcccc caccccaacc gccagcagat ctggggtggg gacccaggcg
ggggctcttg cagccactgc ccggtgcgga ttgcacgcag agacccactc ctccgagcgc
cgaaaaacga gccacggagc cctccgcagc cgaccggcct ccccgcccct gactgccggc

120

240

ttcccggctg cctttgtggc tgcaccttct agctgccgag cagagagccg gctggggcgc

300

ggcgcgcacg gcggagcgat goccagctcgctgtttgcag acctggagcg caacggcago360

ggcggcggcg ggggaggcgg cggcggggga ggcggcggtg gcagcggcgggggagagact

420

ctggatgacc aaagagccct gcagcttgcg ctcgatcagc tctccctgtt gggactggac

480

agtgatgagg gcgcctcttt gtacgacagc gaaccgcgca agaagagcgt gaacatgacc

540

gagtgcgtgc cggtacccag ttccgaacac gtcgcggaga togtagggag gcaaggttgt

600

aaaatcaaag ctttgagggc gaagaccaac acttacatca agaccccagt tcgcggggag

660

gagcctgtct ttgttgtgac gggcaggaag ga

atggctcg gagggagato

720

atctctgccg cagagcactt ctccatgatc cgagcctctc gtaacaagaa cacggctctc

780

aacggagctg tgcccggacc gcccaacctg ccgggacaga ccactatcca agtgagggtg

840

tgtgg

180

US 11,279,935 B2
61

62- continued

ccataccgcg tggtagggct cgtggtgggt ccaaagggcg ccacgatcaa gcgcattcaa
cagcagacac atacatatat tgtgacaccc agccgagaca aggagccagt tttcgaggtg

900

actggcatgc cagagaacgt ggatcgcgct agagaggaga tcgaagctca catcgcgctg

1020

cgcaccggtg gcatcatcga gctgacagac gagaacgact tccatgccaa tggcacagac

1080

gtgggctttg atctgcatca cgggtccggc gggtccgggc cgggcagcct ctggagcaag

1140

cccaccccaa gcatcactcc tacacctggc cgcaagccct tctccagcta tcgcaacgac

1200

agctccagct cgcttggcag cgcatccaca gactcttact tcggtggtgg gaccagcggo

1260

agcgcagctg ctacttcacg cctggcggac tatagccctc ccagccctgc actcagcttt

1320

gctcacaatg ggaacaacaa caataacggc aatggttaca cctacacagc gggggaagcc

1380

tcagtacctt ccccagatgg gggtcctgag ctgcagccta ctttcgaccc agctcccgcc

1440

ccaccacctg ggacacccct tctctgggcc cagttcgagc gctctccagg aggtggatct

1500

gcagcaccag tatcctcttc ctgctcttct tcggcatcct catctgcctc gtcgtcctct
gtggtctttc ccgggggtgg cgccagcagc acaccctcca atgccaatct ggggctgctg
gtgcaccgtc gactgcaccc gggcaccagc tgcccgcgcc tgtctccgcc cttgcacatg

1560

gccacggggg cgggagagca ccaccttgct cgccgcgtgc gcagcgaccc gggcggtgga

1740

ggcctggcct acgctgccta tgctaatggg ctagggacgc agctccctgg cctgccctcg
toggacactt cgggctcctc ctcgtcctct agctcctcct ccagctcttc ctcctcttcc

1800

tctgggctga ggcgcaaagg cagccgcgac tgctctgtgt gottcgagag tgaagtgatc

1920

gccgcgctgg tgccctgtgg ccacaacctc ttctgcatgg agtgtgccaa ccgcatttgt

1980

gagaagagcg agcccgagtg tcccgtctgc cacacggcgg tcactcaggc catccgcatt
ttttcctgaa ggcagtgcgc gcgcgcgcac tgcagggaag gagggtcctc tctcgaccct2040

cattccctag ggtctacctg cccagacgcc tctggtgccc acctctttcc accccaccct2160

catcactctc agagatccca gaggagcttg gaaagctgta gtatccgctc atttttaaaa
tgtcattttt aaacaaagga acttgccagg atctctgcat caggagtact gtagcctccg

2220

aaccacctga attgcatgct ctataaataa taggaacggc gacattctag taacgatagt
ttttacactg tacttaatag gaagcttcca aaagaaaacc ccacaagttt tccattttct

2340

tgaagtagaa aaatgaacag taattatgaa gatgattaat aattgtgcta tgggatgtgt

2460

ggactgtttt gtgtgtttcc ctttgtgggt gggttcctac agcgctcgtt ctagaacaca

2520

agtggatcct ttttgaatgt tcatggaagg gccaggagtt ctgtacagcc aggaccctgc

2580

agctttatta aagttaaaac tgtaacatat ctcttatata ttaaaaaaaa aaacctttaa

2640

aagttttaaa gagaaattgc attaatacag attgaagtat tttattcttt ttttgacttg

2700

aaaaattata tttcatattg caaagatgtt tacaagtatt ttaatttaag ttcagtgaac

2760

ttttttgtag ctgggttaaa tctttttatt tttagtatgg ccttatggca aagaacactg

2820

tattatttta ataatcacac gattgtgacg gattacaaac cataaaatgt ataacgtttt

2880

gagcagtatt ctgttgggat ggagatttta taggttcaga caaatcttct agatctgott

2940

cacccagcat attttctatt cagtgatata aagcatattt tattctatat tattacaaaa
aatggaaatg tataaacatg tcaaaaggaa cagttgatgc tttctaacat ttgtataaat

3000

agaattcagt gcaaattaca gaaattctgt tgcaccgcta tagttttagt gtttctattt

3120

taatacattt gtttaccact cgtttatgta tatgtaggtg acgttacttg agcttaaatg

3180

960

1620
1680

1860

2100

2280

2400

3060

US 11,279,935 B2
64

63- continued
tactttactg agcaaagttt aaaaaaaaac aaagtatatt ttattttatg ataaagggcc
tttaacctca tggtcaaata ctaatattat atttgctgag acaagatttg aaattgtatc

3300

aagagtttta tttttctgac atttaaagtt ctacataata aaggtaaact taagtaatgg

3360

tgctacttca ttttttaagt atttctatat aaataaaaca ttgaagaaaa atcact

3416< 210 > SEQ ID NO 5
< 211 > LENGTH : 569< 212 > TYPE : PRT< 213 > ORGANISM : Human< 400 > SEQUENCE : 5

Met Pro Ser Ser Leu Phe Ala Asp Leu Glu Arg Asn Gly Ser Gly Gly
5

1

15

10

Gly Gly Gly Gly Ser Ser Gly Gly Gly Glu Thr Leu Asp Asp Gin Arg
25

20

30

Ala Leu Gln Leu Ala Leu Asp Gin Leu Ser Leu Leu Gly Leu Asp Ser
35

40

45

Asp Glu Gly Ala Ser Leu Tyr Asp Ser Glu Pro Arg Lys Lys Ser Val
50

55

60

Asn Met Thr Glu Cys Val Pro Val Pro Ser Ser Glu His Val Ala Glu
65

75

70

80

Ile Val Gly Arg Gin Gly Cys Lys Ile Lys Ala Leu Arg Ala Lys Thr
85

95

90

Asn Thr Tyr Ile Lys Thr Pro Val Arg Gly Glu Glu Pro Val Phe Val
100

105

110

Val Thr Gly Arg Lys Glu Asp Val Ala Met Ala Arg Arg Glu Ile Ile
125

120

115

Ser Ala Ala Glu His Phe Ser Met Ile Arg Ala Ser Arg Asn Lys Asn
130

135

140

Thr Ala Leu Asn Gly Ala Val Pro Gly Pro Pro Asn Leu Pro Gly Gln
145

150

155

160

Thr Thr Ile Gin Val Arg Val Pro Tyr Arg Val Val Gly Leu Val Val
170

165

175

Gly Pro Lys Gly Ala Thr Ile Lys Arg Ile Gin Gin Gin Thr His Thr
180

185

190

Tyr Ile Val Thr Pro Ser Arg Asp Lys Glu Pro Val Phe Glu Val Thr
195

205

200

Gly Met Pro Glu Asn Val Asp Arg Ala Arg Glu Glu Ile Glu Ala His
210

220

215

Ile Ala Leu Arg Thr Gly Gly Ile Ile Glu Leu Thr Asp Glu Asn Asp
225

235

230

240

Phe His Ala Asn Gly Thr Asp Val Gly Phe Asp Leu His His Gly Ser
245

255

250

Gly Gly Ser Gly Pro Gly Ser Leu Trp Ser Lys Pro Thr Pro Ser Ile
260

270

265

Thr Pro Thr Pro Gly Arg Lys Pro Phe Ser Ser Tyr Arg Asn Asp Ser
280

275

285

Ser Ser Ser Leu Gly Ser Ala Ser Thr Asp Ser Tyr Phe Gly Gly Gly
295

290

300

Thr Ser Ser Ser Ala Ala Ala Thr Gln Arg Leu Ala Asp Tyr Ser Pro
310

305

315

320

Pro Ser Pro Ala Leu Ser Phe Ala His Asn Gly Asn Asn Asn Asn Asn
325

330

335

3240

US 11,279,935 B2
66

65- continued
Gly Asn Gly Tyr Thr Tyr Thr Ala Gly Gly Glu Ala Ser Val Pro Ser
345

340

350

Pro Asp Gly Cys Pro Glu Leu Gin Pro Thr Phe Asp Pro Ala Pro Ala
360

355

365

Pro Pro Pro Gly Ala Pro Leu Ile Trp Ala Gin Phe Glu Arg Ser Pro
370

375

380

Gly Gly Gly Pro Ala Ala Pro Val Ser Ser Ser Cys Ser Ser Ser Ala
385

395

390

400

Ser Ser Ser Ala Ser Ser Ser Ser Val Val Phe Pro Gly Gly Gly Ala
410

405

415

Ser Ala Pro Ser Asn Ala Asn Leu Gly Leu Leu Val His Arg Arg Leu
425

420

430

His Pro Gly Thr Ser Cys Pro Arg Leu Ser Pro Pro Leu His Met Ala
440

435

445

Pro Gly Ala Gly Glu His His Leu Ala Arg Arg Val Arg Ser Asp Pro
455

450

460

Gly Gly Gly Gly Leu Ala Tyr Ala Ala Tyr Ala Asn Gly Leu Gly Ala
470

465

480

475

Gin Leu Pro Gly Leu Gin Pro Ser Asp Thr Ser Gly Ser Ser Ser Ser
485

495

490

Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Gly Leu Arg Arg
505

500

510

Lys Gly Ser Arg Asp Cys Ser Val Cys Phe Glu Ser Glu Val Ile Ala
515

520

525

Ala Leu Val Pro Cys Gly His Asn Leu Phe Cys Met Glu Cys Ala Asn
530

540

535

Arg Ile Cys Glu Lys Ser Glu Pro Glu Cys Pro Val Cys His Thr Ala
545

555

550

560

Val Thr Gin Ala Ile Arg Ile Phe Ser565< 210 > SEQ ID NO 6
< 211 > LENGTH : 576< 212 > TYPE : PRT< 213 > ORGANISM : Mouse< 400 > SEQUENCE : 6

Met Pro Ser Ser Leu Phe Ala Asp Leu Glu Arg Asn Gly Ser Gly Gly
10

5

1

15

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly
20

30

25

Glu Thr Leu Asp Asp Gin Arg Ala Leu Gin Leu Ala Leu Asp Gln Leu
40

35

45

Ser Leu Leu Gly Leu Asp Ser Asp Glu Gly Ala Ser Leu Tyr Asp Ser
55

50

60

Glu Pro Arg Lys Lys Ser Val Asn Met Thr Glu Cys Val Pro Val Pro
75

70

65

80

Ser Ser Glu His Val Ala Glu Ile Val Gly Arg Gin Gly Cys Lys Ile
90

85

95

Lys Ala Leu Arg Ala Lys Thr Asn Thr Tyr Ile Lys Thr Pro Val Arg
105

100

110

Gly Glu Glu Pro Val Phe Val Val Thr Gly Arg Lys Glu Asp Val Ala
120

115

125

Met Ala Arg Arg Glu Ile Ile Ser Ala Ala Glu His Phe Ser Met Ile
130

135

140

US 11,279,935 B2
68

67- continued
Arg Ala Ser Arg Asn Lys Asn Thr Ala Leu Asn Gly Ala Val Pro Gly
155

150

145

160

Pro Pro Asn Leu Pro Gly Gln Thr Thr Ile Gin Val Arg Val Pro Tyr
165

170

175

Arg Val Val Gly Leu Val Val Gly Pro Lys Gly Ala Thr Ile Lys Arg
180

190

185

Ile Gin Gin Gin Thr His Thr Tyr Ile Val Thr Pro Ser Arg Asp Lys
200

195

205

Glu Pro Val Phe Glu Val Thr Gly Met Pro Glu Asn Val Asp Arg Ala
210

215

220

Arg Glu Glu Ile Glu Ala His Ile Ala Leu Arg Thr Gly Gly Ile Ile
225

235

230

240

Glu Leu Thr Asp Glu Asn Asp Phe His Ala Asn Gly Thr Asp Val Gly
255

250

245

Phe Asp Leu His His Gly Ser Gly Gly Ser Gly Pro Gly Ser Leu Trp
265

260

270

Ser Lys Pro Thr Pro Ser Ile Thr Pro Thr Pro Gly Arg Lys Pro Phe
275

280

285

Ser Ser Tyr Arg Asn Asp Ser Ser Ser Ser Leu Gly Ser Ala Ser Thr
295

290

300

Asp Ser Tyr Phe Gly Gly Gly Thr Ser Gly Ser Ala Ala Ala Thr Ser
305

310

315

320

Arg Leu Ala Asp Tyr Ser Pro Pro Ser Pro Ala Leu Ser Phe Ala His
330

325

335

Asn Gly Asn Asn Asn Asn Asn Gly Asn Gly Tyr Thr Tyr Thr Ala Gly
345

340

350

Glu Ala Ser Val Pro Ser Pro Asp Gly Gly Pro Glu Leu Gin Pro Thr
360

355

365

Phe Asp Pro Ala Pro Ala Pro Pro Pro Gly Thr Pro Leu Leu Trp Ala
375

370

380

Gln Phe Glu Arg Ser Pro Gly Gly Gly Ser Ala Ala Pro Val Ser Ser
385

400

395

390

Ser Cys Ser Ser Ser Ala Ser Ser Ser Ala Ser Ser Ser Ser Val Val
410

405

415

Phe Pro Gly Gly Gly Ala Ser Ser Thr Pro Ser Asn Ala Asn Leu Gly
425

420

430

Leu Leu Val His Arg Arg Leu His Pro Gly Thr Ser Cys Pro Arg Leu
445

440

435

Ser Pro Pro Leu His Met Ala Thr Gly Ala Gly Glu His His Leu Ala
450

455

460

Arg Arg Val Arg Ser Asp Pro Gly Gly Gly Gly Leu Ala Tyr Ala Ala
465

470

480

475

Tyr Ala Asn Gly Leu Gly Thr Gln Leu Pro Gly Leu Pro Ser Ser Asp
485

495

490

Thr Ser Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser
500

505

510

Ser Ser Ser Gly Leu Arg Arg Lys Gly Ser Arg Asp Cys Ser Val Cys
515

525

520

Phe Glu Ser Glu Val Ile Ala Ala Leu Val Pro Cys Gly His Asn Leu
535

530

540

Phe Cys Met Glu Cys Ala Asn Arg Ile Cys Glu Lys Ser Glu Pro Glu
545

550

555

560

US 11,279,935 B2
70

69- continued
Cys Pro Val Cys His Thr Ala Val Thr Gln Ala Ile Arg Ile Phe Ser
565

570

575

< 210 > SEQ ID NO 7
< 211 > LENGTH : 30
< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : MEX3B primer Fw1< 400 > SEQUENCE : 7

cgtcgtcctc tgtggtcttt cccgggggtg

30< 210 > SEQ ID NO 8
< 211 > LENGTH : 30
< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : MEX3B primer Rvi< 400 > SEQUENCE : 8

tcaggaaaaa atgcggatgg cctgagtgac

30< 210 > SEQ ID NO 9
< 211 > LENGTH : 20
< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : Mouse GAPDH primer Fw1< 400 > SEQUENCE : 9

agagacagccgcatcttctt

20< 210 > SEQ ID NO 10< 211 > LENGTH : 20
< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : Mouse GAPDH primer Rv1< 400 > SEQUENCE : 10

gacaagcttc ccattctcgg

20< 210 > SEQ ID NO 11< 211 > LENGTH : 25

< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : Mouse IL - 6 primer Fw1< 400 > SEQUENCE : 11

gctaccaaac tggatataat cagga

25< 210 > SEQ ID NO 12< 211 > LENGTH : 24
< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : Mouse IL - 6 primer Rv1< 400 > SEQUENCE : 12

ccaggtagct atggtactcc agaa
< 210 > SEQ ID NO 13< 211 > LENGTH : 20
< 212 > TYPE : DNA

24

US 11,279,935 B2
72

71- continued< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : Mouse CXCL5 primer Fwl< 400 > SEQUENCE : 13

cagaaggagg tctgtctgga

20< 210 > SEQ ID NO 14< 211 > LENGTH : 20
< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 223 > OTHER INFORMATION : Mouse CXCL5 primer Rv1< 220 > FEATURE :< 400 > SEQUENCE : 14

tgcattccgc ttagctttct

20< 210 > SEQ ID NO 15< 211 > LENGTH : 4368
< 212 > TYPE : DNA< 213 > ORGANISM : Human< 400 > SEQUENCE : 15

tcaagaggcg gggaggagag gaggaaaaag cgctgagtga gggcgggcgg gcgggcggga

60

gggagggagt ggaggagccg gggaggggct ccttaaagaa acgcttctgt cgctcgcctg

120

ctcgcttttc gctcggcatt gcggccagcc agccgggcac tcggggggca cgcgcggcca

180

ccgctagagc tctgccccca ccccacccgc cagcaggtct ggggtgggga cccaggtggg

240

ggctcctgca gccactgccc ggtgcggacc gcacggagcg acccactcct cccagcaccg

300

aggaagaagc aacggagccc tcagcaggcg accggcctcc ccgcccctga ccacccgctt

360

cccggctgcc tttgtggccg cagcttctcgccgccgagcc gagggccggc gggggcgcgg

420

cgcgcacggc cgagcgatgc ccagctcgct gttcgcagac ctggagcgca acggcagcgg

480

cggcggcggc ggcggcagca gcggaggggg agagaccctg gatgaccaaa gagccctgca

540

gctcgcgctc gaccagctct ccctgctggg gctggacagt gacgagggcg cctctctgta

600

cgacagcgag ccgcgcaaga agagcgtgaa catgaccgag tgcgtgccag tacccagttc

660

tgagcatgtc gccgagatcg tggggcggca aggtaggtcg aggagggatg gggaggtggg

720

tgggggtggt gtagggtctc aggccactgc ctcgagggga aggcgtgttc agtatcggga780

tccgcggcgt ggggtgcctt ggggagagag gaagagccgc gcgatgggcc ggcgccccag
acaaagaaag tgcaggcagg ctgtctcccg gagccgcgcc gctgccctgg cgggatgcac

840

tttcttctgc taaaatcact gccttctccc ctaacgccccccaaccagcc cacctccaga
aagacaattt aaatgtaaga tgcttggggg agggggcctt tgatcagtcc tttgggagga

960

1020

ggaaggagga ggagtgagca taggatggga ggaggattct ggatttctgc aaagcggaat

1080

ggagcccaga ggaggaacaa tgggtcccgg gactcacacc ccacccccta cccccagtcg

1140

accagcgctg aggcatcgcg acttcagctg ccttccccga gccccttccc cgtgtcttca

1200

gagctgacgg cgcgcccagc tggatcccag cggcatctcc ccagatgact tttctgggat

1260

tctcgggttt ggttcgggac gactgcagtc actgggggag ggccaggcag ccaatgggct1320

gttcctcgag cgcctcgcgg gtgggatocg ctgcccagcc cgtggcgcgg cccgaggtca

1380

gaga cgcccccttt ccgcccatct cttgtcctgc cgt cgctg tcctggacgc

1440

gggctgtccc cggtccccgc ggttacccca ggataatggg cgtgtctgtc tctctctccc

1500

actccctcct ccicaccctg gttogtaggt tgtaaaatca aagcgctgcg ggcgaagacc

1560

gt

900

US 11,279,935 B2
73

74- continued

aatacttaca tcaagacccc agttcgcggg gaggagcctg tctttgttgt gacgggcagg

1620

aaggaggatg tggccatggc tcggagggag atcatctctg ctgccgagca cttctccatg

1680

atccgcgcct cccggaataa gaacacggca ctcaacggcg cggtgcctgg gccgcccaac

1740

ctgcccgggc agaccaccat ccaagtgcgg gtaccctacc gcgtggtggg gctcgtggtg
gggcccaaag gcgccacaat caagcgcatc cagcagcaga cgcacacgta catcgtgacg

1800

cccagccggg ataaggagcc ggtgttcgag gtgaccggca toccagagaa cgtggatcgc

1920

gctcgagagg agattgaggc gcacattgct ctgcgtaccg goggcatcat tgagctcaca

1980

gacgagaacg acttccacgc caacggcacc gatgtgggct tcgatctgca tcatgggtcc

2040

ggcgggtccg gcccaggcag cctctggagc aagcccaccc ccagcatcac gcccaccccc

2100

ggccgcaagc ctttctctag ctaccgcaac gacagctcca gctcgcttgg cagtgcttcc

2160

acagactctt atttcggcgg cgggaccagc agcagcgcag cggctaccca gcgcctggcg

2220

gactacagcc cccctagccc cgccctgagc tttgcgcaca acggaaacaa taacaataac
ggcaatgggt acacotacac agcgggggga gaagcctcag tgccatcccc cgacggctgc
cccgagctgc agcccacttt tgacccggct cccgctcccc cacctggggc accacttatc

2280

tgggcccagt tcgagcggtc cccgggaggc ggacctgcag ctccggtatc ttcttcctgc

2460

tcttcttctg catcttcgtc tgcttcttcc tcctccgtgg tcttccccgg gggtggcgcc
agtgcgccct ccaacgccaa cctggggcta ttggtgcacc gccggctgca ccctggcacc

2520

agctgcccgc gcctgtctcc acccttgcac atggccccgg gggcgggaga gcaccacctg

2640

gctcgccggg tgcgcagoga cccgggtgga ggaggcctgg cctacgccgc ttatgccaac

2700

gggctggggg cacagctgcc tggcttgcag ccgtcggaca cgtcgggctc ctcctcttcg

2760

tccagctcct cctccagctc ttcatcctct tcctccgggc ttcggcgtaa aggcagccgc
gactgctccg tgtgcttcga gagcgaagtg attgccgcgc tggtgccctg tggccacaac

2820

ctcttctgca tggagtgcgc caatcgcatc tgtgagaaga gcgagcccga gtgcccggtc

2940

toccacaccg cggtcactca ggccatccgc atcttttctt aaaggcagcg ggcgctgcta
gtgcgcaccg tgctggggga agggggaacc cctccccacc ctctttcccc agcgctcgcc

3000

1860

2340
2400

2580

2880

3060

tgcctccctg ggtgcccccc ctctcccttc tccttcccgg ccccaccaac actctgagat
ccgagaggag cttggaaagc tgtagtatcc gctcattttt aaaatttaat ttttaagtaa

3120

aggaatttgc caggatatct gcatcaagag tactgtagcc tgggaaacct gaacacotga

3240

aatgcatgct ctataaataa taggaacggc gacattctag taatgatagt ttttacactg
tacttaatag gaagcttcca aaagaagaaa accccacaag ttttccattt tcttaaagta

3300

ggaaaaaatg aacagtaata attatgatga agatgatagt agtgctatgg gatgtgtgga

3420

ctgtttagtg tgttcccctt tgtgggtggg ttcctatgat acttattata gaacacagtg

3480

gatccttttt gaatgttegt ggaagggcca ggagttcctg tgaaaccagg atactgcago

3540

tttattaaag ttaaagaaac tgtaacatat ctcttatata ttaaaaacgt ttaaaagttt

3600

taaagagaaa ttgcattaat acagattgaa gtattttatt cttttttgac ttgaaaaatt

3660

atatttcata ttgcaaagat gtttacaagt attttaattt aagttcagtg aacttttttg
tagctgggtt aaatcttttt attttagtat ggccttatgg caaagaacac tgtattattt

3720
3780

taataatcac acaattgtga cggaattaca accataaaat gtgtaatgtt ttgaacagta

3840

ttctgttggg atggagattt tataggttca gacaaatctt ctagatctgc ttcacccagc

3900

3180

3360

US 11,279,935 B2
76

75- continued
atattttcta ttcagtgata taaagcatat tttattctat attattacaa aaacggaaat

3960

gtataaacat gtcaaaaaga actgttgatg ctttctaaca tttgtataaa tagaattcag

4020

tgcaagttac aaaaattctg ttgcaccact ctagttttag tatttctatt ttaatacatt4080

tgtttaccac ttgtttatgt atatgtaggt gatgttactt gagcttaaat gtactttact

4140

gagcaaagtt taaaaaacaa agtatatttt attttatgat aaagggcctt taacctcatg

4200

gtcaaatact aatattatat ttgctgagac aagatttgaa attgtatcaa gagttttatt

4260

tttctgacat ttaaagttct acataataaa ggtaaaactt aagtaatggt gctacttcat

4320

tttttaagta tttctatata aataaaatat tgaagaaaat cttaaaaa

4368< 210 > SEQ ID NO 16< 211 > LENGTH : 15
< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : mouseMex3bspecific gapmer< 400 > SEQUENCE : 16

acataaacga gtggt

15< 210 > SEQ ID NO 17< 211 > LENGTH : 20
< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : mouseGAPDH - Fw2< 400 > SEQUENCE : 17tgtgtccgtc gtggatctga

20< 210 > SEQ ID NO 18< 211 > LENGTH : 21
< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : mouseGAPDH - Rv2< 400 > SEQUENCE : 18

ttgctgttga agtcgcagga g

21< 210 > SEQ ID NO 19< 211 > LENGTH : 21
< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : mouse TNF - Fw

< 400 > SEQUENCE : 19

tcttctcatt cctgcttgtg g

21< 210 > SEQ ID NO 20< 211 > LENGTH : 20
< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : mouse TNF - Rv

< 400 > SEQUENCE : 20
aggccattt gggaacttct< 210 > SEQ ID NO 21< 211 > LENGTH : 20

20

US 11,279,935 B2
78

77- continued< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : mouseG - CSF - Fw

< 400 > SEQUENCE : 21

cctggagcaa gtgaggaaga

20< 210 > SEQ ID NO 22< 211 > LENGTH : 20
< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : mouseG - CSF - Rv

< 400 > SEQUENCE : 22

ggggtgacac agcttgtagg

20< 210 > SEQ ID NO 23< 211 > LENGTH : 23
< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : mouseIL - 13 - Fw

< 400 > SEQUENCE : 23

cctctgaccc ttaaggagct tat

23< 210 > SEQ ID NO 24< 211 > LENGTH : 18

< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : mouseIL - 13 - Rv

< 400 > SEQUENCE : 24
cgttgcacag gggagtct

18< 210 > SEQ ID NO 25< 211 > LENGTH : 26
< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : mouseCXCL2 - FW

< 400 > SEQUENCE : 25

aaaatcatcc a aaagatact gaacaa

26< 210 > SEQ ID NO 26< 211 > LENGTH : 20
< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : mouseCXCL2 - Rv

< 400 > SEQUENCE : 26

ctttggttct tccgttgagg< 210 > SEQ ID NO 27< 211 > LENGTH : 20
< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : mouseCXCL1 - Fw

< 400 > SEQUENCE : 27

20

US 11,279,935 B2

79

80- continued
agactccagc cacactccaa

20< 210 > SEQ ID NO 28< 211 > LENGTH : 18
< 212 > TYPE : DNA< 213 > ORGANISM : Artificial Sequence< 220 > FEATURE :< 223 > OTHER INFORMATION : mouseCXCL1 - Rv

< 400 > SEQUENCE : 28

tgacagcgca gctcattg

18

15

The invention claimed is :1. A therapeutic method for a respiratory disease or apulmonary disease, comprising the steps of:administering a substance for decreasing expression of
MEX3B gene to a patient with a respiratory disease or 20
a pulmonary disease , wherein the substance fordecreasing of the MEX3B gene isan artificial nuclease that is CRISPR / Cas nuclease,

wherein the CRISPR / Cas nuclease comprises a guide
RNA having a sequence complementary to a partial
sequence of 15 to 25 bases of the MEX3B gene and Cas
nuclease ;wherein the MEX3B gene is a gene consisting of thesequence described in SEQ ID NO : 1 or 4 of theSequence Listing .

an antisense oligonucleotide which has a sequence
2. The method according to claim 1 , wherein the subcomplementary to an oligonucleotide comprising at stancefor decreasing of the MEX3B gene is administered
least 10 contiguous nucleotides contained in the 25 together with a carrier for lipofection .
MEX3B gene or in an expression control region of the
3. The method according to claim 1 , wherein the respi
ratory or pulmonary disease is severe asthma caused bygene;
a double - stranded RNA containing at least 20 contiguous interleukin 6 , interleukin 13 , TNF, G -CSF, CXCL1 , CXCL2 ,nucleotides in a coding region or an untranslated region 30 or CXCL5.
The method according to claim 1 , wherein the CRISPR /in the sequence of an RNA to be transcribed from the Cas4. nuclease
is CRISPR /Cas9 .
sequence of the MEX3B gene, or a DNA encoding the
double - stranded RNA ; or