U.S. patent application number 14/855046 was filed with the patent office on 2016-03-10 for recombinant virus and preparations thereof.
The applicant listed for this patent is THE BROAD INSTITUTE INC., MASSACHUSETTS INSTITUTE OF TECHNOLOGY, PRESIDENT AND FELLOWS OF HARVARD COLLEGE. Invention is credited to Mark D. Brigham, Le Cong, Silvana Konermann, Feng Zhang.
Application Number | 20160068822 14/855046 |
Document ID | / |
Family ID | 50771583 |
Filed Date | 2016-03-10 |
United States Patent
Application |
20160068822 |
Kind Code |
A1 |
Zhang; Feng ; et
al. |
March 10, 2016 |
RECOMBINANT VIRUS AND PREPARATIONS THEREOF
Abstract
The present invention generally relates to methods and
compositions used delivery of gene editing compositions including
transcriptional effectors with parvovirus and preferred methods for
making same.
Inventors: |
Zhang; Feng; (Cambridge,
MA) ; Brigham; Mark D.; (Somerville, MA) ;
Cong; Le; (Cambridge, MA) ; Konermann; Silvana;
(Zurich, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE BROAD INSTITUTE INC.
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
PRESIDENT AND FELLOWS OF HARVARD COLLEGE |
Cambridge
Cambridge
Cambridge |
MA
MA
MA |
US
US
US |
|
|
Family ID: |
50771583 |
Appl. No.: |
14/855046 |
Filed: |
September 15, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2014/030394 |
Mar 17, 2014 |
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14855046 |
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14213991 |
Mar 14, 2014 |
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PCT/US2014/030394 |
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61799800 |
Mar 15, 2013 |
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Current U.S.
Class: |
506/7 ; 435/455;
435/5 |
Current CPC
Class: |
C12N 2750/14121
20130101; C12N 15/86 20130101; C12N 2750/14043 20130101; C12N
2750/14151 20130101; C12P 19/34 20130101; C12Q 2600/158 20130101;
C12Q 1/701 20130101; C12N 7/00 20130101; C12Q 1/686 20130101; C12N
2750/14152 20130101 |
International
Class: |
C12N 7/00 20060101
C12N007/00; C12Q 1/70 20060101 C12Q001/70; C12N 15/86 20060101
C12N015/86 |
Goverment Interests
FEDERAL FUNDING LEGEND
[0003] This invention was made with government support under grant
numbers NS073124 and MH100706 awarded by the National Institutes of
Health. The government has certain rights in the invention.
Claims
1. A method for obtaining and optionally storing a sample
containing a set amount of rAAV comprising or consisting
essentially of: (a) creating infected or transfected cells by a
process comprising or consisting essentially of one or more methods
selected from: (i) transfecting plasmid(s) containing or consisting
essentially of exogenous DNA including DNA for expression into
AAV-infected cells along with another helper plasmid that provides
AAV rep and/or cap genes which are obligatory for replication and
packaging of the rAAV; or (ii) infecting susceptible cells with a
rAAV containing or consisting essentially of exogenous DNA
including DNA for expression, and helper virus wherein the rAAV
lacks functioning cap and/or rep and the helper virus provides the
cap and/or rev function that the rAAV lacks; or (iii) infecting
susceptible cells with a rAAV containing or consisting essentially
of exogenous DNA including DNA for expression, wherein the
recombinant construct lacks functioning cap and/or rep, and
transfecting said cells with a plasmid supplying cap and/or rep
function that the rAAV lacks; or (iv) infecting susceptible cells
with a rAAV containing or consisting essentially of exogenous DNA
including DNA for expression, wherein the recombinant construct
lacks functioning cap and/or rep, wherein said cells supply cap
and/or rep function that the recombinant construct lacks; or (v)
transfecting the susceptible cells with an AAV lacking functioning
cap and/or rep and plasmids for inserting exogenous DNA into the
recombinant construct so that the exogenous DNA is expressed by the
recombinant construct and for supplying rep and/or cap functions
whereby transfection results in an rAAV containing or consisting
essentially of the exogenous DNA including DNA for expression that
lacks functioning cap and/or rep; and (b) incubating the infected
or transfected cells, whereby there results infected or transfected
cells and supernatant containing the rAAV lacking functioning cap
and/or rep; (c) after incubating, extracting an aliquot from the
supernatant; (d) filtering the aliquot, whereby the filtered
aliquot contains and the method obtains a sample containing set
amount of the rAAV relative to the type and amount of susceptible
cells infected or transfected; and (e) optionally freezing the
filtered aliquot, whereby the method optionally includes storing a
sample containing set amount of the rAAV relative to the type and
amount of susceptible cells infected or transfected.
2. A method for screening rAAV comprising or consisting essentially
of, preparing the filtered aliquot or the stored filtered aliquot
of claim 1, if necessary, thawing the stored filtered aliquot,
contacting the filtered aliquot with cells, and determining whether
the exogenous DNA is expressed in an amount and/or duration
sufficient for an intended use.
3. The method of claim 2 wherein the contacting of the filtered
aliquot with cells comprises or consists essentially of transducing
said cells.
4. The method of claim 3 wherein the contacting is for 5-6
days.
5. The method of claim 2 wherein the rAAV expresses a TALE and the
contacting includes or consists essentially of detecting nuclease,
activator or repressor activity.
6. The method of claim 2 wherein the rAAV expresses a LITE, and the
contacting includes or consists essentially of inducing gene
expression or subjecting the contacted cells to a suitable
stimulus, and detecting whether a transcriptional effector has been
induced.
7. The method of claim 6 wherein detecting whether a
transcriptional effector has been induced includes or consists
essentially of detecting a color change.
8. The method of claim 2 wherein the rAAV expresses a CRISPR
system, and the contacting includes or consists essentially of
detecting gene knockdown or other effects of the CRISPR system.
9. The method of claim 1 wherein the AAV is AAV1, AAV2, AAV5 or an
AAV having a hybrid or mosaic AAV1, AAV2 and/or AAV5 capsid.
10. The method of claim 1 wherein the susceptible cells are 293FT
cells.
11. The method of claim 10 wherein 2.times.10.sup.5 cells are
transfected or infected.
12. The method of claim 11 wherein a 250 .mu.L filtered aliquot
contains the recombinant AAV at a concentration of about
5.6+/-0.24.times.10.sup.5.
13. The method of claim 1 including freezing the filtered
aliquot.
14. The method of claim 13 wherein the filtered aliquot is frozen
at about -80 C.
15. The method of claim 1 including adding a secretion enhancer to
the cells before, during or after and within the incubating.
16. The method of claim 15 wherein the secretion enhancer is
polyethylenimine (PEI).
17. A method of high-throughput screening of a sample comprising or
consisting essentially of contacting the supernatant containing the
rAAV lacking functioning cap and/or rep of claim 1 with the sample
and determining whether the exogenous DNA of claim 1 is present in
the sample.
18. The method of claim 17, wherein the supernatant is thawed from
the filtered aliquot.
Description
RELATED APPLICATIONS AND INCORPORATION BY REFERENCE
[0001] This application is a continuation-in-part of international
patent application Serial No. PCT/US2014/030394, filed Mar. 17,
2014, and published as PCT Publication No. WO2014/145599 on Sep.
18, 2014 and which claims priority to U.S. patent application Ser.
No. 14/213,991 filed on Mar. 14, 2014 which claims priority to U.S.
Provisional Application 61/799,800 filed on Mar. 15, 2013.
Reference is made to US applications having Broad reference
BI-2011/008 to US Provisional Application Nos. 61/736,527 filed
Dec. 12, 2012; 61/748,427 filed Jan. 2, 2013; 61/757,972 filed Jan.
29, 2013, 61/768,959, filed Feb. 25, 2013 and 61/791,409 filed Mar.
15, 2013, titled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE
MANIPULATION; Broad reference BI-2011/020 to US Provisional
Application Nos. 61/675,778 filed Jul. 25, 2012; 61/721,283 filed
Nov. 1, 2012: 61/726,465 filed Dec. 12, 2012 and 61/794,458 filed
Mar. 15, 2013, tided. INDUCIBLE DNA BINDING PROTEINS AND GENOME
PERTURBATION TOOLS AND APPLICATIONS THEREOF; Broad reference
BI-2011/021 to U.S. Provisional Application No. 61/565,171 filed
Nov. 30, 2011 and U.S. application Ser. No. 13/554,922 filed Jul.
30, 2012 and Ser. No. 13/604,945 filed Sep. 6, 2012, titled
NUCLEOTIDE-SPECIFIC RECOGNITION SEQUENCES FOR DESIGNER TAL
EFFECTORS and Broad references BI-2013/003 and BI-2013/004 to U.S.
Provisional Application No. 61/836,123 filed on Jun. 17, 2013 and
U.S. Provisional Application Nos. 61/758,468; 61/769,046;
61/802,174; 61/806,375; 61/814,263; 61/819,803 and 61/828,130 each
entitled ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND
COMPOSITIONS FOR SEQUENCE MANIPULATION, filed on Jan. 30, 2013;
Feb. 25, 2013; Mar. 15, 2013; Mar. 28, 2013; Apr. 20, 2013; May 6,
2013 and May 28, 2013 respectively.
[0002] The foregoing applications, and all documents cited therein
or during their prosecution ("appln cited documents") and all
documents cited or referenced in the appln cited documents, and all
documents cited or referenced herein ("herein cited documents"),
and all documents cited or referenced in herein cited documents,
together with any manufacturer's instructions, descriptions,
product specifications, and product sheets for any products
mentioned herein or in any document incorporated by reference
herein, are hereby incorporated herein by reference, and may be
employed in the practice of the invention. More specifically, all
referenced documents are incorporated by reference to the same
extent as if each individual document was specifically and
individually indicated to be incorporated by reference.
FIELD OF THE INVENTION
[0004] The present invention generally relates to methods for
preparation of viral vector and methods and compositions for
advantageous delivery of nucleic acid molecule(s) for expression of
Transcription Activation Like Effector (TALE) and nucleic acid
molecule(s) for expression of a CRISPR (Clustered Regularly
Interspaced Short Palindromic Repeats) system, or nucleic acid
molecule(s) for expression of a light-inducible transcriptional
effector (LITE), or a cassette or plurality of cassette comprising
or consisting essentially of a promoter and exogenous nucleic acid
molecule encoding same particularly for gene editing in a eukaryote
cell. TALEs, LITEs and CRISPRs expressed via a recombinant
construct, e.g., an AAV, can advantageously provide activator,
repressor or nuclease activity in vivo, in vitro or ex vivo.
[0005] The method of the invention can provide a readily
accessible, reproducible aliquot of recombinant construct that can
be used for testing, e.g., testing whether construction of the
recombinant construct was successful, or whether the recombinant
construct expresses the exogenous DNA in an amount that may be
sufficient for an intended use and/or for a duration that may be
sufficient for an intended use, i.e., for screening, such as high
throughput screening. And hence the invention relates to a method
that may advantageously be for screening or high throughput
screening, wherein the method additionally comprises or consists
essentially of contacting the aliquot with cells and determining
whether the exogenous DNA is expressed in an amount and/or duration
sufficient for an intended use.
BACKGROUND OF THE INVENTION
[0006] Normal gene expression is a dynamic process with carefully
orchestrated temporal and spatial components, the precision of
which are necessary for normal development, homeostasis, and
advancement of the organism. In turn, the dysregulation of required
gene expression patterns, either by increased, decreased, or
altered function of a gene or set of genes, has been linked to a
wide array of pathologies. Technologies capable of modulating gene
expression in a spatiotemporally precise fashion will enable the
elucidation of the genetic cues responsible for normal biological
processes and disease mechanisms. To address this technological
need, Applicants developed molecular tools that may regulate gene
expression.
[0007] There is an evident need for methods and compositions that
allow for efficient and precise spatial and temporal control of a
genomic locus of interest. These methods and compositions may
provide for the regulation and modulation of genomic expression
both in vivo and in vitro as well as provide for novel treatment
methods for a number of disease pathologies.
[0008] Adeno-associated virus (AAV) is a single-stranded DNA
parvovirus which is endogenous to the human population. Although
capable of productive infection in cells from a variety of species,
AAV is a dependovirus, requiring helper functions from either
adenovirus, herpesvirus or a poxvirus such as vaccinia virus for
its own replication. In the absence of helper functions from any of
these helper viruses, AAV will infect cells, uncoat in the nucleus,
and integrate its genome into the host chromosome, but will not
replicate or produce new viral particles. There are at least 12
recognized AAV serotypes, There are recombinant AAVs. A recombinant
AAV can accommodate approximately 4300 bases of exogenous DNA, and
AAVs having a hybrid or mosaic capsid have been produced.
[0009] The genome of AAV has been cloned into bacterial plasmids
and is well characterized. The viral genome consists of 4682 bases
which include two terminal repeats of 145 bases each. These
terminal repeats serve as origins of DNA replication for the virus.
Some investigators have also proposed that they have enhancer
functions. The rest of the genome is divided into two functional
domains. The left portion of the genome codes for the rep functions
which regulate viral DNA replication and vital gene expression. The
right side of the vital genome contains the cap genes that encode
the structural capsid proteins VP1, VP2 and VP3. The proteins
encoded by both the rep and cap genes function in trans during
productive AAV replication.
[0010] Citation or identification of any document in this
application is not an admission that such document is available as
prior art to the present invention.
SUMMARY OF THE INVENTION
[0011] The present invention particularly relates to methods for
preparation of viral vector and methods and compositions for
advantageous delivery of Transcription Activation Like Effector
(TALE) and nucleic acid molecule(s) for expression or a CRISPR
(Clustered Regularly Interspaced Short Palindromic Repeats) system,
or a cassette or plurality of cassette comprising or consisting
essentially of a promoter and exogenous nucleic acid molecule
encoding same particularly for gene editing in a eukaryote
cell.
[0012] The present invention encompasses nucleic acid encoding the
polypeptides of the present invention. The nucleic acid may
comprise a promoter, advantageously human Synapsin I promoter
(hSyn). In one embodiment, the nucleic acid is packaged into a
viral vector. In some embodiments, the nucleic acid is packaged
into a parvovirus-based vector. In some embodiments, the nucleic
acid is packaged into an adeno associated viral vector (AAV).
[0013] The invention further relates to methods of treatment or
therapy that encompass the methods and compositions described
herein.
[0014] As discussed herein, the present invention generally relates
to recombinant parvovirus (Group II viruses according to the
Baltimore classification; e.g., Parvovirus B19, e.g. Dependovirus
(e.g. Adeno-Associated Virus or AAV), Erythrovirus (e.g. Parvovirus
B19) or Bocavirus), advantageously AAV. AAV is a prototypical
Dependovirus, The invention will be discussed with regard to
advantageous AAV embodiments with it understood that the invention
comprehends any of "parvovirus", "Parvovirus B19". "Dependovirus",
"Erythrovirus" or "Bocavirus" or species or serotypes of any of the
foregoing in place of "AAV" in discussion herein. It is also
understood that "AAV", unless specified as being a particular
serotype or specified as having a particular capsid can be any of
the herein identified AAVs.
[0015] There is a need for TALEs and LITEs to be expressed via a
recombinant construct, e.g., an AAV, e.g., to provide activator,
repressor or nuclease activity in vivo, in vitro or ex vivo.
[0016] There is a need for expression of a CRISPR system via a
recombinant construct, e.g., an AAV, e.g., to provide knockdown in
vivo, in vitro or ex vivo by the CRISPR introducing a spacer, which
inhibits a target gene.
[0017] As traditional AAV or rAAV production requires a laborious
production and purification process from cells, e.g., HEK-293FT
cells, and this can make testing many constructs in parallel
impractical. There is a need for a simple yet highly effective
method of preparing AAV or rAAV, including testing or screening
thereof, e.g., high throughput screening, and methods of using the
resulting AAV or rAAV to integrate into the genome of cells
otherwise difficult to infect, such as non-dividing cells, although
AAV is able to infect both dividing and quiescent cells. In one
aspect neuronal cells are targetted e.g., via neuronal
transduction. Means for neuronal transduction also can be
ascertained via Mason et al, "Comparison of AAV Serotypes for Gene
Delivery to Dorsal Root Ganglion Neurons," Mol Ther. 2010 April;
18(4): 715-724 (2010 Feb. 23). All types of AAV and other
Dependovirus are known to infect multiple diverse tissue types, and
various AAV serotypes are known to have natural tropism to
different tissues depending on their capsid proteins. Target
tissues include, but are not limited to, e.g., brain, neurons,
liver, eye, cardiac, muscle, and even cancer. See, e.g., Alam et
al., Mol Cancer. 2011 Aug. 9; 10:97; Bartel et al. Gene Ther. 2012
June; 19(6):694-700.
[0018] There is also a need for a readily accessible, reproducible
aliquot of recombinant construct that can be used for testing
whether construction of the recombinant construct was successful,
or whether the recombinant construct expresses the exogenous DNA in
an amount that may be sufficient for an intended use and/or for a
duration that may be sufficient for an intended use, i.e., for
screening, such as high throughput screening, for therapeutic uses
such as gene therapy, and targeting a broad range of tissues,
whether of dividing or quiescent cells. Thus, there is a need for
methods of the invention including those that may advantageously be
for screening or high throughput screening, wherein the method
includes or consists essentially of contacting the aliquot with
cells and determining whether the exogenous DNA is expressed in an
amount and/or duration sufficient for an intended use, e.g., gene
therapy, genetic engineering or screening.
[0019] AAV is considered an ideal candidate for use as a
transducing vector. Such AAV transducing vectors can comprise
sufficient cis-acting functions to replicate in the presence of
adenovirus or herpesvirus or poxvirus (e.g., vaccinia virus) helper
functions provided in trans. Recombinant AAV (rAAV) can be used to
carry exogenous genes into cells of a variety of lineages. In these
vectors, the AAV cap and/or rep genes are deleted from the viral
genome and replaced with a DNA segment of choice. Current AAV
vectors may accommodate up to 4300 bases of inserted DNA.
[0020] There are a number of ways to produce rAAV, and the
invention provides rAAV compositions and methods for preparing
rAAV. For example, plasmid(s) containing or consisting essentially
of the desired viral construct are transfected into AAV-infected
cells. In addition, a second or additional helper plasmid is
cotransfected into these cells to provide the AAV rep and/or cap
genes which are obligatory for replication and packaging of the
recombinant viral construct. Under these conditions, the rep and/or
cap proteins of AAV act in trans to stimulate replication and
packaging of the rAAV construct. Two to three days after
transfection, rAAV is harvested. Traditionally rAAV is harvested
from the cells along with adenovirus. The contaminating adenovirus
is then inactivated by heat treatment. In the instant invention,
rAAV is advantageously harvested not from the cells themselves, but
from cell supernatant. Accordingly, in an initial aspect the
invention provides for preparing rAAV, and in addition to the
foregoing, rAAV can be prepared by one or more methods that
comprise or consist essentially of, [0021] infecting susceptible
cells with a rAAV containing exogenous DNA including DNA for
expression, and helper virus (e.g., adenovirus, herpesvirus,
poxvirus such as vaccinia virus) wherein the rAAV lacks functioning
cap and/or rep (and the helper virus (e.g., adenovirus,
herpesvirus, poxvirus such as vaccinia virus) provides the cap
and/or rev function that the rAAV lacks); or [0022] infecting
susceptible cells with a rAAV containing exogenous DNA including
DNA for expression, wherein the recombinant construct lacks
functioning cap and/or rep, and transfecting said cells with a
plasmid supplying cap and/or rep function that the rAAV lacks; or
[0023] infecting susceptible cells with a rAAV containing exogenous
DNA including DNA for expression, wherein the recombinant construct
lacks functioning cap and/or rep, wherein said cells supply cap
and/or rep function that the recombinant construct lacks; or [0024]
transfecting the susceptible cells with an AAV lacking functioning
cap and/or rep and plasmids for inserting exogenous DNA into the
recombinant construct so that the exogenous DNA is expressed by the
recombinant construct and for supplying rep and/or cap functions
whereby transfection results in an rAAV containing the exogenous
DNA including DNA for expression that lacks functioning cap and/or
rep. [0025] In addition to methods for preparing rAAV, the
invention provides methods for using such recombinant constructs,
and compositions or preparations of such recombinant constructs,
including without limitation compositions or preparations resulting
from a method for obtaining and optionally storing a sample
containing a set amount of rAAV; and, this method can further
optionally include testing the rAAV.
[0026] The method advantageously may comprise or consist
essentially of, and hence the invention pertains to a method for
obtaining and optionally storing a sample containing a set amount
of rAAV comprising or consisting essentially of: [0027] preparing
the rAAV as herein described, e.g., [0028] plasmid(s) containing or
consisting essentially of the desired viral construct are
transfected into AAV-infected cells along with another helper
plasmid that provide the AAV rep and/or cap genes which are
obligatory for replication and packaging of the recombinant viral
construct; or [0029] infecting susceptible cells with a rAAV
containing exogenous DNA including DNA for expression, and helper
virus (e.g., adenovirus, herpesvirus, poxvirus such as vaccinia
virus) wherein the rAAV lacks functioning cap and/or rep (and the
helper virus (e.g., adenovirus, herpesvirus, poxvirus such as
vaccinia virus) provides the cap and/or rev function that the rAAV
lacks); or [0030] infecting susceptible cells with a rAAV
containing exogenous DNA including DNA for expression, wherein the
recombinant construct lacks functioning cap and/or rep, and
transfecting said cells with a plasmid supplying cap and/or rep
function that the rAAV lacks; or [0031] infecting susceptible cells
with a rAAV containing exogenous DNA including DNA for expression,
wherein the recombinant construct lacks functioning cap and/or rep,
wherein said cells supply cap and/or rep function that the
recombinant lacks; or [0032] transfecting the susceptible cells
with an AAV lacking functioning cap and/or rep and plasmids for
inserting exogenous DNA into the recombinant construct so that the
exogenous DNA is expressed by the recombinant construct and for
supplying rep and/or cap functions whereby transfection results in
an rAAV containing the exogenous DNA including DNA for expression
that lacks functioning cap and/or rep; and [0033] incubating the
infected or transfected cells, whereby there results infected or
transfected cells and supernatant containing the rAAV lacking
functioning cap and/or rep; [0034] after incubating, extracting an
aliquot from the supernatant; [0035] filtering the aliquot, whereby
the filtered aliquot contains and the method obtains a sample
containing set amount of the rAAV relative to the type and amount
of susceptible cells infected or transfected; and [0036] optionally
freezing the filtered aliquot, whereby the method optionally
includes storing a sample containing set amount of the rAAV
relative to the type and amount of susceptible cells infected or
transfected.
[0037] The rAAV can be from an AAV as herein described, and
advantageously can be an rAAV1, rAAV2, AAV5 or rAAV having a hybrid
capsid which may comprise AAV1, AAV2, AAV5 or any combination
thereof. One can select the AAV of the rAAV with regard to the
cells to be targeted by the rAAV; e.g., one can select AAV
serotypes 1, 2, 5 or a hybrid or capsid AAV1, AAV2, AAV5 or any
combination thereof for targeting brain or neuronal cells; and one
can select AAV4 for targeting cardiac tissue.
[0038] The susceptible cells are advantageously 293FT cells. The
method advantageously includes or consists essentially of freezing
(e.g., about -80.degree. C.) the filtered aliquot. A secretion
enhancer (e.g., polyethylenimine (PEI)) may be added to the cells
before, during or after and within the incubating. The incubating
can be typically up to 48 or 72 hours. 2.times.10.sup.5 cells are
advantageously transfected or infected, especially when the cells
are 293FT cells. The filtered aliquot advantageously has a volume
of 250 .mu.L.
[0039] When the cells are 293FT cells and 2.times.10.sup.5 cells
are advantageously transfected or infected, the rAAV concentration
in the filtered 250 .mu.L, aliquot is approximately
5.6+/-0.24.times.10.sup.5. When cells other than 293FT are used,
there should be a linear relationship with regard to the amount of
rAAV in the supernatant, aliquot and filtered aliquot. Thus, from
2.times.10.sup.5 293 FT cells obtaining the rAAV concentration in
the filtered 250 .mu.L aliquot of approximately
5.6+/-0.24.times.10.sup.5, the skilled person can transfect the
same number of other cells and measure the viral output (e.g., via
qPCR) and ascertain the linear relationship amongst cells. Other
cells that can be used in the practice of the invention and the
relative infectivity of certain AAV serotypes in vitro as to these
cells (see Grimm, D. et al, J. Virol. 82: 5887-5911 (2008)) are as
follows:
TABLE-US-00001 Cell Line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8
AAV-9 Huh-7 13 100 2.5 0.0 0.1 10 0.7 0.0 HEK293 25 100 2.5 0.1 0.1
5 0.7 0.1 HeLa 3 100 2.0 0.1 6.7 1 0.2 0.1 HepG2 3 100 16.7 0.3 1.7
5 0.3 ND Hep1A 20 100 0.2 1.0 0.1 1 0.2 0.0 911 17 100 11 0.2 0.1
17 0.1 ND CHO 100 100 14 1.4 333 50 10 1.0 COS 33 100 33 3.3 5.0 14
2.0 0.5 MeWo 10 100 20 0.3 6.7 10 1.0 0.2 NIH3T3 10 100 2.9 2.9 0.3
10 0.3 ND A549 14 100 20 ND 0.5 10 0.5 0.1 HT1180 20 100 10 0.1 0.3
33 0.5 0.1 Monocytes 1111 100 ND ND 125 1429 ND ND Immature DC 2500
100 ND ND 222 2857 ND ND Mature DC 2222 100 ND ND 333 3333 ND
ND
[0040] The invention provides rAAV that contains or consists
essentially of an exogenous nucleic acid molecule encoding a
transcriptional effector such as a Transcription Activation Like
Effector (TALE) and nucleic acid molecule(s) for expression or a
cassette comprising or consisting essentially of a promoter and a
nucleic acid molecule encoding a transcriptional effector such as a
TALE.
[0041] The invention provides rAAV that contains or consists
essentially of an exogenous nucleic acid molecule encoding an
inducible transcriptional effector such as a light-inducible
transcriptional effector (LITE) and nucleic acid molecule(s) for
expression or a cassette comprising or consisting essentially of a
promoter and a nucleic acid molecule encoding an inducible
transcriptional effector such as a LITE.
[0042] The invention provides rAAV that contains or consists
essentially of an exogenous nucleic acid molecule encoding a CRISPR
(Clustered Regularly Interspaced Short Palindromic Repeats) system,
e.g., a plurality of cassettes comprising or consisting a first
cassette comprising or consisting essentially of a promoter, a
nucleic acid molecule encoding a CRISPR-associated (Cas) protein
(putative nuclease or helicase proteins), e.g., Cas9 and a
terminator, and a two, or more, advantageously up to the packaging
size limit of the vector, e.g., in total (including the first
cassette) five, cassettes comprising or consisting essentially of a
promoter, nucleic acid molecule encoding guide RNA (gRNA) and a
terminator (e.g., each cassette schematically represented as
Promoter-gRNA1-terminator, Promoter-gRNA2-terminator . . .
Promoter-gRNA(N)-terminator (where N is a number that can be
inserted that is at an upper limit of the packaging size limit of
the vector), or two or more individual rAAVs, each containing one
or more than one cassette of a CRISPR system, e.g., a first rAAV
containing the first cassette comprising or consisting essentially
of a promoter, a nucleic acid molecule encoding Cas, e.g., Cas9 and
a terminator, and a second rAAV containing a plurality, four,
cassettes comprising or consisting essentially of a promoter,
nucleic acid molecule encoding guide RNA (gRNA) and a terminator
(e.g., each cassette schematically represented as
Promoter-gRNA1-terminator, Promoter-gRNA2-terminator . . .
Promoter-gRNA(N)-terminator (where N is a number that can be
inserted that is at an upper limit of the packaging size limit of
the vector).
[0043] As rAAV is a DNA virus, the nucleic acid molecules in the
herein discussion are advantageously DNA.
[0044] The invention also provides a readily accessible,
reproducible aliquot of rAAV that can be used for testing, e.g.,
testing whether construction of the rAAV was successful, or whether
the rAAV expresses the exogenous DNA in an amount that may be
sufficient for an intended use and/or for a duration that may be
sufficient for an intended use, i.e., for screening, such as high
throughput screening.
[0045] Hence, the invention provides a method for screening or high
throughput screening, wherein the method comprises or consists
essentially of preparing the filtered aliquot or the stored
filtered aliquot as herein described, if necessary, thawing the
stored filtered aliquot, contacting the filtered aliquot with cells
and determining whether the exogenous DNA is expressed in an amount
and/or duration sufficient for an intended use. The contacting with
cells can be transducing said cells (e.g., contacting can take 5-6
days with observation whereby suitable levels of rAAV expression
are reached). For instance, the rAAV can express a TALE and the
contacting can include detecting nuclease, activator or repressor
activity. The rAAV can express an inducible transcriptional
effector such as a LITE, and the contacting can include inducing
gene expression or subjecting the contacted cells to a suitable
stimulus, and if detecting whether transcriptional effector has
been induced, e.g., via detecting a color change. The rAAV can
express a CRISPR system, and the contacting can include detecting
gene knockdown or other effects of the CRISPR system.
[0046] The invention further provides advantageous methods of AAV
or rAAV production. In one aspect, as further described in the
Examples herein, the invention encompasses AAV supernatant
production. The methods of the invention described herein
comprehend varying the DNA ratios of the vectors used, e.g. the
ratios of vector of interest plasmid: AAV serotype plasmid: pHelper
plasmid may be varied. In a preferred embodiment of the invention,
this value may be 1:1.7:2 for AAV supernatant production down to
24-well scale. In another preferred embodiment of the invention,
this value may be 1:2:1 for a 96-well format.
[0047] The invention also comprehends the scaling up of the AAV
supernatant production to higher throughput formats. Aspects of the
invention may be carried out in a 15 cm dish. In a further
embodiment, aspects of the invention comprehend scaling up from a
15 cm dish to 96-well plates for production. In another aspect, the
invention also encompasses scaling up which includes but is not
limited to 384-well plates or 1536-well plates. In a further
embodiment, the invention also comprehends a microfluidic device
capable of maintaining cell cultures in individual chambers. In a
preferred embodiment, the AAV supernatant produced in the methods
of the invention may be produced at the same scale as it may be
applied.
[0048] The invention provides for methods of filtration or
purification of the supernatant containing AAV generated in the
methods described herein. Methods of filtration or purification may
include but are not limited to the use of filters or
centrifugation. In one aspect of the invention, filtration with
specific pore size filters may be employed to remove any potential
293FT cells and large cell debris. In a preferred embodiment, a 22
micron or 45 micron pore size low protein binding filter may be
used. When filtration is utilized the flow-through is harvested and
subsequently used. In another aspect of the invention,
centrifugation may be employed to pellet cells and cell debris. In
a preferred embodiment, centrifugation at speeds in the range of
200 g for 20 min to 6000 g for 1-10 min may be utilized. When
centrifugation is utilized the supernatant is collected and
subsequently used. In a further embodiment of the invention, these
steps may be followed by subsequent purification steps when more
stringent purification is desired. In a preferred embodiment a
sequence of molecular weight cutoff filters (e.g. amicon filters,
Millipore) may be used.
[0049] The invention also provides for methods of AAV supernatant
production which do not use fetal bovine serum (FBS). In a
preferred embodiment, the culture medium used to support AAV
producing 293FT cells may be replaced with a chemically-defined
serum-free medium. e.g. Pro293a.
[0050] The invention also provides for AAV supernatant production
methods being used to generate functional pooled AAV supernatant.
Furthermore, the invention also provides for multiple supernatant
AAV batches being harvested from a single AAV producing 293FT
culture.
[0051] Accordingly, it is an object of the invention not to
encompass within the invention any previously known product,
process of making the product, or method of using the product such
that Applicants reserve the right and hereby disclose a disclaimer
of any previously known product, process, or method. It is further
noted that the invention does not intend to encompass within the
scope of the invention any product, process, or making of the
product or method of using the product, which does not meet the
written description and enablement requirements of the USPTO (35
U.S.C. .sctn.112, first paragraph) or the EPO (Article 83 of the
EPC), such that Applicants reserve the right and hereby disclose a
disclaimer of any previously described product, process of making
the product, or method of using the product.
[0052] It is noted that in this disclosure and particularly in the
claims and/or paragraphs, terms such as "comprises", "comprised",
"comprising" and the like can have the meaning attributed to it in
U.S. Patent law; e.g., they can mean "includes", "included",
"including", and the like; and that terms such as "consisting
essentially of" and "consists essentially of" have the meaning
ascribed to them in U.S. Patent law, e.g., they allow for elements
not explicitly recited, but exclude elements that are found in the
prior art or that affect a basic or novel characteristic of the
invention.
[0053] The invention further also provides other recombinant
constructs, compositions, preparations, and methods described
herein.
[0054] These and other embodiments are disclosed or are obvious
from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] The following detailed description, given by way of example,
but not intended to limit the invention solely to the specific
embodiments described, may best be understood in conjunction with
the accompanying drawings.
[0056] FIG. 1 shows a schematic indicating the need for spatial and
temporal precision.
[0057] FIG. 2 shows transcription activator like effectors (TALEs).
TALEs consist of 34 aa repeats (SEQ ID NO:1) at the core of their
sequence. Each repeat corresponds to a base in the target DNA that
is bound by the TALE, with one example shown as SEQ ID NO:2.
Repeats differ only by 2 variable amino acids at positions 12 and
13. The code of this correspondence has been elucidated (Boch, J et
al., Science, 2009 and Moscou, M et al., Science, 2009) and is
shown in this figure. Applicants have developed a method for the
synthesis of designer TALEs incorporating this code and capable of
binding a sequence of choice within the genome (Zhang, F et al.,
Nature Biotechnology, 2011).
[0058] FIG. 3 shows a design of a LITE: TALE/Cryptochrome
transcriptional activation. Each LITE is a two-component system
which may comprise a TALE fused to CRY2 and the cryptochrome
binding partner CIB1 fused to VP64, a transcription activator. In
the inactive state, the TALE localizes its fused CRY2 domain to the
promoter region of the gene of interest. At this point, CIB1 is
unable to bind CRY2, leaving the CIB1-VP64 unbound in the nuclear
space. Upon stimulation with 488 nm (blue) light, CRY2 undergoes a
conformational change, revealing its CIB1 binding site (Liu, H et
al., Science, 2008). Rapid binding of CIB1 results in recruitment
of the fused VP64 domain, which induces transcription of the target
gene.
[0059] FIG. 4 shows effects of cryptochrome dimer truncations on
LITE activity. Truncations known to alter the activity of CRY2 and
CIB1 (Kennedy M et al., Nature Methods 2010) were compared against
the full length proteins. A LITE targeted to the promoter of
Neurog2 was tested in Neuro-2a cells for each combination of
domains. Following stimulation with 488 nm light, transcript levels
of Neurog2 were quantified using qPCR for stimulated and
unstimulated samples.
[0060] FIG. 5 shows a light-intensity dependent response of KLF4
LITE.
[0061] FIG. 6 shows activation kinetics of Neurog2 LITE and
inactivation kinetics of Neurog2 LITE.
[0062] FIG. 7A shows the base-preference of various RVDs as
determined using the Applicants' RVD screening system.
[0063] FIG. 7B shows the base-preference of additional RVDs as
determined using the Applicants' RVD screening system.
[0064] FIGS. 8A-D show in (a) Natural structure of TALEs derived
from Xanthomonas sp. Each DNA-binding module consists of 34 amino
acids (SEQ ID NO:1), where the RVDs in the 12th and 13th amino acid
positions of each repeat specify the DNA base being targeted (e.g.,
SEQ ID NO:2) according to the cipher NG=T, HD=C, NI=A, and NN=G or
A. The DNA-binding modules are flanked by nonrepetitive N and C
termini, which carry the translocation, nuclear localization (NLS)
and transcription activation (AD) domains. A cryptic signal within
the N terminus specifies a thymine as the first base of the target
site. (b) The TALE toolbox allows rapid and inexpensive
construction of custom TALE-TFs and TALENs. The kit consists of 12
plasmids in total: four monomer plasmids to be used as templates
for PCR amplification, four TALE-TF and four TALEN cloning
backbones corresponding to four different bases targeted by the 0.5
repeat. CMV, cytomegalovirus promoter; N term, nonrepetitive N
terminus from the Hax3 TALE; C term, nonrepetitive C terminus from
the Hax3 TALE; BsaI, type IIs restriction sites used for the
insertion of custom TALE DNA-binding domains; ccdB+CmR, negative
selection cassette containing the ccdB negative selection gene and
chloramphenicol resistance gene; NLS, nuclear localization signal;
VP64, synthetic transcriptional activator derived from VP16 protein
of herpes simplex virus; 2A, 2A self-cleavage linker; EGFP,
enhanced green fluorescent protein; polyA signal, polyadenylation
signal; FokI, catalytic domain from the FokI endonuclease. (c)
TALEs may be used to generate custom TALE-TFs and modulate the
transcription of endogenous genes from the genome. The TALE
DNA-binding domain is fused to the synthetic VP64 transcriptional
activator, which recruits RNA polymerase and other factors needed
to initiate transcription. (d) TALENs may be used to generate
site-specific double-strand breaks to facilitate genome editing
through nonhomologous repair or homology directed repair. Two
TALENs target a pair of binding sites flanking a 16-bp spacer. The
left and right TALENs recognize the top and bottom strands of the
target sites, respectively. Each TALE DNA-binding domain is fused
to the catalytic domain of FokI endonuclease; when FokI dimerizes,
it cuts the DNA in the region between the left and right
TALEN-binding sites.
[0065] FIG. 9A-F shows a table listing monomer sequences (excluding
the RVDs at positions 12 and 13) (SEQ ID NOS:3-74) and the
frequency with which monomers having a particular sequence
occur.
[0066] FIG. 10 shows the comparison of the effect of non-RVD amino
acid on TALE activity (SEQ ID NO:1 and variants thereof).
[0067] FIG. 11 shows an activator screen comparing levels of
activation between VP64, p65 and VP16.
[0068] FIGS. 12A-D show the development of a TALE transcriptional
repressor architecture. (a) Design of SOX2 TALE for TALE repressor
screening. A TALE targeting a 14 bp sequence within the SOX2 locus
of the human genome (SEQ ID NO:75) was synthesized. (b) List of all
repressors screened and their host origin (left). Eight different
candidate repressor domains were fused to the C-term of the SOX2
TALE. (c) The fold decrease of endogenous SOX2 mRNA is measured
using qRTPCR by dividing the SOX2 mRNA levels in mock transfected
cells by SOX2 mRNA levels in cells transfected with each candidate
TALE repressor. (d) Transcriptional repression of endogenous
CACNA1C. TALEs using NN, NK, and NH as the G-targeting RVD were
constructed to target a 18 bp target site (SEQ ID NO:76) within the
human CACNA1C locus. Each TALE is fused to the SID repression
domain. NLS, nuclear localization signal; KRAB, Kruppel-associated
box; SID, mSin interaction domain. All results are collected from
three independent experiments in HEK 293FT cells. Error bars
indicate s.e.m.; n=3. *p<0.05, Student's t test.
[0069] FIGS. 13A-C shows the optimization of TALE transcriptional
repressor architecture using SID and SID4X. (a) Design of p11 TALE
for testing of TALE repressor architecture. A TALE targeting a 20
bp sequence (p11 TALE binding site, SEQ ID NO:77) within the p11
(s100a10) locus of the mouse (Mus musculus) genome was synthesized.
(b) Transcriptional repression of endogenous mouse p11 mRNA. TALEs
targeting the mouse p11 locus harboring two different truncations
of the wild type TALE architecture were fused to different
repressor domains as indicated on the x-axis. The value in the
bracket indicate the number of amino acids at the N- and C-termini
of the TALE DNA binding domain flanking the DNA binding repeats,
followed by the repressor domain used in the construct. The
endogenous p11 mRNA levels were measured using qRT-PCR and
normalized to the level in the negative control cells transfected
with a GFP-encoding construct. (c) Fold of transcriptional
repression of endogenous mouse p11. The fold decrease of endogenous
p11 mRNA is measured using qRT-PCR through dividing the p11 mRNA
levels in cells transfected with a negative control GFP construct
by p11 mRNA levels in cells transfected with each candidate TALE
repressors. The labeling of the constructs along the x-axis is the
same as previous panel. NLS, nuclear localization signal; SID, mSin
interaction domain; SID4X, an optimized four-time tandem repeats of
SID domain linked by short peptide linkers. All results are
collected from three independent experiments in Neuro2A cells.
Error bars indicate s.e.m.; n=3. ***p<0.001, Student's t
test.
[0070] FIG. 14A-D shows a comparison of two different types of TALE
architecture.
[0071] FIGS. 15A-C show a chemically inducible TALE ABA inducible
system. ABI (ABA insensitive 1) and PYL (PYL protein: pyrabactin
resistance (PYR)/PYR1-like (PYL)) are domains from two proteins
listed below that will dimerize upon binding of plant hormone
Abscisic Acid (ABA). This plant hormone is a small molecule
chemical that Applicants used in Applicants' inducible TALE system.
In this system, the TALE DNA-binding polypeptide is fused to the
ABI domain, whereas the VP64 activation domain or SID repressor
domain or any effector domains are linked to the PYL domain. Thus,
upon the induction by the presence of ABA molecule, the two
interacting domains, ABI and PYL, will dimerize and allow the TALE
to be linked to the effector domains to perform its activity in
regulating target gene expression.
[0072] FIGS. 16A-B show a chemically inducible TALE 4OHT inducible
system.
[0073] FIG. 17 depicts an effect of cryptochrome2 heterodimer
orientation on LITE functionality.
[0074] FIG. 18 depicts mGlur2 LITE activity in mouse cortical
neuron culture.
[0075] FIG. 19 depicts transduction of primary mouse neurons with
LITE AAV vectors.
[0076] FIG. 20 depicts expression of LITE component in vivo.
[0077] FIG. 21 depicts an improved design of the construct where
the specific NES peptide sequence used is LDLASLIL.
[0078] FIG. 22 depicts Sox2 mRNA levels in the absence and presence
of 40H tamoxifen.
[0079] FIGS. 23A-E depict a Type II CRISPR locus from Streptococcus
pyogenes SF370 can be reconstituted in mammalian cells to
facilitate targeted DSBs of DNA. (A) Engineering of SpCas9 and
SpRNase III with NLSs enables import into the mammalian nucleus.
(B) Mammalian expression of SpCas9 and SpRNase III are driven by
the EF1a promoter, whereas tracrRNA and pre-crRNA array
(DR-Spacer-DR) are driven by the U6 promoter. A protospacer (blue
highlight) from the human EMX1 locus (SEQ ID NO:78) with PAM is
used as template for the spacer in the pre-crRNA array. (C)
Schematic representation of base pairing between target locus (SEQ
ID NOS:79-80) and EMX1-targeting crRNA (SEQ ID NO:81). Red arrow
indicates putative cleavage site. (D) SURVEYOR assay for
SpCas9-mediated indels. (E) An example chromatogram showing a
micro-deletion, as well as representative sequences of mutated
alleles (SEQ ID NOS:82-89) identified from 187 clonal amplicons.
Red dashes, deleted bases; red bases, insertions or mutations.
Scale bar=10 .mu.m.
[0080] FIGS. 24A-C depict a SpCas9 can be reprogrammed to target
multiple genomic loci in mammalian cells. (A) Schematic of the
human EMX1 locus (SEQ ID NOS:90-91) showing the location of five
protospacers, indicated by blue lines with corresponding PAM in
magenta. (B) Schematic of the pre-crRNA:tracrRNA complex (SEQ ID
NOS:92-93) (top) showing hybridization between the direct repeat
(gray) region of the pre-crRNA and tracrRNA. Schematic of a
chimeric RNA design (SEQ ID NO:94) (M. Jinek et al., A programmable
dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
Science 337, 816 (Aug. 17, 2012)) (bottom). tracrRNA sequence is
shown in red and the 20 bp spacer sequence in blue. (C) SURVEYOR
assay comparing the efficacy of Cas9-mediated cleavage at five
protospacers in the human EMX1 locus. Each protospacer is targeted
using either processed pre-crRNA:tracrRNA complex (crRNA) or
chimeric RNA (chiRNA).
[0081] FIGS. 25A-D depict an evaluation of the SpCas9 specificity
and comparison of efficiency with TALENs. (A) EMX1-targeting
chimeric crRNAs with single point mutations were generated to
evaluate the effects of spacer-protospacer mismatches (SEQ ID
NOS:95-96, 97-108). (B) SURVEYOR assay comparing the cleavage
efficiency of different mutant chimeric RNAs. (C) Schematic showing
the design of TALENs targeting EMX1 (SEQ ID NOS:95-96). (D)
SURVEYOR gel comparing the efficiency of TALEN and SpCas9
(N=3).
[0082] FIGS. 26A-G depict applications of Cas9 for homologous
recombination and multiplex genome engineering. (A) Mutation of the
RuvC I domain converts Cas9 into a nicking enzyme (SpCas9n) (B)
Co-expression of EMX1-targeting chimeric RNA with SpCas9 leads to
indels, whereas SpCas9n does not (N=3). (C) Schematic
representation of the recombination strategy. A repair template is
designed to insert restriction sites into EMX1 locus. Primers used
to amplify the modified region are shown as red arrows. (D)
Restriction fragments length polymorphism gel analysis. Arrows
indicate fragments generated by HindIII digestion. (E) Example
chromatogram showing successful recombination (SEQ ID NO:109). (F)
SpCas9 can facilitate multiplex genome modification using a crRNA
array containing two spacers (SEQ ID NOS:110, 111) targeting EMX1
and PVALB. Schematic showing the design of the crRNA array (top).
Both spacers mediate efficient protospacer cleavage (bottom). (G)
SpCas9 can be used to achieve precise genomic deletion. Two spacers
(SEQ ID NOS:112, 113) targeting EMX1 (top) mediated a 118 bp
genomic deletion (SEQ ID NOS:114-118) (bottom).
[0083] FIG. 27 depicts a schematic of the type II CRISPR-mediated
DNA double-strand break. The type II CRISPR locus from
Streptococcus pyogenes SF370 contains a cluster of four genes,
Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements,
tracrRNA and a characteristic array of repetitive sequences (direct
repeats) interspaced by short stretches of nonrepetitive sequences
(spacers, 30 bp each) (15-18, 30, 31). Each spacer is typically
derived from foreign genetic material (protospacer), and directs
the specificity of CRISPR-mediated nucleic acid cleavage. In the
target nucleic acid, each protospacer is associated with a
protospacer adjacent motif (PAM) whose recognition is specific to
individual CRISPR systems (22, 23). The Type II CRISPR system
carries out targeted DNA double-strand break (DSB) in sequential
steps (M. Jinek et al., Science 337, 816 (Aug. 17, 2012); Gasiunas,
R. et al. Proc Natl Acad Sci USA 109, E2579 (Sep. 25, 2012); J. E.
Garneau et al., Nature 468, 67 (Nov. 4, 2010); R. Sapranauskas et
al., Nucleic Acids Res 39, 9275 (November, 2011); A. H. Magadan et
al. PLoS One 7, e40913 (2012)). First, the pre-crRNA array and
tracrRNA are transcribed from the CRISPR locus. Second, tracrRNA
hybridizes to the direct repeats of pre-crRNA and associates with
Cas9 as a duplex, which mediates the processing of the pre-crRNA
into mature crRNAs containing individual, truncated spacer
sequences. Third, the mature crRNA:tracrRNA duplex directs Cas9 to
the DNA target consisting of the protospacer and the requisite PAM
via heteroduplex formation between the spacer region of the crRNA
and the protospacer DNA. Finally, Cas9 mediates cleavage of target
DNA upstream of PAM to create a DSB within the protospacer.
[0084] FIGS. 28A-C depict a comparison of different tracrRNA
transcripts for Cas9-mediated gene targeting. (A) Schematic showing
the design and sequences of two tracrRNA transcripts (SEQ ID
NOS:119-120) tested (short and long). Each transcript is driven by
a U6 promoter. Transcription start site is marked as +1 and
transcription terminator is as indicated. Blue line indicates the
region whose reverse-complement sequence is used to generate
northern blot probes for tracrRNA detection. (B) SURVEYOR assay
comparing the efficiency of hSpCas9-mediated cleavage of the EMX1
locus. Two biological replicas are shown for each tracrRNA
transcript. (C) Northern blot analysis of total RNA extracted from
293FT cells transfected with U6 expression constructs carrying long
or short tracrRNA, as well as SpCas9 and DR-EMX1(1)-DR. Left and
right panels are from 293FT cells transfected without or with
SpRNase III respectively. U6 indicate loading control blotted with
a probe targeting human U6 snRNA. Transfection of the short
tracrRNA expression construct led to abundant levels of the
processed form of tracrRNA (.about.75 bp) (E. Deltcheva et al.,
Nature 471, 602 (Mar. 31, 2011)). Very low amounts of long tracrRNA
are detected on the Northern blot. As a result of these
experiments, Applicants chose to use short tracrRNA for application
in mammalian cells.
[0085] FIG. 29 depicts a SURVEYOR assay for detection of double
strand break-induced micro insertions and deletions (D. Y. Guschin
et al. Methods Mol Biol 649, 247 (2010)). Schematic of the SURVEYOR
assay used to determine Cas9-mediated cleavage efficiency. First,
genomic PCR (gPCR) is used to amplify the Cas9 target region from a
heterogeneous population of modified and unmodified cells, and the
gPCR products are reannealed slowly to generate heteroduplexes. The
reannealed heteroduplexes are cleaved by SURVEYOR nuclease, whereas
homoduplexes are left intact. Cas9-mediated cleavage efficiency (%
indel) is calculated based on the fraction of cleaved DNA.
[0086] FIG. 30A-B depict a Northern blot analysis of crRNA
processing in mammalian cells. (A) Schematic showing the expression
vector for a single spacer flanked by two direct repeats
(DR-EMX1(1)-DR) (SEQ ID NO: 121). The 30 bp spacer targeting the
human EMX1 locus protospacer 1 (Table 1) is shown in blue and
direct repeats are in shown in gray. Orange line indicates the
region whose reverse complement sequence is used to generate
northern blot probes for EMX1(1) crRNA detection. (B) Northern blot
analysis of total RNA extracted from 293FT cells transfected with
U6 expression constructs carrying DR-EMX1(1)-DR. Left and right
panels are from 293FT cells transfected without or with SpRNase III
respectively. DR-EMX1(1)-DR was processed into mature crRNAs only
in the presence of SpCas9 and short tracrRNA, and was not dependent
on the presence of SpRNase III. The mature crRNA detected from
transfected 293FT total RNA is .about.33 bp and is shorter than the
39-42 bp mature crRNA from S. pyogenes (E. Deltcheva et al., Nature
471, 602 (Mar. 31, 2011)), suggesting that the processed mature
crRNA in human 293FT cells is likely different from the bacterial
mature crRNA in S. pyogenes.
[0087] FIG. 31A-B depict bicistronic expression vectors for
pre-crRNA array or chimeric crRNA with Cas9 (SEQ ID NOS:122-129).
(A) Schematic showing the design of an expression vector for the
pre-crRNA array. Spacers can be inserted between two BbsI sites
using annealed oligonucleotides. Sequence design for the
oligonucleotides are shown below with the appropriate ligation
adapters indicated. (B) Schematic of the expression vector for
chimeric crRNA. The guide sequence can be inserted between two BbsI
sites using annealed oligonucleotides. The vector already contains
the partial direct repeat (gray) and partial tracrRNA (red)
sequences. WPRE, Woodchuck hepatitis virus posttranscriptional
regulatory element.
[0088] FIGS. 32A-B depict a selection of protospacers in the human
PVALB (SEQ ID NOS: 130-131) and mouse (SEQ ID NOS: 132-133) Th
loci. Schematic of the human PVALB (A) and mouse Th (B) loci and
the location of the three protospacers within the last exon of the
PVALB and Th genes, respectively. The 30 bp protospacers are
indicated by black lines and the adjacent PAM sequences are
indicated by the magenta bar. Protospacers on the sense and
anti-sense strands are indicated above and below the DNA sequences
respectively.
[0089] FIGS. 33A-C depict occurrences of PAM sequences in the human
genome. Histograms of distances between adjacent Streptococcus
pyogenes SF370 locus 1 PAM (NGG) (A) and Streptococcus thermophilus
LMD9 locus 1 PAM (NNAGAAW) (B) in the human genome. (C) Distances
for each PAM by chromosome. Chr, chromosome. Putative targets were
identified using both the plus and minus strands of human
chromosomal sequences. Given that there may be chromatin, DNA
methylation-, RNA structure, and other factors that may limit the
cleavage activity at some protospacer targets, it is important to
note that the actual targeting ability might be less than the
result of this computational analysis.
[0090] FIGS. 34A-D depict type II CRISPR from Streptococcus
thermophilus LMD-9 can also function in eukaryotic cells. (A)
Schematic of CRISPR locus 2 from Streptococcus thermophilus LMD-9.
(B) Design of the expression system for the S. thermophilus CRISPR
system. Human codon-optimized hStCas9 is expressed using a
constitutive EF1a promoter. Mature versions of tracrRNA and crRNA
are expressed using the U6 promoter to ensure precise transcription
initiation. Sequences for the mature crRNA and tracrRNA are shown
(SEQ ID NOS: 134-135). A single based indicated by the lower case
"a" in the crRNA sequence was used to remove the polyU sequence,
which serves as a RNA Pol III transcriptional terminator. (C)
Schematic showing protospacer and corresponding PAM sequences
targets in the human EMX1 locus (SEQ ID NOS: 136-137). Two
protospacer sequences are highlighted and their corresponding PAM
sequences satisfying the NNAGAAW motif (SEQ ID NO:138) are
indicated by magenta lines. Both protospacers are targeting the
anti-sense strand. (D) SURVEYOR assay showing StCas9-mediated
cleavage in the target locus. RNA guide spacers 1 and 2 induced 14%
and 6.4% respectively. Statistical analysis of cleavage activity
across biological replica at these two protospacer sites can be
found in Table 1.
[0091] FIGS. 35A-E depict design and optimization of the LITE
system. (A) A TALE DNA-binding domain (SEQ ID NO: 139) is fused to
CRY2 and a transcriptional effector domain is fused to CIB1. In the
inactive state, TALE-CRY2 binds the promoter region of the target
gene while CIB1-effector remains unbound in the nucleus. The VP64
transcriptional activator is shown above. Upon illumination with
blue light, TALE-CRY2 and CIB1-effector rapidly dimerize,
recruiting CIB1-effector to the target promoter. The effector in
turn modulates transcription of the target gene. (B)
Light-dependent upregulation of the endogenous target Ngn2 mRNA
with LITEs containing functional truncations of its light-sensitive
binding partners. LITE-transfected Neuro-2a cells were stimulated
for 24 h with 466 nm light at an intensity of 5 mW/cm.sup.2 and a
duty cycle of 7% (1 s pulses at 0.066 Hz). (C) Ngn2 upregulation
with and without light by LITEs using different transcriptional
activation domains VP16, VP64, and p65. Stimulation parameters are
the same as (b). (D) The transcriptional activity of CRY2PHR-CIB1
LITE was found to vary according to the intensity of 466 nm blue
light. Neuro 2a cells were stimulated for 24 h hours at a 7% duty
cycle (1 s pulses at 0.066 Hz) (E) Light-induced toxicity measured
as the percentage of cells positive for red-fluorescent ethidium
homodimer-1 versus calcein-positive cells. All Ngn2 mRNA levels
were measured relative to cells expressing YFP only
(mean.+-.s.e.m.; n=3-4)
[0092] FIGS. 36A-B depict kinetics of light-induced transcriptional
activation. (A) Time course of light-dependent Ngn2 upregulation by
TALE-CRY2PHR and CIB1-VP64 LITEs. LITE-transfected Neuro-2a cells
were stimulated with 466 nm light at an intensity of 5 mW/cm.sup.2
and a duty cycle of 7% (1 s pulses at 0.066 Hz). (B) Decrease of
Ngn2 mRNA levels after 6 h of light stimulation. All Ngn2 mRNA
levels were measured relative to expressing YFP control cells
(mean.+-.s.e.m.; n=3-4) (*=p<0.05 and ***=p<0.001).
[0093] FIGS. 37A-F depict virus-mediated TALE delivery enabling
bimodal control of endogenous gene expression in neurons (A)
General schematic of constitutive TALE transcriptional activator
and repressor packaged into AAV. Effector domains VP64 and SID4X
are highlighted. (B) Representative images showing transduction
with AAV-TALE-VP64 constructs from (a) in primary cortical neurons.
Cells were stained for virally delivered GFP and neuronal marker
NeuN. Scale bars=25 .mu.m. (C) 6 TALEs were designed, with two
TALEs targeting each of the endogenous mouse loci Grm5, Grin2a, and
Grm2 (SEQ ID NOS:140-145). TALEs were fused to the transcriptional
activator domain VP64 or the repressor domain SID4X and virally
transduced into primary neurons. Both the target gene upregulation
via VP64 and downregulation via SID4X are shown for each TALE
relative to levels in neurons expressing GFP only. (D) Efficient
delivery of TALE-VP64 by AAV into the ILC of mice. Scale bar=100
um. (Cg1=cingulate cortex, PLC=prelimbic cortex, ILC=infralimbic
cortex). (E) Higher magnification image of efficient transduction
of neurons in ILC. (F) Grm2 mRNA upregulation by TALE-VP64 in vivo
in ILC (mean.+-.s.e.m.; n=3).
[0094] FIGS. 38A-J depict light-mediated manipulation of Grm2
expression in primary neurons and in vivo (A) AAV LITE activator
construct with switched CRY2PHR and CIB1 architecture. (B)
Representative images showing co-transduction of AAV-delivered LITE
constructs in primary neurons. Cells were stained for GFP, HA-tag,
and DAPI. (Scale bars=25 .mu.m). (C) Light-induced activation of
Grm2 expression in primary neurons after 24 h of stimulation with
0.8% duty cycle pulsed 466 nm light (250 ms pulses at 0.033 Hz or
500 ms pulses at 0.016 Hz; 5 mW/cm.sup.2). (D) Upregulation of Grm2
mRNA in primary cortical neurons with and without light stimulation
at 4 h and 24 h time points. Expression levels are shown relative
to neurons transduced with GFP only. (E) Quantification of mGluR2
protein levels in GFP only control transductions, unstimulated
neurons with LITEs, and light-stimulated neurons with LITEs. A
representative western blot is shown with .beta.-tubulin-III as a
loading control. (F) LITE repressor construct highlighting SID4X
repressor domain. (G) Light-induced repression of endogenous Grm2
expression in primary cortical neurons using Grm2 T1-LITE and Grm2
T2-LITE. Fold downregulation is shown relative to neurons
transduced with GFP only (mean.+-.s.e.m.; n=3-4 for all subpanels).
(H) Schematic showing transduction of ILC with the LITE system, the
optical fiber implant, and the 0.35 mm diameter brain punch used
for tissue isolation. (I) Representative images of ILC
co-transduced with both LITE components. Stains are shown for
HA-tag (red), GFP (green), and DAPI (blue). (Scale bar=25 .mu.m).
(J) Light-induced activation of endogenous Grm2 expression using
LITEs transduced into ILC.
[0095] FIG. 39 depicts an activation Ratio of CRY2 and CIB1
truncations. Fold activation of Ngn2 expression by LITEs was
calculated as the ratio of mRNA levels in stimulated cells versus
unstimulated cells (light/no light; experiment and data
corresponding to FIG. 35B), for each CRY2 and CIB1 truncation
pair.
[0096] FIG. 40 depicts an impact of illumination duty cycle on
LITE-mediated gene expression. Varying duty cycles (illumination as
percentage of total time) were used to stimulate HEK293FT cells
expressing LITEs targeting the KLF4 gene, in order to investigate
the effect of duty cycle on LITE activity. KLF4 expression levels
were compared to cells expressing GFP only. Stimulation parameters
were: 466 nm, 5 mW/cm.sup.2 for 24 h. Pulses were performed at
0.067 Hz with the following durations: 1.7%=0.25 s pulse, 7%=1 s
pulse, 27%=4 s pulse, 100%=constant illumination.
[0097] FIG. 41 depicts an illustration of the absorption spectrum
of CRY2 in vitro. Cryptochrome 2 was optimally activated by 350-475
nm light'. A sharp drop in absorption and activation was seen for
wavelengths greater than 480 nm. Spectrum was adapted from
Banerjee, R. et al. The Signaling State of Arabidopsis Cryptochrome
2 Contains Flavin Semiquinone. Journal of Biological Chemistry 282,
14916-14922, doi:10.1074/jbc.M700616200 (2007).
[0098] FIGS. 42A-C depict AAV supernatant production. (A)
Lentiviral and AAV vectors carrying GFP were used to test
transduction efficiency. (B) Primary embryonic cortical neurons
were transduced with 250 .mu.L supernatant derived from the same
number of AAV or lentivirus-transfected 293FT cells. Representative
images of GFP expression were collected at 7 d.p.i. Scale bars=50
.mu.m. (C) The depicted process was developed for the production of
AAV supernatant and subsequent transduction of primary neurons.
293FT cells were transfected with an AAV vector carrying the gene
of interest, the AAV1 serotype packaging vector (pAAV1), and helper
plasmid (pDF6) using PEI. 48 h later, the supernatant was harvested
and filtered through a 0.45 .mu.m PVDF membrane. Primary neurons
were then transduced with supernatant and remaining aliquots were
stored at -80.degree. C. Stable levels of AAV construct expression
were reached after 5-6 days.
[0099] FIG. 43 depicts a selection of TALE target sites guided by
DNaseI-sensitive chromatin regions. High DNaseI sensitivity based
on mouse cortical tissue data from ENCODE (at the website of
genome.ucsc.edu) was used to identify open chromatin regions. The
peak with the highest amplitude within the region 2 kb upstream of
the transcriptional start site was selected for targeting. TALE
binding targets were then picked within a 200 bp region at the
center of the peak.
[0100] FIG. 44 depicts a TALE SID4X repressor characterization. A
synthetic repressor was constructed by concatenating 4 SID domains
(SID4X). To identify the optimal TALE-repressor architecture, SID
or SID4X was fused to a TALE designed to target the mouse p11 gene
(SEQ ID NO:146). Fold decrease in p11 mRNA was assayed using
qRT-PCR.
[0101] FIGS. 45A-B depict exchanging CRY2PHR and CIB1 components.
(A) TALE-CIB1::CRY2PHR-VP64 was able to activate Ngn2 at higher
levels than TALE-CRY2PHR::CIB1-VP64. (B) Fold activation ratios
(light versus no light) ratios of Ngn2 LITEs show similar
efficiency for both designs. Stimulation parameters were the same
as those used in FIG. 35B.
[0102] FIG. 46 depicts an impact of light duty cycle on primary
neuron health. The effect of light stimulation on primary cortical
neuron health was compared for duty cycles of 7%, 0.8%, and no
light conditions. Calcein was used to evaluate neuron viability.
Bright-field images were captured to show morphology and cell
integrity. Primary cortical neurons were stimulated with the
indicated duty cycle for 24 h with 5 mW/cm.sup.2 of 466 nm light.
Representative images, scale bar=50 .mu.m. Pulses were performed in
the following manner: 7% duty cycle=1 s pulse at 0.067 Hz, 0.8%
duty cycle=0.5 s pulse at 0.0167 Hz.
[0103] FIGS. 47A-B depict a contribution of individual LITE
components to baseline transcription modulation. (A) Grm2 mRNA
levels were determined in primary neurons transfected with
individual LITE components. Primary neurons expressing T6-CIB1
alone led to a similar increase in Grm2 mRNA levels as unstimulated
cells expressing the complete LITE system. (B) Transcription
repression by individual LITE repressor components targeting the
Grm2 gene was compared.
[0104] FIG. 48 depicts a co-transduction efficiency of LITE
components by AAV1/2 in mouse infralimbic cortex. Cells transduced
by T6-CIB1 alone, CRY2PHR-VP64 alone, or co-transduced were
calculated as a percentage of all transduced cells.
[0105] FIG. 49 shows a schematic of an AAV-promotor-TALE-effector
construct. In the construct: hSyn=human synapsin 1 promoter;
N+136=TALE N-term, AA+136 truncation; C63=TALE C-term, AA+63
truncation; vp=VP64 effector domain; GFP=green fluorescent protein;
WPRE=Woodchuck Hepatitis Virus Posttranscriptional Regulatory
Element; bGH=bovine growth hormone polyA; ITR=AAV inverted terminal
repeat; AmpR=ampicillin resistance gene.
DETAILED DESCRIPTION OF THE INVENTION
[0106] The term "nucleic acid" or "nucleic acid sequence" refers to
a deoxyribonucleic or ribonucleic oligonucleotide in either single-
or double-stranded form. The term encompasses nucleic acids, i.e.,
oligonucleotides, containing known analogues of natural
nucleotides. The term also encompasses nucleic-acid-like structures
with synthetic backbones, see, e.g., Eckstein, 1991; Baserga et
al., 1992; Milligan, 1993; WO 97/03211; WO 96/39154; Mata, 1997;
Strauss-Soukup, 1997; and Samstag, 1996.
[0107] As used herein, "recombinant" refers to a non-naturally
occurring composition comprising materials from more than one
origin and, in some embodiments, materials derived from more than
one organism. A "recombinant construct" may be a polynucleotide
synthesized or otherwise manipulated in vitro (e.g., "recombinant
polynucleotide"), and the invention includes methods of using
recombinant polynucleotides to produce gene products in cells or
other biological systems, or to a polypeptide ("recombinant
protein") encoded by a recombinant polynucleotide. "Recombinant
means" encompasses methods of recombining compositions, e.g.,
ligation of nucleic acids having various coding regions or domains
or promoter sequences from different sources into an expression
cassette or vector for expression of, e.g., inducible or
constitutive expression of polypeptide coding sequences in the
vectors of invention.
[0108] The term "heterologous" when used with reference to a
nucleic acid, indicates that the nucleic acid is in a cell or a
virus where it is not normally found in nature; or, comprises two
or more subsequences that are not found in the same relationship to
each other as normally found in nature, or is recombinantly
engineered so that its level of expression, or physical
relationship to other nucleic acids or other molecules in a cell,
or structure, is not normally found in nature. A similar term used
in this context is "exogenous". For instance, a heterologous
nucleic acid is typically recombinantly produced, having two or
more sequences from unrelated genes arranged in a manner not found
in nature; e.g., a human gene operably linked to a promoter
sequence inserted into an adenovirus-based vector of the invention.
As an example, a heterologous nucleic acid of interest may encode
an immunogenic gene product, wherein the adenovirus is administered
therapeutically or prophylactically as a carrier or drug-vaccine
composition. Heterologous sequences may comprise various
combinations of promoters and sequences, examples of which are
described in detail herein.
[0109] A "therapeutic ligand" may be a substance which may bind to
a receptor of a target cell with therapeutic effects.
[0110] A "therapeutic effect" may be a consequence of a medical
treatment of any kind, the results of which are judged by one of
skill in the field to be desirable and beneficial. The "therapeutic
effect" may be a behavioral or physiologic change which occurs as a
response to the medical treatment. The result may be expected,
unexpected, or even an unintended consequence of the medical
treatment. A "therapeutic effect" may include, for example, a
reduction of symptoms in a subject suffering from infection by a
pathogen.
[0111] A "target cell" may be a cell in which an alteration in its
activity may induce a desired result or response.
[0112] A "ligand" may be any substance that binds to and forms a
complex with a biomolecule to serve a biological purpose. As used
herein, "ligand" may also refer to an "antigen" or "immunogen". As
used herein "antigen" and "immunogen" are used interchangeably.
[0113] "Expression" of a gene or nucleic acid encompasses not only
cellular gene expression, but also the transcription and
translation of nucleic acid(s) in cloning systems and in any other
context.
[0114] As used herein, a "vector" is a tool that allows or
facilitates the transfer of an entity from one environment to
another. By way of example, some vectors used in recombinant DNA
techniques allow entities, such as a segment of DNA (such as a
heterologous DNA segment, such as a heterologous cDNA segment), to
be transferred into a target cell. The present invention
comprehends recombinant vectors that may include viral vectors,
bacterial vectors, protozoan vectors, DNA vectors, or recombinant
constructs thereof.
[0115] With respect to exogenous DNA for expression in a vector
(e.g., encoding an epitope of interest and/or an antigen and/or a
therapeutic) and documents providing such exogenous DNA, as well as
with respect to the expression of transcription and/or translation
factors for enhancing expression of nucleic acid molecules, and as
to terms such as "epitope of interest", "therapeutic", "immune
response", "immunological response", "protective immune response",
"immunological composition", "immunogenic composition", and
"vaccine composition", inter alia, reference is made to U.S. Pat.
No. 5,990,091 issued Nov. 23, 1999, and WO 98/00166 and WO
99/60164, and the documents cited therein and the documents of
record in the prosecution of that patent and those PCT
applications; all of which are incorporated herein by reference.
Thus, U.S. Pat. No. 5,990,091 and WO 98/00166 and WO 99/60164 and
documents cited therein and documents of record in the prosecution
of that patent and those PCT applications, and other documents
cited herein or otherwise incorporated herein by reference, may be
consulted in the practice of this invention; and, all exogenous
nucleic acid molecules, promoters, and vectors cited therein may be
used in the practice of this invention. In this regard, mention is
also made of U.S. Pat. Nos. 6,706,693; 6,716,823; 6,348,450; U.S.
patent application Ser. Nos. 10/424,409; 10/052,323; 10/116,963;
10/346,021; and WO 99/08713, published Feb. 25, 1999, from
PCT/US98/16739.
[0116] As used herein, the terms "drug composition" and "drug",
"vaccinal composition", "vaccine", "vaccine composition",
"therapeutic composition" and "therapeutic-immunologic composition"
cover any composition that induces protection against an antigen or
pathogen. In some embodiments, the protection may be due to an
inhibition or prevention of infection by a pathogen. In other
embodiments, the protection may be induced by an immune response
against the antigen(s) of interest, or which efficaciously protects
against the antigen; for instance, after administration or
injection into the subject, elicits a protective immune response
against the targeted antigen or immunogen or provides efficacious
protection against the antigen or immunogen expressed from the
inventive adenovirus vectors of the invention. The term
"pharmaceutical composition" means any composition that is
delivered to a subject. In some embodiments, the composition may be
delivered to inhibit or prevent infection by a pathogen.
[0117] A "therapeutically effective amount" is an amount or
concentration of the recombinant vector encoding the gene of
interest, that, when administered to a subject, produces a
therapeutic response or an immune response to the gene product of
interest.
[0118] The term "viral vector" as used herein includes but is not
limited to retroviruses, adenoviruses, adeno-associated viruses,
alphaviruses, and herpes simplex virus.
[0119] The present invention enables spatiotemporal control of
endogenous gene expression using a form of energy. The form of
energy by include but is not limited to electromagnetic radiation,
sound energy, chemical energy and thermal energy. In a preferred
embodiment of the invention, the form of energy is electromagnetic
radiation, preferably, light energy. Previous approaches to control
expression of endogenous genes, such as transcription activators
linked to DNA binding zinc finger proteins provided no mechanism
for temporal or spatial control. The capacity for photoactivation
of the system described herein allows the induction of gene
expression modulation to begin at a precise time within a localized
population of cells.
[0120] Two key molecular tools were leveraged in the design of the
photoresponsive transcription activator-like (TAL) effector system.
First, the DNA binding specificity of engineered TAL effectors is
utilized to localize the complex to a particular region in the
genome. Second, light-induced protein dimerization is used to
attract an activating or repressing domain to the region specified
by the TAL effector, resulting in modulation of the downstream
gene.
[0121] Inducible effectors are contemplated for in vitro or in vivo
application in which temporally or spatially specific gene
expression control is desired. In vitro examples: temporally
precise induction/suppression of developmental genes to elucidate
the timing of developmental cues, spatially controlled induction of
cell fate reprogramming factors for the generation of cell-type
patterned tissues. In vivo examples: combined temporal and spatial
control of gene expression within specific brain regions.
[0122] In a preferred embodiment of the invention, the inducible
effector is a Light Inducible Transcriptional Effector (LITE). The
modularity of the LITE system allows for any number of effector
domains to be employed for transcriptional modulation. In a
particularly advantageous embodiment, transcription activator like
effector (TALE) and the activation domain VP64 are utilized in the
present invention.
[0123] LITEs are designed to modulate or alter expression of
individual endogenous genes in a temporally and spatially precise
manner. Each LITE may comprise a two component system consisting of
a customized DNA-binding transcription activator like effector
(TALE) protein, a light-responsive cryptochrome heterodimer from
Arabadopsis thaliana, and a transcriptional activation/repression
domain. The TALE is designed to bind to the promoter sequence of
the gene of interest. The TALE protein is fused to one half of the
cryptochrome heterodimer (cryptochrome-2 or CIB1), while the
remaining cryptochrome partner is fused to a transcriptional
effector domain. Effector domains may be either activators, such as
VP16, VP64, or p65, or repressors, such as KRAB, EnR, or SID. In a
LITE's unstimulated state, the TALE-cryptochrome2 protein localizes
to the promoter of the gene of interest, but is not bound to the
CIB1-effector protein. Upon stimulation of a LITE with blue
spectrum light, cryptochrome-2 becomes activated, undergoes a
conformational change, and reveals its binding domain. CIB1, in
turn, binds to cryptochrome-2 resulting in localization of the
effector domain to the promoter region of the gene of interest and
initiating gene overexpression or silencing.
[0124] Activator and repressor domains may selected on the basis of
species, strength, mechanism, duration, size, or any number of
other parameters. Preferred effector domains include, but are not
limited to, a transposase domain, integrase domain, recombinase
domain, resolvase domain, invertase domain, protease domain, DNA
methyltransferase domain, DNA demethylase domain, histone acetylase
domain, histone deacetylases domain, nuclease domain, repressor
domain, activator domain, nuclear-localization signal domains,
transcription-protein recruiting domain, cellular uptake activity
associated domain, nucleic acid binding domain or antibody
presentation domain.
[0125] Gene targeting in a LITE or in any other inducible effector
may be achieved via the specificity of customized TALE DNA binding
proteins. A target sequence in the promoter region of the gene of
interest is selected and a TALE customized to this sequence is
designed. The central portion of the TALE consists of tandem
repeats 34 amino acids in length. Although the sequences of these
repeats are nearly identical, the 12th and 13th amino acids (termed
repeat variable diresidues) of each repeat vary, determining the
nucleotide-binding specificity of each repeat. Thus, by
synthesizing a construct with the appropriate ordering of TALE
monomer repeats, a DNA binding protein specific to the target
promoter sequence is created.
[0126] In advantageous embodiments of the invention, the methods
provided herein use isolated, non-naturally occurring, recombinant
or engineered DNA binding proteins that comprise TALE monomers or
TALE monomers or half monomers as a part of their organizational
structure that enable the targeting of nucleic acid sequences with
improved efficiency and expanded specificity.
[0127] Naturally occurring TALEs or "wild type TALEs" are nucleic
acid binding proteins secreted by numerous species of
proteobacteria. TALE polypeptides contain a nucleic acid binding
domain composed of tandem repeats of highly conserved monomer
polypeptides that are predominantly 33, 34 or 35 amino acids in
length and that differ from each other mainly in amino acid
positions 12 and 13. In advantageous embodiments the nucleic acid
is DNA. As used herein, the term "polypeptide monomers", "TALE
monomers" or "monomers" will be used to refer to the highly
conserved repetitive polypeptide sequences within the TALE nucleic
acid binding domain and the term "repeat variable di-residues" or
"RVD" will be used to refer to the highly variable amino acids at
positions 12 and 13 of the polypeptide monomers. As provided
throughout the disclosure, the amino acid residues of the RVD are
depicted using the IUPAC single letter code for amino acids. A
general representation of a TALE monomer which is comprised within
the DNA binding domain is X1-11-(X12X13)-X14-33 or 34 or 35, where
the subscript indicates the amino acid position and X represents
any amino acid. X12X13 indicate the RVDs. In some polypeptide
monomers, the variable amino acid at position 13 is missing or
absent and in such monomers, the RVD consists of a single amino
acid. In such cases the RVD may be alternatively represented as X*,
where X represents X12 and (*) indicates that X13 is absent. The
DNA binding domain comprises several repeats of TALE monomers and
this may be represented as (X1-11-(X12X13)-X14-33 or 34 or 35)z,
where in an advantageous embodiment, z is at least 5 to 40. In a
further advantageous embodiment, z is at least 10 to 26.
[0128] The TALE monomers have a nucleotide binding affinity that is
determined by the identity of the amino acids in its RVD. For
example, polypeptide monomers with an RVD of NI preferentially bind
to adenine (A), monomers with an RVD of NG preferentially bind to
thymine (T), monomers with an RVD of HD preferentially bind to
cytosine (C) and monomers with an RVD of NN preferentially bind to
both adenine (A) and guanine (G). In yet another embodiment of the
invention, monomers with an RVD of IG preferentially bind to T.
Thus, the number and order of the polypeptide monomer repeats in
the nucleic acid binding domain of a TALE determines its nucleic
acid target specificity. In still further embodiments of the
invention, monomers with an RVD of NS recognize all four base pairs
and may bind to A, T, G or C. The structure and function of TALEs
is further described in, for example, Moscou et al., Science
326:1501 (2009); Boch et al., Science 326:1509-1512 (2009); and
Zhang et al., Nature Biotechnology 29:149-153 (2011), each of which
is incorporated by reference in its entirety.
[0129] The polypeptides used in methods of the invention are
isolated, non-naturally occurring, recombinant or engineered
nucleic acid-binding proteins that have nucleic acid or DNA binding
regions containing polypeptide monomer repeats that are designed to
target specific nucleic acid sequences.
[0130] As described herein, polypeptide monomers having an RVD of
HN or NH preferentially bind to guanine and thereby allow the
generation of TALE polypeptides with high binding specificity for
guanine containing target nucleic acid sequences. In a preferred
embodiment of the invention, polypeptide monomers having RVDs RN,
NN, NK, SN, NH, KN, HN, NQ, HH, RG, KH, RH and SS preferentially
bind to guanine. In a much more advantageous embodiment of the
invention, polypeptide monomers having RVDs RN, NK, NQ, HH, KH, RH,
SS and SN preferentially bind to guanine and thereby allow the
generation of TALE polypeptides with high binding specificity for
guanine containing target nucleic acid sequences. In an even more
advantageous embodiment of the invention, polypeptide monomers
having RVDs HH, KH, NH, NK, NQ, RH, RN and SS preferentially bind
to guanine and thereby allow the generation of TALE polypeptides
with high binding specificity for guanine containing target nucleic
acid sequences. In a further advantageous embodiment, the RVDs that
have high binding specificity for guanine are RN, NH RH and KH.
Furthermore, polypeptide monomers having an RVD of NV
preferentially bind to adenine and guanine. In more preferred
embodiments of the invention, monomers having RVDs of H*, HA, KA,
N*, NA, NC, NS, RA, and S* bind to adenine, guanine, cytosine and
thymine with comparable affinity.
[0131] The predetermined N-terminal to C-terminal order of the one
or more polypeptide monomers of the nucleic acid or DNA binding
domain determines the corresponding predetermined target nucleic
acid sequence to which the polypeptides of the invention will bind.
As used herein the monomers and at least one or more half monomers
are "specifically ordered to target" the genomic locus or gene of
interest. In plant genomes, the natural TALE-binding sites always
begin with a thymine (T), which may be specified by a cryptic
signal within the nonrepetitive N-terminus of the TALE polypeptide;
in some cases this region may be referred to as repeat 0. In animal
genomes, TALE binding sites do not necessarily have to begin with a
thymine (T) and polypeptides of the invention may target DNA
sequences that begin with T, A, G or C. The tandem repeat of TALE
monomers always ends with a half-length repeat or a stretch of
sequence that may share identity with only the first 20 amino acids
of a repetitive full length TALE monomer and this half repeat may
be referred to as a half-monomer (FIG. 8). Therefore, it follows
that the length of the nucleic acid or DNA being targeted is equal
to the number of full monomers plus two.
[0132] As described in Zhang et al., Nature Biotechnology
29:149-153 (2011), TALE polypeptide binding efficiency may be
increased by including amino acid sequences from the "capping
regions" that are directly N-terminal or C-terminal of the DNA
binding region of naturally occurring TALEs into the engineered
TALEs at positions N-terminal or C-terminal of the engineered TALE
DNA binding region. Thus, in certain embodiments, the TALE
polypeptides described herein further comprise an N-terminal
capping region and/or a C-terminal capping region.
[0133] An exemplary amino acid sequence of a N-terminal capping
region is:
TABLE-US-00002 (SEQ ID NO: 147) M D P I R S R T P S P A R E L L S G
P Q P D G V Q P T A D R G V S P P A G G P L D G L P A R R T M S R T
R L P S P P A P S P A F S A D S F S D L L R Q F D P S L F N T S L F
D S L P P F G A H H T E A A T G E W D E V Q S G L R A A D A P P P T
M R V A V T A A R P P R A K P A P R R R A A Q P S D A S P A A Q V D
L R T L G Y S Q Q Q Q E K I K P K V R S T V A Q H H E A L V G H G F
T H A H I V A L S Q H P A A L G T V A V K Y Q D M I A A L P E A T H
E A I V G V G K Q W S G A R A L E A L L T V A G E L R G P P L Q L D
T G Q L L K I A K R G G V T A V E A V H A W R N A L T G A P L N
[0134] An exemplary amino acid sequence of a C-terminal capping
region is:
TABLE-US-00003 (SEQ ID NO: 148) R P A L E S I V A Q L S R P D P A L
A A L T N D H L V A L A C L G G R P A L D A V K K G L P H A P A L I
K R T N R R I P E R T S H R V A D H A Q V V R V L G F F Q C H S H P
A Q A F D D A M T Q F G M S R H G L L Q L F R R V G V T E L E A R S
G T L P P A S Q R W D R I L Q A S G M K R A K P S P T S T Q T P D Q
A S L H A F A D S L E R D L D A P S P M H E G D Q T R A S
[0135] As used herein the predetermined "N-terminus" to "C
terminus" orientation of the N-terminal capping region, the DNA
binding domain comprising the repeat TALE monomers and the
C-terminal capping region provide structural basis for the
organization of different domains in the d-TALEs or polypeptides of
the invention.
[0136] The entire N-terminal and/or C-terminal capping regions are
not necessary to enhance the binding activity of the DNA binding
region. Therefore, in certain embodiments, fragments of the
N-terminal and/or C-terminal capping regions are included in the
TALE polypeptides described herein.
[0137] In certain embodiments, the TALE polypeptides described
herein contain a N-terminal capping region fragment that included
at least 10, 20, 30, 40, 50, 54, 60, 70, 80, 87, 90, 94, 100, 102,
110, 117, 120, 130, 140, 147, 150, 160, 170, 180, 190, 200, 210,
220, 230, 240, 250, 260 or 270 amino acids of an N-terminal capping
region. In certain embodiments, the N-terminal capping region
fragment amino acids are of the C-terminus (the DNA-binding region
proximal end) of an N-terminal capping region. As described in
Zhang et al., Nature Biotechnology 29:149-153 (2011), N-terminal
capping region fragments that include the C-terminal 240 amino
acids enhance binding activity equal to the full length capping
region, while fragments that include the C-terminal 147 amino acids
retain greater than 80% of the efficacy of the full length capping
region, and fragments that include the C-terminal 117 amino acids
retain greater than 50% of the activity of the full-length capping
region.
[0138] In some embodiments, the TALE polypeptides described herein
contain a C-terminal capping region fragment that included at least
6, 10, 20, 30, 37, 40, 50, 60, 68, 70, 80, 90, 100, 110, 120, 127,
130, 140, 150, 155, 160, 170, 180 amino acids of a C-terminal
capping region. In certain embodiments, the C-terminal capping
region fragment amino acids are of the N-terminus (the DNA-binding
region proximal end) of a C-terminal capping region. As described
in Zhang et al., Nature Biotechnology 29:149-153 (2011), C-terminal
capping region fragments that include the C-terminal 68 amino acids
enhance binding activity equal to the full length capping region,
while fragments that include the C-terminal 20 amino acids retain
greater than 50% of the efficacy of the full length capping
region.
[0139] In certain embodiments, the capping regions of the TALE
polypeptides described herein do not need to have identical
sequences to the capping region sequences provided herein. Thus, in
some embodiments, the capping region of the TALE polypeptides
described herein have sequences that are at least 50%, 60%, 70%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical or share identity to the capping region amino acid
sequences provided herein. Sequence identity is related to sequence
homology. Homology comparisons may be conducted by eye, or more
usually, with the aid of readily available sequence comparison
programs. These commercially available computer programs may
calculate percent (%) homology between two or more sequences and
may also calculate the sequence identity shared by two or more
amino acid or nucleic acid sequences. In some preferred
embodiments, the capping region of the TALE polypeptides described
herein have sequences that are at least 95% identical or share
identity to the capping region amino acid sequences provided
herein.
[0140] Sequence homologies may be generated by any of a number of
computer programs known in the art, which include but are not
limited to BLAST or FASTA. Suitable computer program for carrying
out alignments like the GCG Wisconsin Bestfit package may also be
used. Once the software has produced an optimal alignment, it is
possible to calculate % homology, preferably % sequence identity.
The software typically does this as part of the sequence comparison
and generates a numerical result.
[0141] In advantageous embodiments described herein, the TALE
polypeptides of the invention include a nucleic acid binding domain
linked to the one or more effector domains. The terms "effector
domain" or "regulatory and functional domain" refer to a
polypeptide sequence that has an activity other than binding to the
nucleic acid sequence recognized by the nucleic acid binding
domain. By combining a nucleic acid binding domain with one or more
effector domains, the polypeptides of the invention may be used to
target the one or more functions or activities mediated by the
effector domain to a particular target DNA sequence to which the
nucleic acid binding domain specifically binds.
[0142] In some embodiments of the TALE polypeptides described
herein, the activity mediated by the effector domain is a
biological activity. For example, in some embodiments the effector
domain is a transcriptional inhibitor (i.e., a repressor domain),
such as an mSin interaction domain (SID). SID4X domain or a
Kruppel-associated box (KRAB) or fragments of the KRAB domain. In
some embodiments the effector domain is an enhancer of
transcription (i.e. an activation domain), such as the VP16, VP64
or p65 activation domain. In some embodiments, the nucleic acid
binding is linked, for example, with an effector domain that
includes but is not limited to a transposase, integrase,
recombinase, resolvase, invertase, protease, DNA methyltransferase,
DNA demethylase, histone acetylase, histone deacetylase, nuclease,
transcriptional repressor, transcriptional activator, transcription
factor recruiting, protein nuclear-localization signal or cellular
uptake signal.
[0143] In some embodiments, the effector domain is a protein domain
which exhibits activities which include but are not limited to
transposase activity, integrase activity, recombinase activity,
resolvase activity, invertase activity, protease activity, DNA
methyltransferase activity, DNA demethylase activity, histone
acetylase activity, histone deacetylase activity, nuclease
activity, nuclear-localization signaling activity, transcriptional
repressor activity, transcriptional activator activity,
transcription factor recruiting activity, or cellular uptake
signaling activity. Other preferred embodiments of the invention
may include any combination the activities described herein.
[0144] As described in Zhang et al., Nature Biotechnology
29:149-153 (2011), a TALE polypeptide having a nucleic acid binding
domain and an effector domain may be used to target the effector
domain's activity to a genomic position having a predetermined
nucleic acid sequence recognized by the nucleic acid binding
domain. In some embodiments of the invention described herein, TALE
polypeptides are designed and used for targeting gene regulatory
activity, such as transcriptional or translational modifier
activity, to a regulatory, coding, and/or intergenic region, such
as enhancer and/or repressor activity, that may affect
transcription upstream and downstream of coding regions, and may be
used to enhance or repress gene expression. For example, TALEs
polypeptide may comprise effector domains having DNA-binding
domains from transcription factors, effector domains from
transcription factors (activators, repressors, co-activators,
co-repressors), silencers, nuclear hormone receptors, and/or
chromatin associated proteins and their modifiers (e.g.,
methylases, kinases, phosphatases, acetylases and deacetylases). In
a preferred embodiment, the TALE polypeptide may comprise a
nuclease domain. In a more preferred embodiment the nuclease domain
is a non-specific FokI endonucleases catalytic domain.
[0145] In a further embodiment, useful domains for regulating gene
expression may also be obtained from the gene products of
oncogenes. In yet further advantageous embodiments of the
invention, effector domains having integrase or transposase
activity may be used to promote integration of exogenous nucleic
acid sequence into specific nucleic acid sequence regions,
eliminate (knock-out) specific endogenous nucleic acid sequence,
and/or modify epigenetic signals and consequent gene regulation,
such as by promoting DNA methyltransferase, DNA demethylase,
histone acetylase and histone deacetylase activity. In other
embodiments, effector domains having nuclease activity may be used
to alter genome structure by nicking or digesting target sequences
to which the polypeptides of the invention specifically bind, and
may allow introduction of exogenous genes at those sites. In still
further embodiments, effector domains having invertase activity may
be used to alter genome structure by swapping the orientation of a
DNA fragment.
[0146] In particularly advantageous embodiments, the polypeptides
used in the methods of the invention may be used to target
transcriptional activity. As used herein, the term "transcription
factor" refers to a protein or polypeptide that binds specific DNA
sequences associated with a genomic locus or gene of interest to
control transcription. Transcription factors may promote (as an
activator) or block (as a repressor) the recruitment of RNA
polymerase to a gene of interest. Transcription factors may perform
their function alone or as a part of a larger protein complex.
Mechanisms of gene regulation used by transcription factors include
but are not limited to a) stabilization or destabilization of RNA
polymerase binding, b) acetylation or deacetylation of histone
proteins and c) recruitment of co-activator or co-repressor
proteins. Furthermore, transcription factors play roles in
biological activities that include but are not limited to basal
transcription, enhancement of transcription, development, response
to intercellular signaling, response to environmental cues,
cell-cycle control and pathogenesis. With regards to information on
transcriptional factors, mention is made of Latchman and DS (1997)
Int. J. Biochem. Cell Biol. 29 (12): 1305-12; Lee T I, Young R A
(2000) Annu Rev. Genet. 34: 77-137 and Mitchell P J, Tjian R (1989)
Science 245 (4916): 371-8, herein incorporated by reference in
their entirety.
[0147] Light responsiveness of a LITE is achieved via the
activation and binding of cryptochrome-2 and CIB1. As mentioned
above, blue light stimulation induces an activating conformational
change in cryptochrome-2, resulting in recruitment of its binding
partner CIB1. This binding is fast and reversible, achieving
saturation in <15 sec following pulsed stimulation and returning
to baseline <15 min after the end of stimulation. These rapid
binding kinetics result in a LITE system temporally bound only by
the speed of transcription/translation and transcript/protein
degradation, rather than uptake and clearance of inducing agents.
Cryptochrome-2 activation is also highly sensitive, allowing for
the use of low light intensity stimulation and mitigating the risks
of phototoxicity. Further, in a context such as the intact
mammalian brain, variable light intensity may be used to control
the size of a LITE stimulated region, allowing for greater
precision than vector delivery alone may offer.
[0148] The modularity of the LITE system allows for any number of
effector domains to be employed for transcriptional modulation.
Thus, activator and repressor domains may be selected on the basis
of species, strength, mechanism, duration, size, or any number of
other parameters.
[0149] Applicants next present two prototypical manifestations of
the LITE system. The first example is a LITE designed to activate
transcription of the mouse gene NEUROG2. The sequence
TGAATGATGATAATACGA (SEQ ID NO:149), located in the upstream
promoter region of mouse NEUROG2, was selected as the target and a
TALE was designed and synthesized to match this sequence. The TALE
sequence was linked to the sequence for cryptochrome-2 via a
nuclear localization signal (amino acids: SPKKKRKVEAS; SEQ ID NO:
150) to facilitate transport of the protein from the cytosol to the
nuclear space. A second vector was synthesized comprising the CIB1
domain linked to the transcriptional activator domain VP64 using
the same nuclear localization signal. This second vector, also a
GFP sequence, is separated from the CIB1-VP64 fusion sequence by a
2A translational skip signal. Expression of each construct was
driven by a ubiquitous, constitutive promoter (CMV or EF1-.alpha.).
Mouse neuroblastoma cells from the Neuro 2A cell line were
co-transfected with the two vectors. After incubation to allow for
vector expression, samples were stimulated by periodic pulsed blue
light from an array of 488 nm LEDs. Unstimulated co-transfected
samples and samples transfected only with the fluorescent reporter
YFP were used as controls. At the end of each experiment, mRNA was
purified from the samples analyzed via qPCR.
[0150] Truncated versions of cryptochrome-2 and CIB1 were cloned
and tested in combination with the full-length versions of
cryptochrome-2 and CIB1 in order to determine the effectiveness of
each heterodimer pair. The combination of the CRY2PHR domain,
consisting of the conserved photoresponsive region of the
cryptochrome-2 protein, and the full-length version of CIB1
resulted in the highest upregulation of Neurog2 mRNA levels
(.about.22 fold over YFP samples and .about.7 fold over
unstimulated co-transfected samples). The combination of
full-length cryptochrome-2 (CRY2) with full-length CIB1 resulted in
a lower absolute activation level (.about.4.6 fold over YFP), but
also a lower baseline activation (.about.1.6 fold over YFP for
unstimulated co-transfected samples). These cryptochrome protein
pairings may be selected for particular uses depending on absolute
level of induction required and the necessity to minimize baseline
"leakiness" of the LITE system.
[0151] Speed of activation and reversibility are critical design
parameters for the LITE system. The invention contemplates energy
sources such as electromagnetic radiation, sound energy or thermal
energy.
[0152] The cells of the present invention are preferably a
eukaryotic cell, advantageously an animal cell, more advantageously
a mammalian cell.
[0153] The present invention also contemplates a multiplex genome
engineering using CRISPR/Cas systems. Functional elucidation of
causal genetic variants and elements requires precise genome
editing technologies. The type II prokaryotic CRISPR (clustered
regularly interspaced short palindromic repeats) adaptive immune
system has been shown to facilitate RNA-guided site-specific DNA
cleavage. Applicants engineered two different type II CRISPR
systems and demonstrate that Cas9 nucleases can be directed by
short RNAs to induce precise cleavage at endogenous genomic loci in
human and mouse cells. Cas9 can also be converted into a nicking
enzyme to facilitate homology-directed repair with minimal
mutagenic activity. Finally, multiple guide sequences can be
encoded into a single CRISPR array to enable simultaneous editing
of several sites within the mammalian genome, demonstrating easy
programmability and wide applicability of the CRISPR
technology.
[0154] In general, "CRISPR system" refers collectively to
transcripts and other elements involved in the expression of or
directing the activity of CRISPR-associated ("Cas") genes,
including sequences encoding a Cas gene, a tracr (trans-activating
CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a
tracr-mate sequence (encompassing a "direct repeat" and a
tracrRNA-processed partial direct repeat in the context of an
endogenous CRISPR system), a guide sequence (also referred to as a
"spacer" in the context of an endogenous CRISPR system), or other
sequences and transcripts from a CRISPR locus. In some embodiments,
one or more elements of a CRISPR system is derived from a type I,
type II, or type III CRISPR system. In some embodiments, one or
more elements of a CRISPR system is derived from a particular
organism comprising an endogenous CRISPR system, such as
Streptococcus pyogenes. In general, a CRISPR system is
characterized by elements that promote the formation of a CRISPR
complex at the site of a target sequence (also referred to as a
protospacer in the context of an endogenous CRISPR system). In the
context of formation of a CRISPR complex, "target sequence" refers
to a sequence to which a guide sequence is designed to have
complementarity, where hybridization between a target sequence and
a guide sequence promotes the formation of a CRISPR complex. A
target sequence may comprise any polynucleotide, such as DNA or RNA
polynucleotides. In some embodiments, a target sequence is located
in the nucleus or cytoplasm of a cell.
[0155] Typically, in the context of an endogenous CRISPR system,
formation of a CRISPR complex (comprising a guide sequence
hybridized to a target sequence and complexed with one or more Cas
proteins) results in cleavage of one or both strands in or near
(e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base
pairs from) the target sequence. Without wishing to be bound by
theory, all or a portion of the tracr sequence may also form part
of a CRISPR complex, such as by hybridization to all or a portion
of a tracr mate sequence that is operably linked to the guide
sequence. In some embodiments, one or more vectors driving
expression of one or more elements of a CRISPR system are
introduced into a host cell such that expression of the elements of
the CRISPR system direct formation of a CRISPR complex at one or
more target sites. For example, a Cas enzyme, a guide sequence
linked to a tracr-mate sequence, and a tracr sequence could each be
operably linked to separate regulatory elements on separate
vectors. Alternatively, two or more of the elements expressed from
the same or different regulatory elements, may be combined in a
single vector, with one or more additional vectors providing any
components of the CRISPR system not included in the first vector.
CRISPR system elements that are combined in a single vector may be
arranged in any suitable orientation, such as one element located
5' with respect to ("upstream" of) or 3' with respect to
("downstream" of) a second element. The coding sequence of one
element may be located on the same or opposite strand of the coding
sequence of a second element, and oriented in the same or opposite
direction. In some embodiments, a single promoter drives expression
of a transcript encoding a CRISPR enzyme and one or more of the
guide sequence, tracr mate sequence (optionally operably linked to
the guide sequence), and a tracr sequence embedded within one or
more intron sequences (e.g. each in a different intron, two or more
in at least one intron, or all in a single intron). In some
embodiments, the CRISPR enzyme, guide sequence, tracr mate
sequence, and tracr sequence are operably linked to and expressed
from the same promoter.
[0156] In some embodiments, a vector comprises one or more
insertion sites, such as a restriction endonuclease recognition
sequence (also referred to as a "cloning site"). In some
embodiments, one or more insertion sites (e.g. about or more than
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are
located upstream and/or downstream of one or more sequence elements
of one or more vectors. In some embodiments, a vector comprises an
insertion site upstream of a tracr mate sequence, and optionally
downstream of a regulatory element operably linked to the tracr
mate sequence, such that following insertion of a guide sequence
into the insertion site and upon expression the guide sequence
directs sequence-specific binding of a CRISPR complex to a target
sequence in a eukaryotic cell. In some embodiments, a vector
comprises two or more insertion sites, each insertion site being
located between two tracr mate sequences so as to allow insertion
of a guide sequence at each site. In such an arrangement, the two
or more guide sequences may comprise two or more copies of a single
guide sequence, two or more different guide sequences, or
combinations of these. When multiple different guide sequences are
used, a single expression construct may be used to target CRISPR
activity to multiple different, corresponding target sequences
within a cell. For example, a single vector may comprise about or
more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more
guide sequences. In some embodiments, about or more than about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containing
vectors may be provided, and optionally delivered to a cell.
[0157] In some embodiments, a vector comprises a regulatory element
operably linked to an enzyme-coding sequence encoding a CRISPR
enzyme, such as a Cas protein. Non-limiting examples of Cas
proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7,
Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3,
Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6,
Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14,
Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4,
homologues thereof, or modified versions thereof. In some
embodiments, the unmodified CRISPR enzyme has DNA cleavage
activity, such as Cas9. In some embodiments, the CRISPR enzyme
directs cleavage of one or both strands at the location of a target
sequence, such as within the target sequence and/or within the
complement of the target sequence. In some embodiments, the CRISPR
enzyme directs cleavage of one or both strands within about 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more
base pairs from the first or last nucleotide of a target sequence.
In some embodiments, a vector encodes a CRISPR enzyme that is
mutated to with respect to a corresponding wild-type enzyme such
that the mutated CRISPR enzyme lacks the ability to cleave one or
both strands of a target polynucleotide containing a target
sequence. For example, an aspartate-to-alanine substitution (D10A)
in the RuvC I catalytic domain of Cas9 from S. pyogenes converts
Cas9 from a nuclease that cleaves both strands to a nickase
(cleaves a single strand). Other examples of mutations that render
Cas9 a nickase include, without limitation, H840A, N854A, and
N863A. As a further example, two or more catalytic domains of Cas9
(RuvC I, RuvC II, and RuvC III) may be mutated to produce a mutated
Cas9 substantially lacking all DNA cleavage activity. In some
embodiments, a D10A mutation is combined with one or more of H840A,
N854A, or N863A mutations to produce a Cas9 enzyme substantially
lacking all DNA cleavage activity. In some embodiments, a CRISPR
enzyme is considered to substantially lack all DNA cleavage
activity when the DNA cleavage activity of the mutated enzyme is
less than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or lower with
respect to its non-mutated form.
[0158] In some embodiments, an enzyme coding sequence encoding a
CRISPR enzyme is codon optimized for expression in particular
cells, such as eukaryotic cells. The eukaryotic cells may be those
of or derived from a particular organism, such as a mammal,
including but not limited to human, mouse, rat, rabbit, dog, or
non-human primate. In general, codon optimization refers to a
process of modifying a nucleic acid sequence for enhanced
expression in the host cells of interest by replacing at least one
codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25,
50, or more codons) of the native sequence with codons that are
more frequently or most frequently used in the genes of that host
cell while maintaining the native amino acid sequence. Various
species exhibit particular bias for certain codons of a particular
amino acid. Codon bias (differences in codon usage between
organisms) often correlates with the efficiency of translation of
messenger RNA (mRNA), which is in turn believed to be dependent on,
among other things, the properties of the codons being translated
and the availability of particular transfer RNA (tRNA) molecules.
The predominance of selected tRNAs in a cell is generally a
reflection of the codons used most frequently in peptide synthesis.
Accordingly, genes can be tailored for optimal gene expression in a
given organism based on codon optimization. Codon usage tables are
readily available, for example, at the "Codon Usage Database"
available at www.kazusa.orjp/codon/ (visited Jul. 9, 2002), and
these tables can be adapted in a number of ways. See Nakamura, Y.,
et al. "Codon usage tabulated from the international DNA sequence
databases: status for the year 2000''Nucl. Acids Res. 28:292
(2000). Computer algorithms for codon optimizing a particular
sequence for expression in a particular host cell are also
available, such as Gene Forge (Aptagen; Jacobus, PA), are also
available. In some embodiments, one or more codons (e.g. 1, 2, 3,
4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence
encoding a CRISPR enzyme correspond to the most frequently used
codon for a particular amino acid.
[0159] In some embodiments, a vector encodes a CRISPR enzyme
comprising one or more nuclear localization sequences (NLSs), such
as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
NLSs. In some embodiments, the CRISPR enzyme comprises about or
more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or
near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a
combination of these (e.g. one or more NLS at the amino-terminus
and one or more NLS at the carboxy terminus). When more than one
NLS is present, each may be selected independently of the others,
such that a single NLS may be present in more than one copy and/or
in combination with one or more other NLSs present in one or more
copies. In some embodiments, an NLS is considered near the N- or
C-terminus when the nearest amino acid of the NLS is within about
1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids
along the polypeptide chain from the N- or C-terminus. Non-limiting
examples of NLSs include an NLS sequence derived from: the NLS of
the SV40 virus large T-antigen, having the amino acid sequence
PKKKRKV (SEQ ID NO: 151); the NLS from nucleoplasmin (e.g. the
nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK; SEQ
ID NO: 152); the c-myc NLS having the amino acid sequence PAAKRVKLD
(SEQ ID NO: 153) or RQRRNELKRSP (SEQ ID NO: 154); the hRNPA1 M9 NLS
having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID
NO: 155); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV
(SEQ ID NO: 156) of the IBB domain from importin-alpha; the
sequences VSRKRPRP (SEQ ID NO: 157) and PPKKARED (SEQ ID NO: 158)
of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 159) of
human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 160) of mouse
c-abl IV; the sequences DRLRR (SEQ ID NO: 161) and PKQKKRK (SEQ ID
NO: 162) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ
ID NO: 163) of the Hepatitis virus delta antigen; the sequence
REKKKFLKRR (SEQ ID NO: 164) of the mouse Mx1 protein; the sequence
KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 165) of the human poly(ADP-ribose)
polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 166) of
the steroid hormone receptors (human) glucocorticoid.
[0160] In general, the one or more NLSs are of sufficient strength
to drive accumulation of the CRISPR enzyme in a detectable amount
in the nucleus of a eukaryotic cell. In general, strength of
nuclear localization activity may derive from the number of NLSs in
the CRISPR enzyme, the particular NLS(s) used, or a combination of
these factors. Detection of accumulation in the nucleus may be
performed by any suitable technique. For example, a detectable
marker may be fused to the CRISPR enzyme, such that location within
a cell may be visualized, such as in combination with a means for
detecting the location of the nucleus (e.g. a stain specific for
the nucleus such as DAPI). Cell nuclei may also be isolated from
cells, the contents of which may then be analyzed by any suitable
process for detecting protein, such as immunohistochemistry,
Western blot, or enzyme activity assay. Accumulation in the nucleus
may also be determined indirectly, such as by an assay for the
effect of CRISPR complex formation (e.g. assay for DNA cleavage or
mutation at the target sequence, or assay for altered gene
expression activity affected by CRISPR complex formation and/or
CRISPR enzyme activity), as compared to a control no exposed to the
CRISPR enzyme or complex, or exposed to a CRISPR enzyme lacking the
one or more NLSs.
[0161] The present invention also encompasses nucleic acid encoding
the polypeptides of the present invention. The nucleic acid may
comprise a promoter, advantageously human Synapsin I promoter
(hSyn). In a particularly advantageous embodiment, the nucleic acid
may be packaged into an adeno associated viral vector (AAV).
[0162] Also contemplated by the present invention are recombinant
vectors and recombinant adenoviruses that may comprise subviral
particles from more than one adenovirus serotype. For example, it
is known that adenovirus vectors may display an altered tropism for
specific tissues or cell types (Havenga, M. J. E. et al., 2002),
and therefore, mixing and matching of different adenoviral capsids,
i.e., fiber, or penton proteins from various adenoviral serotypes
may be advantageous. Modification of the adenoviral capsids,
including fiber and penton may result in an adenoviral vector with
a tropism that is different from the unmodified adenovirus.
Adenovirus vectors that are modified and optimized in their ability
to infect target cells may allow for a significant reduction in the
therapeutic or prophylactic dose, resulting in reduced local and
disseminated toxicity.
[0163] Viral vector gene delivery systems are commonly used in gene
transfer and gene therapy applications. Different viral vector
systems have their own unique advantages and disadvantages. Viral
vectors that may be used to express the pathogen-derived ligand of
the present invention include but are not limited to adenoviral
vectors, adeno-associated viral vectors, alphavirus vectors, herpes
simplex viral vectors, and retroviral vectors, described in more
detail below.
[0164] Additional general features of adenoviruses are such that
the biology of the adenovirus is characterized in detail; the
adenovirus is not associated with severe human pathology; the
adenovirus is extremely efficient in introducing its DNA into the
host cell; the adenovirus may infect a wide variety of cells and
has a broad host range; the adenovirus may be produced in large
quantities with relative ease; and the adenovirus may be rendered
replication defective and/or non-replicating by deletions in the
early region 1 ("E1") of the viral genome.
[0165] Adenovirus is a non-enveloped DNA virus. The genome of
adenovirus is a linear double-stranded DNA molecule of
approximately 36,000 base pairs ("bp") with a 55-kDa terminal
protein covalently bound to the 5'-terminus of each strand. The
adenovirus DNA contains identical inverted terminal repeats
("ITRs") of about 100 bp, with the exact length depending on the
serotype. The viral origins of replication are located within the
ITRs exactly at the genome ends. DNA synthesis occurs in two
stages. First, replication proceeds by strand displacement,
generating a daughter duplex molecule and a parental displaced
strand. The displaced strand is single stranded and may form a
"panhandle" intermediate, which allows replication initiation and
generation of a daughter duplex molecule. Alternatively,
replication may proceed from both ends of the genome
simultaneously, obviating the requirement to form the panhandle
structure.
[0166] During the productive infection cycle, the viral genes are
expressed in two phases: the early phase, which is the period up to
viral DNA replication, and the late phase, which coincides with the
initiation of viral DNA replication. During the early phase, only
the early gene products, encoded by regions E1, E2, E3 and E4, are
expressed, which carry out a number of functions that prepare the
cell for synthesis of viral structural proteins (Berk, A. J.,
1986). During the late phase, the late viral gene products are
expressed in addition to the early gene products and host cell DNA
and protein synthesis are shut off. Consequently, the cell becomes
dedicated to the production of viral DNA and of viral structural
proteins (Tooze, J., 1981).
[0167] The E1 region of adenovirus is the first region of
adenovirus expressed after infection of the target cell. This
region consists of two transcriptional units, the E1A and E1B
genes, both of which are required for oncogenic transformation of
primary (embryonal) rodent cultures. The main functions of the E1A
gene products are to induce quiescent cells to enter the cell cycle
and resume cellular DNA synthesis, and to transcriptionally
activate the E1B gene and the other early regions (E2, E3 and E4)
of the viral genome. Transfection of primary cells with the E1A
gene alone may induce unlimited proliferation (immortalization),
but does not result in complete transformation. However, expression
of E1A, in most cases, results in induction of programmed cell
death (apoptosis), and only occasionally is immortalization
obtained (Jochemsen et al., 1987). Co-expression of the E1B gene is
required to prevent induction of apoptosis and for complete
morphological transformation to occur. In established immortal cell
lines, high-level expression of E1A may cause complete
transformation in the absence of E1B (Roberts, B. E. et al.,
1985).
[0168] The E1B encoded proteins assist E1A in redirecting the
cellular functions to allow viral replication. The E1B 55 kD and E4
33 kD proteins, which form a complex that is essentially localized
in the nucleus, function in inhibiting the synthesis of host
proteins and in facilitating the expression of viral genes. Their
main influence is to establish selective transport of viral mRNAs
from the nucleus to the cytoplasm, concomitantly with the onset of
the late phase of infection. The E1B 21 kD protein is important for
correct temporal control of the productive infection cycle, thereby
preventing premature death of the host cell before the virus life
cycle has been completed. Mutant viruses incapable of expressing
the E1B 21 kD gene product exhibit a shortened infection cycle that
is accompanied by excessive degradation of host cell chromosomal
DNA (deg-phenotype) and in an enhanced cytopathic effect
(cyt-phenotype; Telling et al., 1994). The deg and cyt phenotypes
are suppressed when in addition the E1A gene is mutated, indicating
that these phenotypes are a function of E1A (White, E. et al.,
1988). Furthermore, the E1B 21 kDa protein slows down the rate by
which E1A switches on the other viral genes. It is not yet known by
which mechanisms E1B 21 kD quenches these E1A dependent
functions.
[0169] In contrast to, for example, retroviruses, adenoviruses do
not efficiently integrate into the host cell's genome, are able to
infect non-dividing cells, and are able to efficiently transfer
recombinant genes in vivo (Brody et al., 1994). These features make
adenoviruses attractive candidates for in vivo gene transfer of,
for example, an antigen or immunogen of interest into cells,
tissues or subjects in need thereof.
[0170] Adenovirus vectors containing multiple deletions are
preferred to both increase the carrying capacity of the vector and
reduce the likelihood of recombination to generate replication
competent adenovirus (RCA). Where the adenovirus contains multiple
deletions, it is not necessary that each of the deletions, if
present alone, would result in a replication defective and/or
non-replicating adenovirus. As long as one of the deletions renders
the adenovirus replication defective or non-replicating, the
additional deletions may be included for other purposes, e.g., to
increase the carrying capacity of the adenovirus genome for
heterologous nucleotide sequences. Preferably, more than one of the
deletions prevents the expression of a functional protein and
renders the adenovirus replication defective and/or non-replicating
and/or attenuated. More preferably, all of the deletions are
deletions that would render the adenovirus replication-defective
and/or non-replicating and/or attenuated. However, the invention
also encompasses adenovirus and adenovirus vectors that are
replication competent and/or wild-type, i.e. comprises all of the
adenoviral genes necessary for infection and replication in a
subject.
[0171] Embodiments of the invention employing adenovirus
recombinants may include E1-defective or deleted, or E3-defective
or deleted, or E4-defective or deleted or adenovirus vectors
comprising deletions of E1 and E3, or E1 and E4, or E3 and E4, or
E1, E3, and E4 deleted, or the "gutless" adenovirus vector in which
all viral genes are deleted. The adenovirus vectors may comprise
mutations in E1, E3, or E4 genes, or deletions in these or all
adenoviral genes. The E1 mutation raises the safety margin of the
vector because E1-defective adenovirus mutants are said to be
replication-defective and/or non-replicating in non-permissive
cells, and are, at the very least, highly attenuated. The E3
mutation enhances the immunogenicity of the antigen by disrupting
the mechanism whereby adenovirus down-regulates MHC class I
molecules. The E4 mutation reduces the immunogenicity of the
adenovirus vector by suppressing the late gene expression, thus may
allow repeated re-vaccination utilizing the same vector. The
present invention comprehends adenovirus vectors of any serotype or
serogroup that are deleted or mutated in E1, or E3, or E4, or E1
and E3, or E1 and E4. Deletion or mutation of these adenoviral
genes result in impaired or substantially complete loss of activity
of these proteins.
[0172] The "gutless" adenovirus vector is another type of vector in
the adenovirus vector family. Its replication requires a helper
virus and a special human 293 cell line expressing both E1a and
Cre, a condition that does not exist in a natural environment; the
vector is deprived of all viral genes, thus the vector as a vaccine
carrier is non-immunogenic and may be inoculated multiple times for
re-vaccination. The "gutless" adenovirus vector also contains 36 kb
space for accommodating antigen or immunogen(s) of interest, thus
allowing co-delivery of a large number of antigen or immunogens
into cells.
[0173] Adeno-associated virus (AAV) is a single-stranded DNA
parvovirus which is endogenous to the human population. Although
capable of productive infection in cells from a variety of species,
AAV is a dependovirus, requiring helper functions from either
adenovirus or herpes virus for its own replication. In the absence
of helper functions from either of these helper viruses, AAV will
infect cells, uncoat in the nucleus, and integrate its genome into
the host chromosome, but will not replicate or produce new viral
particles.
[0174] The genome of AAV has been cloned into bacterial plasmids
and is well characterized. The viral genome consists of 4682 bases
which include two terminal repeats of 145 bases each. These
terminal repeats serve as origins of DNA replication for the virus.
Some investigators have also proposed that they have enhancer
functions. The rest of the genome is divided into two functional
domains. The left portion of the genome codes for the rep functions
which regulate viral DNA replication and vital gene expression. The
right side of the vital genome contains the cap genes that encode
the structural capsid proteins VP1, VP2 and VP3. The proteins
encoded by both the rep and cap genes function in trans during
productive AAV replication.
[0175] AAV is considered an ideal candidate for use as a
transducing vector, and it has been used in this manner. Such AAV
transducing vectors comprise sufficient cis-acting functions to
replicate in the presence of adenovirus or herpes virus helper
functions provided in trans. Recombinant AAV (rAAV) have been
constructed in a number of laboratories and have been used to carry
exogenous genes into cells of a variety of lineages. In these
vectors, the AAV cap and/or rep genes are deleted from the viral
genome and replaced with a DNA segment of choice. Current vectors
may accommodate up to 4300 bases of inserted DNA.
[0176] To produce rAAV, plasmids containing the desired vital
construct are transfected into adenovirus-infected cells. In
addition, a second helper plasmid is cotransfected into these cells
to provide the AAV rep and cap genes which are obligatory for
replication and packaging of the recombinant viral construct. Under
these conditions, the rep and cap proteins of AAV act in trans to
stimulate replication and packaging of the rAAV construct. Three
days after transfection, rAAV is harvested from the cells along
with adenovirus. The contaminating adenovirus is then inactivated
by heat treatment.
[0177] Herpes Simplex Virus 1 (HSV-1) is an enveloped,
double-stranded DNA virus with a genome of 153 kb encoding more
than 80 genes. Its wide host range is due to the binding of viral
envelope glycoproteins to the extracellular heparin sulphate
molecules found in cell membranes (WuDunn & Spear, 1989).
Internalization of the virus then requires envelope glycoprotein gD
and fibroblast growth factor receptor (Kaner, 1990). HSV is able to
infect cells lytically or may establish latency. HSV vectors have
been used to infect a wide variety of cell types (Lowenstein, 1994;
Huard, 1995; Miyanohara, 1992; Liu, 1996; Goya, 1998).
[0178] There are two types of HSV vectors, called the recombinant
HSV vectors and the amplicon vectors. Recombinant HSV vectors are
generated by the insertion of transcription units directly into the
HSV genome, through homologous recombination events. The amplicon
vectors are based on plasmids bearing the transcription unit of
choice, an origin of replication, and a packaging signal.
[0179] HSV vectors have the obvious advantages of a large capacity
for insertion of foreign genes, the capacity to establish latency
in neurons, a wide host range, and the ability to confer transgene
expression to the CNS for up to 18 months (Carpenter & Stevens,
1996).
[0180] Retroviruses are enveloped single-stranded RNA viruses,
which have been widely used in gene transfer protocols.
Retroviruses have a diploid genome of about 7-10 kb, composed of
four gene regions termed gag, pro, pol and env. These gene regions
encode for structural capsid proteins, viral protease, integrase
and viral reverse transcriptase, and envelope glycoproteins,
respectively. The genome also has a packaging signal and cis-acting
sequences, termed long-terminal repeats (LTRs), at each end, which
have a role in transcriptional control and integration.
[0181] The viral vectors of the present invention are useful for
the delivery of nucleic acids expressing antigens or immunogens to
cells both in vitro and in vivo. In particular, the inventive
vectors may be advantageously employed to deliver or transfer
nucleic acids to cells, more preferably mammalian cells. Nucleic
acids of interest include nucleic acids encoding peptides and
proteins, preferably therapeutic (e.g., for medical or veterinary
uses) or immunogenic (e.g., for vaccines) peptides or proteins.
[0182] Preferably, the codons encoding the antigen or immunogen of
interest are "optimized" codons, i.e., the codons are those that
appear frequently in, e.g., highly expressed genes in the subject's
species, instead of those codons that are frequently used by, for
example, an influenza virus. Such codon usage provides for
efficient expression of the antigen or immunogen in animal cells.
In other embodiments, for example, when the antigen or immunogen of
interest is expressed in bacteria, yeast or another expression
system, the codon usage pattern is altered to represent the codon
bias for highly expressed genes in the organism in which the
antigen or immunogen is being expressed. Codon usage patterns are
known in the literature for highly expressed genes of many species
(e.g., Nakamura et al., 1996; Wang et al., 1998; McEwan et al.
1998).
[0183] As a further alternative, the viral vectors may be used to
infect a cell in culture to express a desired gene product, e.g.,
to produce a protein or peptide of interest. Preferably, the
protein or peptide is secreted into the medium and may be purified
therefrom using routine techniques known in the art. Signal peptide
sequences that direct extracellular secretion of proteins are known
in the art and nucleotide sequences encoding the same may be
operably linked to the nucleotide sequence encoding the peptide or
protein of interest by routine techniques known in the art.
Alternatively, the cells may be lysed and the expressed recombinant
protein may be purified from the cell lysate. Preferably, the cell
is an animal cell, more preferably a mammalian cell. Also preferred
are cells that are competent for transduction by particular viral
vectors of interest. Such cells include PER.C6 cells, 911 cells,
and HEK293 cells.
[0184] A culture medium for culturing host cells includes a medium
commonly used for tissue culture, such as M199-earle base, Eagle
MEM (E-MEM), Dulbecco MEM (DMEM), SC-UCM102, UP-SFM (GIBCO BRL),
EX-CELL302 (Nichirei), EX-CELL293-S(Nichirei), TFBM-01 (Nichirei),
ASF104, among others. Suitable culture media for specific cell
types may be found at the American Type Culture Collection (ATCC)
or the European Collection of Cell Cultures (ECACC). Culture media
may be supplemented with amino acids such as L-glutamine, salts,
anti-fungal or anti-bacterial agents such as Fungizone.RTM.,
penicillin-streptomycin, animal serum, and the like. The cell
culture medium may optionally be serum-free.
[0185] The present invention also relates to cell lines or
transgenic animals which are capable of expressing or
overexpressing LITEs or at least one agent useful in the present
invention. Preferably the cell line or animal expresses or
overexpresses one or more LITEs.
[0186] The transgenic animal is typically a vertebrate, more
preferably a rodent, such as a rat or a mouse, but also includes
other mammals such as human, goat, pig or cow etc.
[0187] Such transgenic animals are useful as animal models of
disease and in screening assays for new useful compounds. By
specifically expressing one or more polypeptides, as defined above,
the effect of such polypeptides on the development of disease may
be studied. Furthermore, therapies including gene therapy and
various drugs may be tested on transgenic animals. Methods for the
production of transgenic animals are known in the art. For example,
there are several possible routes for the introduction of genes
into embryos. These include (i) direct transfection or retroviral
infection of embryonic stem cells followed by introduction of these
cells into an embryo at the blastocyst stage of development; (ii)
retroviral infection of early embryos; and (iii) direct
microinjection of DNA into zygotes or early embryo cells. The gene
and/or transgene may also include genetic regulatory elements
and/or structural elements known in the art. A type of target cell
for transgene introduction is the embryonic stem cell (ES). ES
cells may be obtained from pre-implantation embryos cultured in
vitro and fused with embryos (Evans et al., 1981, Nature
292:154-156; Bradley et al., 1984, Nature 309:255-258; Gossler et
al., 1986, Proc. Natl. Acad. Sci. USA 83:9065-9069; and Robertson
et al., 1986 Nature 322:445-448). Transgenes may be efficiently
introduced into the ES cells by a variety of standard techniques
such as DNA transfection, microinjection, or by retrovirus-mediated
transduction. The resultant transformed ES cells may thereafter be
combined with blastocysts from a non-human animal. The introduced
ES cells thereafter colonize the embryo and contribute to the germ
line of the resulting chimeric animal (Jaenisch, 1988, Science 240:
1468-1474).
[0188] LITEs may also offer valuable temporal precision in vivo.
LITEs may be used to alter gene expression during a particular
stage of development, for example, by repressing a particular
apoptosis gene only during a particular stage of C elegans growth.
LITEs may be used to time a genetic cue to a particular
experimental window. For example, genes implicated in learning may
be overexpressed or repressed only during the learning stimulus in
a precise region of the intact rodent or primate brain. Further,
LITEs may be used to induce gene expression changes only during
particular stages of disease development. For example, an oncogene
may be overexpressed only once a tumor reaches a particular size or
metastatic stage. Conversely, proteins suspected in the development
of Alzheimer's may be knocked down only at defined time points in
the animal's life and within a particular brain region. Although
these examples do not exhaustively list the potential applications
of the LITE system, they highlight some of the areas in which LITEs
may be a powerful technology.
[0189] Therapeutic or diagnostic compositions of the invention are
administered to an individual in amounts sufficient to treat or
diagnose disorders. The effective amount may vary according to a
variety of factors such as the individual's condition, weight, sex
and age. Other factors include the mode of administration.
[0190] The pharmaceutical compositions may be provided to the
individual by a variety of routes such as subcutaneous, topical,
oral and intramuscular.
[0191] Compounds identified according to the methods disclosed
herein may be used alone at appropriate dosages. Alternatively,
co-administration or sequential administration of other agents may
be desirable.
[0192] The present invention also has the objective of providing
suitable topical, oral, systemic and parenteral pharmaceutical
formulations for use in the novel methods of treatment of the
present invention. The compositions containing compounds identified
according to this invention as the active ingredient may be
administered in a wide variety of therapeutic dosage forms in
conventional vehicles for administration. For example, the
compounds may be administered in such oral dosage forms as tablets,
capsules (each including timed release and sustained release
formulations), pills, powders, granules, elixirs, tinctures,
solutions, suspensions, syrups and emulsions, or by injection.
Likewise, they may also be administered in intravenous (both bolus
and infusion), intraperitoneal, subcutaneous, topical with or
without occlusion, or intramuscular form, all using forms well
known to those of ordinary skill in the pharmaceutical arts.
[0193] Advantageously, compounds of the present invention may be
administered in a single daily dose, or the total daily dosage may
be administered in divided doses of two, three or four times daily.
Furthermore, compounds for the present invention may be
administered in intranasal form via topical use of suitable
intranasal vehicles, or via transdermal routes, using those forms
of transdermal skin patches well known to those of ordinary skill
in that art. To be administered in the form of a transdermal
delivery system, the dosage administration will, of course, be
continuous rather than intermittent throughout the dosage
regimen.
[0194] For combination treatment with more than one active agent,
where the active agents are in separate dosage formulations, the
active agents may be administered concurrently, or they each may be
administered at separately staggered times.
[0195] The dosage regimen utilizing the compounds of the present
invention is selected in accordance with a variety of factors
including type, species, age, weight, sex and medical condition of
the patient; the severity of the condition to be treated; the route
of administration; the renal, hepatic and cardiovascular function
of the one patient; and the particular compound thereof employed. A
physician of ordinary skill may readily determine and prescribe the
effective amount of the drug required to prevent, counter or arrest
the progress of the condition. Optimal precision in achieving
concentrations of drug within the range that yields efficacy
without toxicity requires a regimen based on the kinetics of the
drug's availability to target sites. This involves a consideration
of the distribution, equilibrium, and elimination of a drug.
[0196] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations may be made herein without departing
from the spirit and scope of the invention as defined in the
appended claims.
[0197] The present invention will be further illustrated in the
following Examples which are given for illustration purposes only
and are not intended to limit the invention in any way.
EXAMPLES
Example 1
[0198] The ability to directly modulate gene expression from the
endogenous mammalian genome is critical for elucidating normal gene
function and disease mechanism. Advances that further refine the
spatial and temporal control of gene expression within cell
populations have the potential to expand the utility of gene
modulation. Applicants previously developed transcription
activator-like effectors (TALEs) from Xanthamonas oryze to enable
the rapid design and construction of site-specific DNA binding
proteins. Applicants developed a set of molecular tools for
enabling light-regulated gene expression in the endogenous
mammalian genome. The system consists of engineered artificial
transcription factors linked to light-sensitive dimerizing protein
domains from Arabidopsis thaliana. The system responds to light in
the range of 450 nm-500 nm and is capable of inducing a significant
increase in the expression of pluripotency factors after
stimulation with light at an intensity of 6.2 mW/cm.sup.2 in
mammalian cells. Applicants are developing tools for the targeting
of a wide range of genes. Applicants believe that a toolbox for the
light-mediated control of gene expression would complement the
existing optogenetic methods and may in the future help elucidate
the timing-, cell type- and concentration dependent role of
specific genes in the brain.
[0199] The ability to directly modulate gene expression from the
endogenous mammalian genome is critical for elucidating normal gene
function and disease mechanisms. Applicants present the development
of a set of molecular tools for enabling light-regulated gene
expression in the endogenous mammalian genome. This system consists
of a transcription activator like effector (TALE) and the
activation domain VP64 linked to the light-sensitive dimerizing
protein domains cryptochrome 2 (CRY2) and CIB1 from Arabidopsis
thaliana. Applicants show that blue-light stimulation of HEK293FT
and Neuro-2a cells transfected with these LITE constructs designed
to target the promoter region of KLF4 and Neurog2 results in a
significant increase in target expression, demonstrating the
functionality of TALE-based optical gene expression modulation
technology.
[0200] FIG. 2 shows transcription activator like effectors (TALEs).
TALEs consist of 34 aa repeats (SEQ ID NO:1) at the core of their
sequence. Each repeat corresponds to a base in the target DNA that
is bound by the TALE. Repeats differ only by 2 variable amino acids
at positions 12 and 13. The code of this correspondence has been
elucidated (Boch, J et al., Science, 2009 and Moscou, M et al.,
Science, 2009) and is shown in this figure. One example of a
binding site is shown as SEQ ID NO: 2. Applicants developed a
method for the synthesis of designer TALEs incorporating this code
and capable of binding a sequence of choice within the genome
(Zhang, F et al., Nature Biotechnology, 2011).
[0201] FIG. 3 depicts a design of a LITE: TALE/Cryptochrome
transcriptional activation. Each LITE is a two-component system
which may comprise a TALE fused to CRY2 and the cryptochrome
binding partner CIB1 fused to VP64, a transcription activator. In
the inactive state, the TALE localizes its fused CRY2 domain to the
promoter region of the gene of interest. At this point, CIB1 is
unable to bind CRY2, leaving the CIB1-VP64 unbound in the nuclear
space. Upon stimulation with 488 nm (blue) light, CRY2 undergoes a
conformational change, revealing its CIB1 binding site (Liu, H et
al., Science, 2008). Rapid binding of CIB1 results in recruitment
of the fused VP64 domain, which induces transcription of the target
gene.
Example 2
[0202] Normal gene expression is a dynamic process with carefully
orchestrated temporal and spatial components, the precision of
which are necessary for normal development, homeostasis, and
advancement of the organism. In turn, the dysregulation of required
gene expression patterns, either by increased, decreased, or
altered function of a gene or set of genes, has been linked to a
wide array of pathologies. Technologies capable of modulating gene
expression in a spatiotemporally precise fashion will enable the
elucidation of the genetic cues responsible for normal biological
processes and disease mechanisms. To address this technological
need, Applicants developed light-inducible transcriptional
effectors (LITEs), which provide light-mediated control of
endogenous gene expression.
[0203] Inducible gene expression systems have typically been
designed to allow for chemically inducible activation of an
inserted open reading frame or shRNA sequence, resulting in gene
overexpression or repression, respectively. Disadvantages of using
open reading frames for overexpression include loss of splice
variation and limitation of gene size. Gene repression via RNA
interference, despite its transformative power in human biology,
may be hindered by complicated off-target effects. Certain
inducible systems including estrogen, ecdysone, and FKBP12/FRAP
based systems are known to activate off-target endogenous genes.
The potentially deleterious effects of long-term antibiotic
treatment may complicate the use of tetracycline transactivator
(TET) based systems. In vivo, the temporal precision of these
chemically inducible systems is dependent upon the kinetics of
inducing agent uptake and elimination. Further, because inducing
agents are generally delivered systemically, the spatial precision
of such systems is bounded by the precision of exogenous vector
delivery.
[0204] In response to these limitations, LITEs are designed to
modulate expression of individual endogenous genes in a temporally
and spatially precise manner. Each LITE is a two component system
consisting of a customized DNA-binding transcription activator like
effector (TALE) protein, a light-responsive cryptochrome
heterodimer from Arabadopsis thaliana, and a transcriptional
activation/repression domain. The TALE is designed to bind to the
promoter sequence of the gene of interest. The TALE protein is
fused to one half of the cryptochrome heterodimer (cryptochrome-2
or CIB1), while the remaining cryptochrome partner is fused to a
transcriptional effector domain. Effector domains may be either
activators, such as VP16, VP64, or p65, or repressors, such as
KRAB, EnR, or SID. In a LITE's unstimulated state, the
TALE-cryptochrome2 protein localizes to the promoter of the gene of
interest, but is not bound to the CIB1-effector protein. Upon
stimulation of a LITE with blue spectrum light, cryptochrome-2
becomes activated, undergoes a conformational change, and reveals
its binding domain. CIB1, in turn, binds to cryptochrome-2
resulting in localization of the effector domain to the promoter
region of the gene of interest and initiating gene overexpression
or silencing.
[0205] Gene targeting in a LITE is achieved via the specificity of
customized TALE DNA binding proteins. A target sequence in the
promoter region of the gene of interest is selected and a TALE
customized to this sequence is designed. The central portion of the
TALE consists of tandem repeats 34 amino acids in length. Although
the sequences of these repeats are nearly identical, the 12th and
13th amino acids (termed repeat variable diresidues) of each repeat
vary, determining the nucleotide-binding specificity of each
repeat. Thus, by synthesizing a construct with the appropriate
ordering of TALE monomer repeats, a DNA binding protein specific to
the target promoter sequence is created.
[0206] Light responsiveness of a LITE is achieved via the
activation and binding of cryptochrome-2 and CIB1. As mentioned
above, blue light stimulation induces an activating conformational
change in cryptochrome-2, resulting in recruitment of its binding
partner CIB1. This binding is fast and reversible, achieving
saturation in <15 sec following pulsed stimulation and returning
to baseline <15 min after the end of stimulation. These rapid
binding kinetics result in a LITE system temporally bound only by
the speed of transcription/translation and transcript/protein
degradation, rather than uptake and clearance of inducing agents.
Cryptochrome-2 activation is also highly sensitive, allowing for
the use of low light intensity stimulation and mitigating the risks
of phototoxicity. Further, in a context such as the intact
mammalian brain, variable light intensity may be used to control
the size of a LITE stimulated region, allowing for greater
precision than vector delivery alone may offer.
[0207] The modularity of the LITE system allows for any number of
effector domains to be employed for transcriptional modulation.
Thus, activator and repressor domains may be selected on the basis
of species, strength, mechanism, duration, size, or any number of
other parameters.
[0208] Applicants next present two prototypical manifestations of
the LITE system. The first example is a LITE designed to activate
transcription of the mouse gene NEUROG2. The sequence
TGAATGATGATAATACGA (SEQ ID NO:149), located in the upstream
promoter region of mouse NEUROG2, was selected as the target and a
TALE was designed and synthesized to match this sequence. The TALE
sequence was linked to the sequence for cryptochrome-2 via a
nuclear localization signal (amino acids: SPKKKRKVEAS; SEQ ID NO:
150) to facilitate transport of the protein from the cytosol to the
nuclear space. A second vector was synthesized comprising the CIB1
domain linked to the transcriptional activator domain VP64 using
the same nuclear localization signal. This second vector, also a
GFP sequence, is separated from the CIB1-VP64 fusion sequence by a
2A translational skip signal. Expression of each construct was
driven by a ubiquitous, constitutive promoter (CMV or EF1-.alpha.).
Mouse neuroblastoma cells from the Neuro 2A cell line were
co-transfected with the two vectors. After incubation to allow for
vector expression, samples were stimulated by periodic pulsed blue
light from an array of 488 nm LEDs. Unstimulated co-transfected
samples and samples transfected only with the fluorescent reporter
YFP were used as controls. At the end of each experiment, mRNA was
purified from the samples analyzed via qPCR.
[0209] Truncated versions of cryptochrome-2 and CIB1 were cloned
and tested in combination with the full-length versions of
cryptochrome-2 and CIB1 in order to determine the effectiveness of
each heterodimer pair. The combination of the CRY2PHR domain,
consisting of the conserved photoresponsive region of the
cryptochrome-2 protein, and the full-length version of CIB1
resulted in the highest upregulation of Neurog2 mRNA levels
(.about.22 fold over YFP samples and .about.7 fold over
unstimulated co-transfected samples). The combination of
full-length cryptochrome-2 (CRY2) with full-length CIB1 resulted in
a lower absolute activation level (.about.4.6 fold over YFP), but
also a lower baseline activation (.about.1.6 fold over YFP for
unstimulated co-transfected samples). These cryptochrome protein
pairings may be selected for particular uses depending on absolute
level of induction required and the necessity to minimize baseline
"leakiness" of the LITE system.
[0210] Speed of activation and reversibility are critical design
parameters for the LITE system. To characterize the kinetics of the
LITE system, constructs consisting of the Neurog2 TALE-CRY2PHR and
CIB1-VP64 version of the system were tested to determine its
activation and inactivation speed. Samples were stimulated for as
little as 0.5 h to as long as 24 h before extraction. Upregulation
of Neurog2 expression was observed at the shortest, 0.5 h, time
point (.about.5 fold vs YFP samples). Neurog2 expression peaked at
12 h of stimulation (.about.19 fold vs YFP samples). Inactivation
kinetics were analyzed by stimulating co-transfected samples for 6
h, at which time stimulation was stopped, and samples were kept in
culture for 0 to 12 h to allow for mRNA degradation. Neurog2 mRNA
levels peaked at 0.5 h after the end of stimulation (.about.16 fold
vs. YFP samples), after which the levels degraded with an .about.3
h half-life before returning to near baseline levels by 12 h.
[0211] The second prototypical example is a LITE designed to
activate transcription of the human gene KLF4. The sequence
TTCTTACTTATAAC (SEQ ID NO: 167), located in the upstream promoter
region of human KLF4, was selected as the target and a TALE was
designed and synthesized to match this sequence. The TALE sequence
was linked to the sequence for CRY2PHR via a nuclear localization
signal (amino acids: SPKKKRKVEAS; SEQ ID NO: 150). The identical
CIB1-VP64 activator protein described above was also used in this
manifestation of the LITE system. Human embryonal kidney cells from
the HEK293FT cell line were co-transfected with the two vectors.
After incubation to allow for vector expression, samples were
stimulated by periodic pulsed blue light from an array of 488 nm
LEDs. Unstimulated co-transfected samples and samples transfected
only with the fluorescent reporter YFP were used as controls. At
the end of each experiment, mRNA was purified from the samples
analyzed via qPCR.
[0212] The light-intensity response of the LITE system was tested
by stimulating samples with increased light power (0-9 mW/cm2).
Upregulation of KLF4 mRNA levels was observed for stimulation as
low as 0.2 mW/cm2. KLF4 upregulation became saturated at 5 mW/cm2
(2.3 fold vs. YFP samples). Cell viability tests were also
performed for powers up to 9 mW/cm2 and showed >98% cell
viability. Similarly, the KLF4 LITE response to varying duty cycles
of stimulation was tested (1.6-100%). No difference in KLF4
activation was observed between different duty cycles indicating
that a stimulation paradigm of as low as 0.25 sec every 15 sec
should result in maximal activation.
[0213] There are potential applications for which LITEs represent
an advantageous choice for gene expression control. There exist a
number of in vitro applications for which LITEs are particularly
attractive. In all these cases, LITEs have the advantage of
inducing endogenous gene expression with the potential for correct
splice variant expression.
[0214] Because LITE activation is photoinducible, spatially defined
light patterns, created via masking or rasterized laser scanning,
may be used to alter expression levels in a confined subset of
cells. For example, by overexpressing or silencing an intercellular
signaling molecule only in a spatially constrained set of cells,
the response of nearby cells relative to their distance from the
stimulation site may help elucidate the spatial characteristics of
cell non-autonomous processes. Additionally, recent advances in
cell reprogramming biology have shown that overexpression of sets
of transcription factors may be utilized to transform one cell
type, such as fibroblasts, into another cell type, such as neurons
or cardiomyocytes. Further, the correct spatial distribution of
cell types within tissues is critical for proper organotypic
function. Overexpression of reprogramming factors using LITEs may
be employed to reprogram multiple cell lineages in a spatially
precise manner for tissue engineering applications.
[0215] The rapid transcriptional response and endogenous targeting
of LITEs make for an ideal system for the study of transcriptional
dynamics. For example, LITEs may be used to study the dynamics of
mRNA splice variant production upon induced expression of a target
gene. On the other end of the transcription cycle, mRNA degradation
studies are often performed in response to a strong extracellular
stimulus, causing expression level changes in a plethora of genes.
LITEs may be utilized to reversibly induce transcription of an
endogenous target, after which point stimulation may be stopped and
the degradation kinetics of the unique target may be tracked.
[0216] The temporal precision of LITEs may provide the power to
time genetic regulation in concert with experimental interventions.
For example, targets with suspected involvement in long-term
potentiation (LTP) may be modulated in organotypic or dissociated
neuronal cultures, but only during stimulus to induce LTP, so as to
avoid interfering with the normal development of the cells.
Similarly, in cellular models exhibiting disease phenotypes,
targets suspected to be involved in the effectiveness of a
particular therapy may be modulated only during treatment.
Conversely, genetic targets may be modulated only during a
pathological stimulus. Any number of experiments in which timing of
genetic cues to external experimental stimuli is of relevance may
potentially benefit from the utility of LITE modulation.
[0217] The in vivo context offers equally rich opportunities for
the use of LITEs to control gene expression. As mentioned above,
photoinducibility provides the potential for previously
unachievable spatial precision. Taking advantage of the development
of optrode technology, a stimulating fiber optic lead may be placed
in a precise brain region. Stimulation region size may then be
tuned by light intensity. This may be done in conjunction with the
delivery of LITEs via viral vectors, or, if transgenic LITE animals
were to be made available, may eliminate the use of viruses while
still allowing for the modulation of gene expression in precise
brain regions. LITEs may be used in a transparent organism, such as
an immobilized zebrafish, to allow for extremely precise laser
induced local gene expression changes.
[0218] LITEs may also offer valuable temporal precision in vivo.
LITEs may be used to alter gene expression during a particular
stage of development, for example, by repressing a particular
apoptosis gene only during a particular stage of C elegans growth.
LITEs may be used to time a genetic cue to a particular
experimental window. For example, genes implicated in learning may
be overexpressed or repressed only during the learning stimulus in
a precise region of the intact rodent or primate brain. Further,
LITEs may be used to induce gene expression changes only during
particular stages of disease development. For example, an oncogene
may be overexpressed only once a tumor reaches a particular size or
metastatic stage. Conversely, proteins suspected in the development
of Alzheimer's may be knocked down only at defined time points in
the animal's life and within a particular brain region. Although
these examples do not exhaustively list the potential applications
of the LITE system, they highlight some of the areas in which LITEs
may be a powerful technology.
Example 3
Development of Mammalian TALE ToolBox
[0219] Customized TALEs may be used for a wide variety of genome
engineering applications, including transcriptional modulation and
genome editing. Here, Applicants describe a toolbox for rapid
construction of custom TALE transcription factors (TALE-TFs) and
nucleases (TALENs) using a hierarchical ligation procedure. This
toolbox facilitates affordable and rapid construction of custom
TALE-TFs and TALENs within 1 week and may be easily scaled up to
construct TALEs for multiple targets in parallel. Applicants also
provide details for testing the activity in mammalian cells of
custom TALE-TFs and TALENs using quantitative reverse-transcription
PCR and Surveyor nuclease, respectively. The TALE toolbox will
enable a broad range of biological applications.
[0220] TALEs are natural bacterial effector proteins used by
Xanthomonas sp. to modulate gene transcription in host plants to
facilitate bacterial colonization (7, 8). The central region of the
protein contains tandem repeats of 34-aa sequences (termed
monomers; e.g., SEQ ID NO: 1) that are required for DNA recognition
and binding (9, 10, 11, 12) (FIG. 8). Naturally occurring TALEs
have been found to have a variable number of monomers, ranging from
1.5 to 33.5 (7). Although the sequence of each monomer is highly
conserved, they differ primarily in two positions termed the repeat
variable diresidues (RVDs, 12th and 13th positions). Recent reports
have found that the identity of these two residues determines the
nucleotide-binding specificity of each TALE repeat and that a
simple cipher specifies the target base of each RVD (NI=A, HD=C,
NG=T, NN=G or A) (1, 2). Thus, each monomer targets one nucleotide
and the linear sequence of monomers in a TALE specifies the target
DNA sequence in the 5' to 3' orientation. The natural TALE-binding
sites within plant genomes always begin with a thymine (1, 2),
which is presumably specified by a cryptic signal within the
nonrepetitive N terminus of TALEs. The tandem repeat DNA-binding
domain always ends with a half-length repeat (0.5 repeat, FIG. 8).
Therefore, the length of the DNA sequence being targeted is equal
to the number of full repeat monomers plus two.
[0221] Applicants have further improved the TALE assembly system
with a few optimizations, including maximizing the dissimilarity of
ligation adaptors to minimize misligations and combining separate
digest and ligation steps into single Golden Gate (13, 14, 15)
reactions. Briefly, each nucleotide-specific monomer sequence is
amplified with ligation adaptors that uniquely specify the monomer
position within the TALE tandem repeats. Once this monomer library
is produced, it may conveniently be reused for the assembly of many
TALEs. For each TALE desired, the appropriate monomers are first
ligated into hexamers, which are then amplified via PCR. Then, a
second Golden Gate digestion-ligation with the appropriate TALE
cloning backbone (FIG. 8) yields a fully assembled,
sequence-specific TALE. The backbone contains a ccdB negative
selection cassette flanked by the TALE N and C termini, which is
replaced by the tandem repeat DNA-binding domain when the TALE has
been successfully constructed. ccdB selects against cells
transformed with an empty backbone, thereby yielding clones with
tandem repeats inserted (5).
[0222] Assemblies of monomeric DNA-binding domains may be inserted
into the appropriate TALE-TF or TALEN cloning backbones to
construct customized TALE-TFs and TALENs. TALE-TFs are constructed
by replacing the natural activation domain within the TALE C
terminus with the synthetic transcription activation domain VP64
(3; FIG. 8).
REFERENCES
[0223] 1. Boch, J. et al. Breaking the code of DNA binding
specificity of TAL-type III effectors. Science 326, 1509-1512
(2009). [0224] 2. Moscou, M. J. & Bogdanove, A. J. A simple
cipher governs DNA recognition by TAL effectors. Science 326, 1501
(2009). [0225] 3. Zhang, F. et al. Efficient construction of
sequence-specific TAL effectors for modulating mammalian
transcription. Nat. Biotechnol. 29, 149-153 (2011). [0226] 4.
Miller, J. C. et al. A TALE nuclease architecture for efficient
genome editing. Nat. Biotechnol. 29, 143-148 (2011). [0227] 5.
Cermak, T. et al. Efficient design and assembly of custom TALEN and
other TAL effector-based constructs for DNA targeting. Nucleic
Acids Res. 39, e82 (2011). [0228] 6. Hockemeyer, D. et al. Genetic
engineering of human pluripotent cells using TALE nucleases. Nat.
Biotechnol. 29, 731-734 (2011). [0229] 7. Boch, J. & Bonas, U.
Xanthomonas AvrBs3 family-type III effectors: discovery and
function. Annu. Rev. Phytopathol. 48, 419-436 (2010). [0230] 8.
Bogdanove, A. J., Schornack, S. & Lahaye, T. TAL effectors:
finding plant genes for disease and defense. Curr. Opin. Plant
Biol. 13, 394-401 (2010). [0231] 9. Romer, P. et al. Plant pathogen
recognition mediated by promoter activation of the pepper Bs3
resistance gene. Science 318, 645-648 (2007). [0232] 10. Kay, S.,
Hahn, S., Marois, E., Hause, G. & Bonas, U. A bacterial
effector acts as a plant transcription factor and induces a cell
size regulator. Science 318, 648-651 (2007). [0233] 11. Kay, S.,
Hahn, S., Marois, E., Wieduwild, R. & Bonas, U. Detailed
analysis of the DNA recognition motifs of the Xanthomonas type III
effectors AvrBs3 and AvrBs3Deltarep16. Plant J. 59, 859-871 (2009).
[0234] 12. Romer, P. et al. Recognition of AvrBs3-like proteins is
mediated by specific binding to promoters of matching pepper Bs3
alleles. Plant Physiol. 150, 1697-1712 (2009). [0235] 13. Engler,
C., Kandzia, R. & Marillonnet, S. A one pot, one step,
precision cloning method with high throughput capability. PLoS ONE
3, e3647 (2008). [0236] 14. Engler, C., Gruetzner, R., Kandzia, R.
& Marillonnet, S. Golden gate shuffling: a one-pot DNA
shuffling method based on type IIs restriction enzymes. PLoS ONE 4,
e5553 (2009). [0237] 15. Weber, E., Engler, C., Gruetzner, R.,
Werner, S. & Marillonnet, S. A modular cloning system for
standardized assembly of multigene constructs. PLoS ONE 6, e16765
(2011). [0238] 16. Huertas, P. DNA resection in eukaryotes:
deciding how to fix the break. Nat. Struct. Mol. Biol. 17, 11-16
(2010).
Example 4
[0239] FIG. 17 depicts an effect of cryptochrome2 heterodimer
orientation on LITE functionality. Two versions of the Neurogenin 2
(Neurog2) LITE were synthesized to investigate the effects of
cryptochrome 2 photolyase homology region (CRY2PHR)/calcium and
integrin-binding protein 1 (CIB1) dimer orientation. In one
version, the CIB1 domain was fused to the C-terminus of the TALE
(Neurog2) domain, while the CRY2PHR domain was fused to the
N-terminus of the VP64 domain. In the converse version, the CRY2PHR
domain was fused to the C-terminus of the TALE (Neurog2) domain,
while the CIB1 domain was fused to the N-terminus of the VP64
domain. Each set of plasmids were transfected in Neuro2a cells and
stimulated (466 nm, 5 mW/cm.sup.2, 1 sec pulse per 15 sec, 12 h)
before harvesting for qPCR analysis. Stimulated LITE and
unstimulated LITE Neurog2 expression levels were normalized to
Neurog2 levels from stimulated GFP control samples. The
TALE-CRY2PHR/CIB1-VP64 LITE exhibited elevated basal activity and
higher light induced Neurog2 expression, and suggested its
suitability for situations in which higher absolute activation is
required. Although the relative light inducible activity of the
TALE-CIB1/CRY2PHR-VP64 LITE was lower that its counterpart, the
lower basal activity suggested its utility in applications
requiring minimal baseline activation. Further, the TALE-CIB1
construct was smaller in size, compared to the TALE-CRY2PHR
construct, a potential advantage for applications such as viral
packaging.
[0240] FIG. 18 depicts metabotropic glutamate receptor 2 (mGlur2)
LITE activity in mouse cortical neuron culture. A mGluR2 targeting
LITE was constructed via the plasmids pAAV-human Synapsin I
promoter (hSyn)-HA-TALE(mGluR2)-CIB1 and
pAAV-hSyn-CRY2PHR-VP64-2A-GFP. These fusion constructs were then
packaged into adeno associated viral vectors (AAV). Additionally,
AAV carrying hSyn-TALE-VP64-2A-GFP and GFP only were produced.
Embryonic mouse (E16) cortical cultures were plated on
Poly-L-lysine coated 24 well plates. After 5 days in vitro neural
cultures were co-transduced with a mixture of TALE(mGluR2)-CIB1 and
CRY2PHR-VP64 AAV stocks. Control samples were transduced with
either TALE(mGluR2)-VP64 AAV or GFP AAV. 6 days after AAV
transduction, experimental samples were stimulated using either of
two light pulsing paradigms: 0.5 s per min and 0.25 sec per 30 sec.
Neurons were stimulated for 24 h and harvested for qPCR analysis.
All mGluR2 expression levels were normalized to the respective
stimulated GFP control. The data suggested that the LITE system
could be used to induce the light-dependent activation of a target
gene in primary neuron cultures in vitro.
[0241] FIG. 19 depicts transduction of primary mouse neurons with
LITE AAV vectors. Primary mouse cortical neuron cultures were
co-transduced at 5 days in vitro with AAV vectors encoding
hSyn-CRY2PHR-VP64-2A-GFP and hSyn-HA-TALE-CIB1, the two components
of the LITE system. Left panel: at 6 days after transduction,
neural cultures exhibited high expression of GFP from the
hSyn-CRY2PHR-VP64-2A-GFP vector. Right panel: Co-transduced neuron
cultures were fixed and stained with an antibody specific to the HA
epitope on the N-terminus of the TALE domain in hSyn-HA-TALE-CIB1.
Red signal indicated HA expression, with particularly strong
nuclear signal (DNA stained by DAPI in blue channel). Together
these images suggested that the expression of each LITE component
could be achieved in primary mouse neuron cultures. (scale bars=50
um).
[0242] FIG. 20 depicts expression of a LITE component in vivo. An
AAV vector of serotype 1/2 carrying hSyn-CRY2PHR-VP64 was produced
via transfection of HEK293FT cells and purified via heparin column
binding. The vector was concentrated for injection into the intact
mouse brain. 1 uL of purified AAV stock was injected into the
hippocampus and infralimbic cortex of an 8 week old male C57BL/6
mouse by steroeotaxic surgery and injection. 7 days after in vivo
transduction, the mouse was euthanized and the brain tissue was
fixed by paraformaldehyde perfusion. Slices of the brain were
prepared on a vibratome and mounted for imaging. Strong and
widespread GFP signals in the hippocampus and infralimbic cortex
suggested efficient transduction and high expression of the LITE
component CRY2PHR-VP64.
Example 5
Multiplex Genome Engineering Using CRISPR/Cas Systems
[0243] Functional elucidation of causal genetic variants and
elements requires precise genome editing technologies. The type II
prokaryotic CRISPR (clustered regularly interspaced short
palindromic repeats) adaptive immune system has been shown to
facilitate RNA-guided site-specific DNA cleavage. Applicants
engineered two different type II CRISPR systems and demonstrate
that Cas9 nucleases can be directed by short RNAs to induce precise
cleavage at endogenous genomic loci in human and mouse cells. Cas9
can also be converted into a nicking enzyme to facilitate
homology-directed repair with minimal mutagenic activity. Finally,
multiple guide sequences can be encoded into a single CRISPR array
to enable simultaneous editing of several sites within the
mammalian genome, demonstrating easy programmability and wide
applicability of the CRISPR technology.
[0244] Prokaryotic CRISPR adaptive immune systems can be
reconstituted and engineered to mediate multiplex genome editing in
eukaryote cells, advantageously mammalian cells.
[0245] Precise and efficient genome targeting technologies are
needed to enable systematic reverse engineering of causal genetic
variations by allowing selective perturbation of individual genetic
elements. Although genome-editing technologies such as designer
zinc fingers (ZFs) (1-4), transcription activator-like effectors
(TALEs) (4-10), and homing meganucleases (11) have begun to enable
targeted genome modifications, there remains a need for new
technologies that are scalable, affordable, and easy to engineer.
Here, Applicants report the development of a new class of precision
genome engineering tools based on the RNA-guided Cas9 nuclease
(12-14) from the type II prokaryotic CRISPR adaptive immune system
(15-18).
[0246] The Streptococcus pyogenes SF370 type II CRISPR locus
consists of four genes, including the Cas9 nuclease, as well as two
non-coding RNAs: tracrRNA and a pre-crRNA array containing nuclease
guide sequences (spacers) interspaced by identical direct repeats
(DRs) (FIG. 27) (19). Applicants sought to harness this prokaryotic
RNA-programmable nuclease system to introduce targeted double
stranded breaks (DSBs) in mammalian chromosomes through
heterologous expression of the key components. It has been
previously shown that expression of tracrRNA, pre-crRNA, host
factor RNase III, and Cas9 nuclease are necessary and sufficient
for cleavage of DNA in vitro (12, 13) and in prokaryotic cells (20,
21). Applicants codon optimized the S. pyogenes Cas9 (SpCas9) and
RNase III (SpRNase III) and attached nuclear localization signals
(NLS) to ensure nuclear compartmentalization in mammalian cells.
Expression of these constructs in human 293FT cells revealed that
two NLSs are required for targeting SpCas9 to the nucleus (FIG.
23A). To reconstitute the non-coding RNA components of CRISPR,
Applicants expressed an 89-nucleotide (nt) tracrRNA (FIG. 28) under
the RNA polymerase III U6 promoter (FIG. 23B). Similarly,
Applicants used the U6 promoter to drive the expression of a
pre-crRNA array comprising a single guide spacer flanked by DRs
(FIG. 23B). Applicants designed an initial spacer to target a
30-basepair (bp) site (protospacer) in the human EMX1 locus that
precedes an NGG, the requisite protospacer adjacent motif (PAM)
(FIG. 23C and FIG. 27) (22, 23).
[0247] To test whether heterologous expression of the CRISPR system
(SpCas9, SpRNase III, tracrRNA, and pre-crRNA) can achieve targeted
cleavage of mammalian chromosomes, Applicants transfected 293FT
cells with different combinations of CRISPR components. Since DSBs
in mammalian DNA are partially repaired by the indel-forming
non-homologous end joining (NHEJ) pathway, Applicants used the
SURVEYOR assay to detect endogenous target cleavage (FIG. 23D).
Co-transfection of all four required CRISPR components resulted in
efficient cleavage of the protospacer (FIG. 23D), which is
subsequently verified by Sanger sequencing (FIG. 23E). Removing any
of the remaining RNA or Cas9 components abolished the genome
cleavage activity of the CRISPR system (FIG. 23D). These results
define a minimal three-component system for efficient
CRISPR-mediated genome modification in mammalian cells.
Example 6
Optical Control of Endogenous Mammalian Transcription
[0248] The ability to directly modulate transcription of the
endogenous mammalian genome is critical for elucidating normal gene
function and disease mechanisms. Here, Applicants describe the
development of Light-Inducible Transcriptional Effectors (LITEs), a
two-component system integrating the customizable TALE DNA-binding
domain with the light-sensitive cryptochrome 2 protein and its
interacting partner CIB1 from Arabidopsis thaliana. LITEs can be
engineered and delivered to mediate positive and negative
regulation of endogenous mammalian gene expression in a reversible
manner, and changes in mRNA levels occur within minutes after
optical illumination. Applicants have applied this system in cell
lines, primary mouse neurons, as well as in the brain of awake,
behaving mice in vivo.
[0249] An ideal optogenetic approach for controlling endogenous
gene transcription would be readily generalizable to target any
gene locus, would not require manipulation of the endogenous
genomic sequence, would not depend on the addition of exogenous
chemical co-factors, and would exhibit fast and reversible
kinetics. The DNA-binding domain of transcription activator-like
effectors (TALEs) (13, 14) from Xanthomonas sp. can be easily
customized to bind specific DNA sequences in mammalian cells
(15-17). TALE DNA-binding domains are modular and can be fused with
a variety of effector domains, including nucleases, transcriptional
activators, and transcriptional repressors to edit or modulate
endogenous mammalian genomic loci (15-18). Applicants sought to
combine TALEs with light-sensitive proteins to create a suite of
tools for enabling spatiotemporally precise control of endogenous
gene transcription.
[0250] Here, Applicants report the development of Light-Inducible
Transcriptional Effectors (LITEs), a two-component system
integrating the customizable TALE DNA-binding domain with the
light-sensitive cryptochrome 2 protein and its interacting partner
CIB1 from Arabidopsis thaliana (8, 19). LITEs can be engineered to
mediate positive and negative regulation of endogenous mammalian
gene expression in a reversible manner, and changes in transcript
levels occur within minutes after stimulation. Like other
optogenetic tools, LITEs can be packaged into viral vectors and
genetically targeted to probe gene function within specific cell
populations. Applicants demonstrate the application of this system
in primary neurons as well as in the mouse brain in vivo.
[0251] In the design of the LITE system, Applicants sought to use
light-inducible heterodimeric proteins to mediate the recruitment
of transcriptional effector domains to a TALE targeted to an
endogenous genomic locus. While several plant-based light-sensitive
proteins have been developed for mammalian applications, some
suffer from slow or irreversible kinetics while others depend on
the supplementation of exogenous co-factors that are not present in
mammalian cells (5, 6, 9). The Arabidopsis thaliana cryptochrome 2
(CRY2) was previously shown to employ flavin adenine
dinucleotide--an abundant biomolecule in mammalian cells--as its
light-sensing chromophore.sup.19. The flavin chromophore is reduced
upon photoexcitation with blue light (peak .about.450 nm),
triggering a conformational change in CRY2 that allows dimerization
with its interacting protein partner CIB1.sup.19. The dimerization
between CRY2 and CIB1 occurs within seconds and is reversible
within a few minutes following withdrawal of light
illumination.sup.8. Based on these properties, Applicants selected
CRY2 and CIB1 as light-sensing components for constructing
LITEs.
[0252] Manipulating endogenous gene expression presents various
challenges, as the rate of expression depends on many factors,
including regulatory elements, mRNA processing, and transcript
stability (22, 23). Applicants sought to investigate the
feasibility of using the system to modulate endogenous gene
expression in primary neurons and the intact brain. To this end,
Applicants pursued viral transduction as an effective method for
TALE and LITE gene delivery into neurons. However, lentiviral
delivery can compromise TALE integrity due to recombination of the
tandem repeat DNA-binding domains during reverse transcription
(26). To overcome this challenge, Applicants developed an
adeno-associated virus (AAV)-based vector for the delivery of TALE
genes and efficient process for AAV production (FIGS. 37A-B, FIG.
42, and Example 7). AAV has an ssDNA-based genome and is therefore
less susceptible to recombination (27-29).
[0253] AAV1/2 (serotype AAV1/2, i.e., hybrid or mosaic AAV1/AAV2
capsid AAV) heparin purified concentrated virus protocol
[0254] Media: D10+HEPES
500 ml bottle DMEM high glucose+Glutamax (GIBCO) 50 ml Hyclone FBS
(heat-inactivated) (Thermo Fischer) 5.5 ml HEPES solution (1M,
GIBCO) Cells: low passage HEK293FT (passage <10 at time of virus
production, thaw new cells of passage 2-4 for virus production,
grow up for 3-5 passages)
[0255] Transfection Reagent: Polyethylenimine (PEI) "Max"
Dissolve 50 mg PEI "Max" in 50 ml sterile Ultrapure H.sub.2O
Adjust pH to 7.1
[0256] Filter with 0.22 um fliptop filter Seal tube and wrap with
parafilm Freeze aliquots at -20.degree. C. (for storage, can also
be used immediately)
[0257] Cell Culture
Culture low passage HEK293FT in D10+HEPES Passage everyday between
1:2 and 1:2.5 Advantageously do not allow cells to reach more than
85% confluency
[0258] For T75
[0259] Warm 10 ml HBSS (--Mg2+, --Ca2+, GIBCO)+1 ml TrypLE Express
(GIBCO) per flask to 37.degree. C. (Waterbath)
[0260] Aspirate media fully
[0261] Add 10 ml warm HBSS gently (to wash out media
completely)
[0262] Add 1 ml TrypLE per Flask
[0263] Place flask in incubator (37.degree. C.) for 1 min
[0264] Rock flask to detach cells
[0265] Add 9 ml D10+HEPES media (37.degree. C.)
[0266] Pipette up and down 5 times to generate single cell
suspension
[0267] Split at 1:2-1:2.5 (12 ml media for T75) ratio (if cells are
growing more slowly, discard and thaw a new batch, they are not in
optimal growth)
[0268] transfer to T225 as soon as enough cells are present (for
ease of handling large amounts of cells)
[0269] AAV Production (5*15 cm Dish Scale Per Construct):
Plate 10 million cells in 21.5 ml media into a 15 cm dish Incubate
for 18-22 hours at 37.degree. C. Transfection is ideal at 80%
confluence
[0270] Per Plate
Prewarm 22 ml media (D10+HEPES) Prepare Tube with DNA Mixture (Use
Endofree Maxiprep DNA): 5.2 ug vector of interest plasmid 4.35 ug
AAV 1 serotype plasmid 4.35 ug AAV 2 serotype plasmid 10.4 ug pDF6
plasmid (adenovirus helper genes)
.fwdarw.Vortex to mix
[0271] Add 434 uL DMEM (no serum!) Add 130 ul PEI solution Vortex
5-10 seconds Add DNA/DMEM/PEI mixture to prewarmed media
.fwdarw.Vortex briefly to mix Replace media in 15 cm dish with
DNA/DMEM/PEI mixture .fwdarw.Return to 37.degree. C. incubator
.fwdarw.Incubate 48 h before harvesting (make sure medium isn't
turning too acidic)
[0272] Virus Harvest:
1. aspirate media carefully from 15 cm dish dishes (advantageously
do not dislodge cells) 2. Add 25 ml RT DPBS (Invitrogen) to each
plate and gently remove cells with a cell scraper. Collect
suspension in 50 ml tubes. 3. Pellet cells at 800.times.g for 10
minutes. 4. Discard supernatant .fwdarw.pause point: freeze cell
pellet at -80 C if desired 5. resuspend pellet in 150 mM NaCl, 20
mM Tris pH 8.0, use 10 ml per tissue culture plate. 6. Prepare a
fresh solution of 10% sodium deoxycholate in dH2O. Add 1.25 ml of
this per tissue culture plate for a final concentration of 0.5%.
Add benzonase nuclease to a final concentration of 50 units per ml.
Mix tube thoroughly. 7. Incubate at 37.degree. C. for 1 hour
(Waterbath). 8. Remove cellular debris by centrifuging at
3000.times.g for 15 mins. Transfer to fresh 50 ml tube and ensure
all cell debris has been removed to prevent blocking of heparin
columns.
[0273] Heparin Column Purification of AAV1/2:
1. Set up HiTrap heparin columns using a peristaltic pump so that
solutions flow through the column at 1 ml per minute. It is
important to ensure no air bubbles are introduced into the heparin
column. 2. Equilibrate the column with 10 ml 150 mM NaCl, 20 mM
Tris, pH 8.0 using the peristaltic pump. 3. Binding of virus: Apply
50 ml virus solution to column and allow to flow through. 4. Wash
step 1: column with 20 ml 100 mM NaCl, 20 mM Tris, pH 8.0. (using
the peristaltic pump) 5. Wash step 2: Using a 3 ml or 5 ml syringe
continue to wash the column with 1 ml 200 mM NaCl, 20 mM Tris, pH
8.0, followed by 1 ml 300 mM NaCl, 20 mM Tris, pH 8.0.
.fwdarw.Discard the flow-through. (prepare the syringes with
different buffers during the 50 min flow through of virus solution
above) 6. Elution Using 5 ml syringes and gentle pressure (flow
rate of <1 ml/min) elute the virus from the column by
applying:
1.5 ml 400 mM NaCl, 20 mM Tris, pH 8.0
3.0 ml 450 mM NaCl, 20 mM Tris, pH 8.0
1.5 ml 500 mM NaCl, 20 mM Tris, pH 8.0
[0274] Collect these in a 15 ml centrifuge tube.
[0275] Concentration of AAV1/2:
1. Concentration step 1: Concentrate the eluted virus using Amicon
ultra 15 ml centrifugal filter units with a 100,000 molecular
weight cutoff. Load column eluate into the concentrator and
centrifuge at 2000.times.g for 2 minutes (at room temperature.
Check concentrated volume--it should be approximately 500 .mu.l. If
necessary, centrifuge in 1 min intervals until correct volume is
reached. 2. buffer exchange: Add 1 ml sterile DPBS to filter unit,
centrifuge in 1 min intervals until correct volume (500 ul) is
reached. 3. Concentration step 2: Add 500 ul concentrate to an
Amicon Ultra 0.5 ml 100K filter unit. Centrifuge at 6000 g for 2
min. Check concentrated volume--it should be approximately 100
.mu.l. If necessary, centrifuge in 1 min intervals until correct
volume is reached. 4. Recovery: Invert filter insert and insert
into fresh collection tube. Centrifuge at 1000 g for 2 min.
.fwdarw.Aliquot and freeze at -80.degree. C. .fwdarw.1 ul is
typically required per injection site, small aliquots (e.g. 5 ul)
are therefore recommended (avoid freeze-thaw of virus).
.fwdarw.determine DNaseI-resistant GC particle titer using qPCR
(see separate protocol)
[0276] Materials
Amicon Ultra, 0.5 ml, 100K; MILLIPORE; UFC510024
Amicon Ultra, 15 ml, 100K; MILLIPORE; UFC910024
[0277] Benzonase nuclease; Sigma-Aldrich, E1014 HiTrap Heparin
cartridge; Sigma-Aldrich; 54836 Sodium deoxycholate; Sigma-Aldrich;
D5670
[0278] AAV1 Supernatant Production Protocol
Media: D10+HEPES
[0279] 500 ml bottle DMEM high glucose+Glutamax (Invitrogen) 50 ml
Hyclone FBS (heat-inactivated) (Thermo Fischer) 5.5 ml HEPES
solution (1M, GIBCO)
[0280] Cells: low passage HEK293FT (passage <10 at time of virus
production)
Thaw new cells of passage 2-4 for virus production, grow up for 2-5
passages Transfection reagent: Polyethylenimine (PEI) "Max"
Dissolve 50 mg PEI "Max" in 50 ml sterile Ultrapure H.sub.2O
Adjust pH to 7.1
[0281] Filter with 0.22 um fliptop filter Seal tube and wrap with
parafilm Freeze aliquots at -20.degree. C. (for storage, can also
be used immediately)
[0282] Cell Culture
Culture low passage HEK293FT in D10+HEPES Passage everyday between
1:2 and 1:2.5 Advantageously do let cells reach more than 85%
confluency
For T75
[0283] Warm 10 ml HBSS (--Mg2+, --Ca2+, GIBCO)+1 ml TrypLE Express
(GIBCO) per flask to 37.degree. C. (Waterbath)
[0284] Aspirate media fully
[0285] Add 10 ml warm HBSS gently (to wash out media
completely)
[0286] Add 1 ml TrypLE per Flask
[0287] Place flask in incubator (37.degree. C.) for 1 min
[0288] Rock flask to detach cells
[0289] Add 9 ml D10+HEPES media (37.degree. C.)
[0290] Pipette up and down 5 times to generate single cell
suspension
[0291] Split at 1:2-1:2.5 (12 ml media for T75) ratio (if cells are
growing more slowly, discard and thaw a new batch, they are not in
optimal growth)
[0292] transfer to T225 as soon as enough cells are present (for
ease of handling large amounts of cells)
[0293] AAV Production (Single 15 cm Dish Scale)
[0294] Plate 10 million cells in 21.5 ml media into a 15 cm
dish
[0295] Incubate for 18-22 hours at 37.degree. C.
[0296] Transfection is ideal at 80% confluence per plate
[0297] Prewarm 22 ml media (D10+HEPES)
[0298] Prepare tube with DNA mixture (use endofree maxiprep DNA):
[0299] 5.2 ug vector of interest plasmid [0300] 8.7 ug AAV 1
serotype plasmid [0301] 10.4 ug DF6 plasmid (adenovirus helper
genes)
[0302] Vortex to mix
[0303] Add 434 uL DMEM (no serum!)
[0304] Add 130 ul PEI solution
[0305] Vortex 5-10 seconds
[0306] Add DNA/DMEM/PEI mixture to prewarmed media
[0307] Vortex briefly to mix
[0308] Replace media in 15 cm dish with DNA/DMEM/PEI mixture
[0309] Return to 37.degree. C. incubator
[0310] Incubate 48 h before harvesting (advantageously monitor to
ensure medium is not turning too acidic)
[0311] Virus Harvest:
[0312] Remove supernatant from 15 cm dish
[0313] Filter with 0.45 um filter (low protein binding) Aliquot and
freeze at -80.degree. C.
[0314] Transduction (primary neuron cultures in 24-well format,
5DIV)
[0315] Replace complete neurobasal media in each well of neurons to
be transduced with fresh neurobasal (usually 400 ul out of 500 ul
per well is replaced)
[0316] Thaw AAV supernatant in 37.degree. C. waterbath
[0317] Let equilibrate in incubator for 30 min
[0318] Add 250 ul AAV supernatant to each well
[0319] Incubate 24 h at 37.degree. C.
[0320] Remove media/supernatant and replace with fresh complete
neurobasal
[0321] Expression starts to be visible after 48 h, saturates around
6-7 Days Post Infection
[0322] Constructs for pAAV plasmid with GOI should not exceed 4.8
kb including both ITRS
[0323] AAV Supernatant Production
[0324] HEK 293FT cells (Life Technologies) were grown in
antibiotic-free D10 media (DMEM high glucose with GlutaMax and
Sodium Pyruvate, 10% heat-inactivated Hyclone FBS, and 1% 1M HEPES)
and passaged daily at 1:2-2.5. The total number of passages was
kept below 10 and cells were never grown beyond 85% confluence. The
day before transfection, 1.times.10.sup.6 cells in 21.5 mL of D10
media were plated onto 15 cm dishes and incubated for 18-22 hours
or until .about.80% confluence. For use as a transfection reagent,
1 mg/mL of PEI "Max" (Polysciences) was dissolved in water and the
pH of the solution was adjusted to 7.1. For AAV production, 10.4
.mu.g of pDF6 helper plasmid, 8.7 .mu.g of pAAV1 serotype packaging
vector, and 5.2 .mu.g of pAAV vector carrying the gene of interest
were added to 434 .mu.L of serum-free DMEM and 1304, of PEI "Max"
solution was added to the DMEM-diluted DNA mixture. The
DNA/DMEM/PEI cocktail was vortexed and incubated at room
temperature for 15 min. After incubation, the transfection mixture
was added to 22 mL of complete media, vortexed briefly, and used to
replace the media for a 15 cm dish of 293FT cells. For supernatant
production, transfection supernatant was harvested at 48 hours,
filtered through a 0.45 micron PVDF filter (Millipore), distributed
into aliquots, and frozen for storage at -80.degree. C.
[0325] To test the efficacy of AAV-mediated TALE delivery for
modulating transcription in primary mouse cortical neurons,
Applicants constructed six TALE-DNA binding domains targeting the
genetic loci of three mouse neurotransmitter receptors: Grm5,
Grin2a, and Grm2, which encode mGluR5, NMDA subunit 2A and mGluR2,
respectively (FIG. 37C). To increase the likelihood of a target
site accessibility, Applicants used mouse cortex DNase I
sensitivity data from the UCSC genome browser to identify putative
open chromatin regions. DNase I sensitive regions in the promoter
of each target gene provided a guide for the selection of TALE
binding sequences (FIG. 43). For each TALE, Applicants employed
VP64 as a transcriptional activator or a quadruple tandem repeat of
the mSin3 interaction domain (SID) (20, 30) as a repressor.
Applicants have previously shown that a single SID fused to TALE
downregulated a target gene effectively in 293FT cells (18). Hoping
to further improve this TALE repressor, Applicants reasoned that
four repeats of SID--analogous to the successful quadruple VP16
repeat architecture of VP64 (20)--might augment its repressive
activity. This was indeed the case, as TALE-SID4X constructs
enhanced repression .about.2-fold over TALE-SID in 293FT cells
(FIG. 44).
[0326] Applicants found that four out of six TALE-VP64 constructs
(T1, T2, T5 and T6) efficiently activated their target genes Grm5
and Grm2 in AAV-transduced primary neurons by up to 3- and 8-fold,
respectively (FIG. 37C). Similarly, four out of six TALE-SID4X
repressors (T9, T10, T11, T12) reduced the expression of their
endogenous targets Grin2a and Grm2 by up to 2- and 8-fold,
respectively (FIG. 37C). Together, these results indicate that
constitutive TALEs can positively or negatively modulate endogenous
target gene expression in neurons. Notably, efficient activation or
repression by a given TALE did not predict its efficiency at
transcriptional modulation in the opposite direction. Therefore,
multiple TALEs may need to be screened to identify the most
effective TALE for a particular locus.
[0327] As a confirmation of TALE expression and activity in vivo,
Applicants performed stereotactic injection of concentrated AAV
vectors into the mouse prefrontal cortex. Delivery of constitutive
TALE-VP64 AAV vectors resulted in robust TALE expression in the
mouse prefrontal cortex (FIG. 37D-E). Tissue punches from the
AAV-transduced brain regions showed that a TALE-VP64 targeting the
Grm2 gene locus is able to activate mRNA levels by up to 2.5-fold
(FIG. 37F).
[0328] In order to deliver LITEs into neurons using AAV, Applicants
had to ensure that the total viral genome size, with the LITE
transgenes included, did not exceed 4.8 kb.sup.31,32. To that end,
Applicants shortened the TALE N- and C-termini (keeping 136 aa in
the N-terminus and 63 aa in the C-terminus) and exchanged the
CRY2PHR and CIB1 domains (TALE-CIB1 and CRY2PHR-VP64; FIG. 38A).
This switch allowed each component of LITE to fit into AAV vectors
and did not reduce the efficacy of light-mediated transcription
modulation (FIG. 45). These LITEs can be efficiently delivered into
primary cortical neurons via co-transduction by a combination of
two AAV vectors (FIG. 38B; delivery efficiencies of 83-92% for
individual components with >80% co-transduction efficiency).
[0329] When implementing a neuron specific light-stimulation
protocol, cultured neurons proved to be much more sensitive to blue
light than Neuro-2a cells. Stimulation parameters that Applicants
previously optimized for Neuro 2a cells (466 nm, 5 mW/cm.sup.2
intensity, 7% duty cycle with 1 s light pulse at 0.067 Hz for a
total of 24 h) caused >50% toxicity in primary neurons.
Applicants therefore tested survival with a lower duty cycle, as
Applicants had previously observed that a wide range of duty cycles
had little effect on LITE-mediated transcriptional activation (FIG.
40).
[0330] For a neuronal application of LITEs, Applicants selected the
Grm2 TALE (T6), which exhibited the strongest level of target
upregulation in primary neurons, based on Applicants' comparison of
6 constitutive TALE activators (FIG. 37C). Applicants investigated
its function using 2 light pulsing frequencies with the same duty
cycle of 0.8%. Both stimulation conditions achieved a .about.7-fold
light-dependent increase in Grm2 mRNA levels (FIG. 38C). Further
study confirmed that, significant target gene expression increases
could be attained quickly (4-fold upregulation within 4 h; FIG.
38D). In addition, Applicants observed significant upregulation of
mGluR2 protein after stimulation, demonstrating that changes
effected by LITEs at the mRNA level are translated to the protein
domain (FIG. 38E). Taken together, these results confirm that LITEs
enable temporally precise optical control of endogenous gene
expression in neurons.
[0331] As a compliment to Applicants' previously implemented LITE
activators, Applicants next engineered a LITE repressor based on
the TALE-SID4X constructs. Constitutive Grm2 TALEs (T11 and T12,
FIG. 38F) mediated the highest level of transcription repression,
and were chosen as LITE repressors (FIG. 38F-G). Both light-induced
repressors mediated significant downregulation of Grm2 expression,
with 1.95-fold and 1.75-fold reductions for T11 and T12,
respectively, demonstrating the feasibility of optically controlled
repression in neurons (FIG. 38G).
[0332] Light-mediated control of gene expression would be
particularly desirable in vivo. In contrast to current chemically
inducible expression systems, LITEs have the potential for finer
anatomical localization. Moreover, the kinetics of the system do
not depend on drug diffusion, metabolism, or clearance, and
stimulation can be achieved without drug-related side effects. To
apply the LITE system in vivo, Applicants stereotactically
delivered a 1:1 mixture of high concentration AAV vectors
(10.sup.12 DNAseI resistant particles/mL) carrying the
Grm2-targeting T6-CIB1 and CRY2PHR-VP64 LITE components into the
infralimbic cortex (ILC) of wildtype C57BL/6N mice. To provide
optical stimulation of LITE-expressing neurons in vivo, Applicants
also implanted a fiber optic cannula at the injection site (FIG.
38H).sup.33. Neurons in the injection site were efficiently
co-transduced by both viruses, with >80% of transduced cells
expressing both TALE12-CIB1 and CRY2PHR-VP64 (FIGS. 381 and 48). 8
days post-surgery, Applicants stimulated the ILC by connecting a
solid-state 473 nm laser to the implanted fiber cannula. Following
a 12 h stimulation period (5 mW, 0.8% duty cycle using 0.5 s light
pulses at 0.0167 Hz), brain tissue from the fiber optic cannula
implantation site was analyzed (FIG. 38H) for changes in Grm2 mRNA.
Applicants observed a significant increase in Grm2 mRNA after light
stimulation compared with unstimulated ILC (2.1-fold, p<0.01 vs.
1.3-fold background FIG. 38J), successfully demonstrating the
utility of the LITE system for altering gene expression in vivo.
This experiment suggests the potential value of LITEs for probing
gene functions in the brain.
[0333] The investigation of dynamic transcriptional networks in
heterogeneous tissues such as the brain would benefit greatly from
spatiotemporally precise in vivo gene regulation. Such a system
would allow researchers to ask questions about the role of dynamic
gene regulation in processes as diverse as development, learning,
memory, and disease progression. LITEs can be used to enable
temporally precise, spatially-targeted, and bi-modal control of
endogenous gene expression in cell lines, primary neurons, and in
the mouse brain in vivo. The TALE DNA binding component of LITEs
can be customized to target a wide range of genomic loci.
Independently, novel functionalities can be achieved via alteration
of the LITE effector domain. This system provides a powerful
addition to existing optogenetic platforms, establishing a highly
generalizable mode of altering endogenous gene transcription using
light. Future work will increase the potency of LITE-mediated
transcription modulation, reduce the level of background activity,
and expand the range of wavelengths through which LITEs may be
controlled. This may be achieved through exploration of other
naturally occurring light-sensitive proteins.sup.34-37 or through
directed evolution.sup.38-41 of cryptochrome proteins. Finally, the
modular design of the LITE system provides the opportunity for the
development of a broad array of light-switchable tools for
reverse-engineering genetic and epigenetic functions in a variety
of biological systems.
[0334] LITE constructs were transfected into in Neuro 2A cells
using GenJetAAV vectors carrying TALE or LITE constructs were used
to transduce mouse primary embryonic cortical neurons as well as
the mouse brain in vivo. RNA was extracted and reverse transcribed
and mRNA levels were measured using TaqMan-based RT-qPCR. Light
emitting diodes or solid-state lasers were used for light delivery
in tissue culture and in vivo respectively.
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[0377] Neuro 2a cells (Sigma-Aldrich) were grown in media
containing a 1:1 ratio of OptiMEM (Life Technologies) to
high-glucose DMEM with GlutaMax and Sodium Pyruvate (Life
Technologies) supplemented with 5% HyClone heat-inactivated FBS
(Thermo Scientific), 1% penicillin/streptomycin (Life
Technologies), and passaged at 1:5 every 2 days. 120,000 cells were
plated in each well of a 24-well plate 18-20 h prior to
transfection. 1 h before transfection, media was changed to DMEM
supplemented with 5% HyClone heat-inactivated FBS and 1%
penicillin/streptomycin. Cells were transfected with 1.0 .mu.g
total of construct DNA (at equimolar ratios) per well with 1.5
.mu.L of GenJet (SignaGen Laboratories) transfection reagent
according to the manufacturer's instructions. Media was exchanged
24 h and 44 h post-transfection and light stimulation was started
at 48 h. Stimulation parameters were: 5 mW/cm2, 466 nm, 7% duty
cycle (1 s light pulse 0.067 Hz) for 24 h unless indicated
otherwise in figure legends. RNA was extracted using the RNeasy kit
(Qiagen) according to manufacturer's instructions and 1 .mu.g of
RNA per sample was reverse-transcribed using qScript (Quanta
Biosystems). Relative mRNA levels were measured by quantitative
real-time PCR (qRT-PCR) using TaqMan probes specific for the
targeted gene as well as GAPDH as an endogenous control (Life
Technologies, see Table 2 for Taqman probe IDs). .DELTA..DELTA.Ct
analysis was used to obtain fold-changes relative to negative
controls transduced with GFP only and subjected to light
stimulation. Toxicity experiments were conducted using the
LIVE/DEAD assay kit (Life Technologies) according to
instructions.
[0378] 293FT cells (Life Technologies) were grown in
antibiotic-free D10 media (DMEM high glucose with GlutaMax and
Sodium Pyruvate, 10% heat-inactivated Hyclone FBS, and 1% 1M HEPES)
and passaged daily at 1:2-2.5. The total number of passages was
kept below 10 and cells were never grown beyond 85% confluence. The
day before transfection, 1.times.10.sup.6 cells in 21.5 mL of D10
media were plated onto 15 cm dishes and incubated for 18-22 hours
or until .about.80% confluence. For use as a transfection reagent,
1 mg/mL of PEI "Max" (Polysciences) was dissolved in water and the
pH of the solution was adjusted to 7.1. For AAV production, 10.4
.mu.g of pDF6 helper plasmid, 8.7 .mu.g of pAAV1 serotype packaging
vector, and 5.2 .mu.g of pAAV vector carrying the gene of interest
were added to 434 .mu.L of serum-free DMEM and 130 .mu.L of PEI
"Max" solution was added to the DMEM-diluted DNA mixture. The
DNA/DMEM/PEI cocktail was vortexed and incubated at room
temperature for 15 min. After incubation, the transfection mixture
was added to 22 mL of complete media, vortexed briefly, and used to
replace the media for a 15 cm dish of 293FT cells. For supernatant
production, transfection supernatant was harvested at 48 h,
filtered through a 0.45 .mu.m PVDF filter (Millipore), distributed
into aliquots, and frozen for storage at -80.degree. C.
[0379] Dissociated cortical neurons were prepared from C57BL/6N
mouse embryos on E16 (Charles River Labs). Cortical tissue was
dissected in ice-cold HBSS--(50 mL 10.times.HBSS, 435 mL dH.sub.2O,
0.3 M HEPES pH 7.3, and 1% penicillin/streptomycin). Cortical
tissue was washed 3.times. with 20 mL of ice-cold HBSS and then
digested at 37.degree. C. for 20 min in 8 mL of HBSS with 240 .mu.L
of 2.5% trypsin (Life Technologies). Cortices were then washed 3
times with 20 mL of warm HBSS containing 1 mL FBS. Cortices were
gently triturated in 2 ml of HBSS and plated at 150,000 cells/well
in poly-D-lysine coated 24-well plates (BD Biosciences). Neurons
were maintained in Neurobasal media (Life Technologies),
supplemented with 1.times.B27 (Life Technologies), GlutaMax (Life
Technologies) and 1% penicillin/streptomycin.
[0380] Primary cortical neurons were transduced with 250 .mu.L of
AAV1 supernatant on DIV 5. The media and supernatant were replaced
with regular complete neurobasal the following day. Neurobasal was
exchanged with Minimal Essential Medium (Life Technologies)
containing 1.times.B27, GlutaMax (Life Technologies) and 1%
penicillin/streptomycin 6 days after AAV transduction to prevent
formation of phototoxic products from HEPES and riboflavin
contained in Neurobasal during light stimulation.
[0381] Light stimulation was started 6 days after AAV transduction
(DIV 11) with an intensity of 5 mW/cm.sup.2, duty cycle of 0.8%
(250 ms pulses at 0.033 Hz or 500 ms pulses at 0.016 Hz), 466 nm
blue light for 24 h unless indicated otherwise in figure legends.
RNA extraction and reverse transcription were performed using the
Cells-to-Ct kit according to the manufacturers instructions (Life
Technologies). Relative mRNA levels were measured by quantitative
real-time PCR (qRT-PCR) using TaqMan probes as described above for
Neuro 2a cells.
[0382] For immunohistochemistry of primary neurons, cells were
plated on poly-D-lysine/laminin coated coverslips (BD Biosciences)
after harvesting. AAV1-transductions were performed as described
above. Neurons were fixed 7 days post-transduction with 4%
paraformaldehyde (Sigma Aldrich) for 15 min at RT. Blocking and
permeabilization were performed with 10% normal goat serum (Life
Technologies) and 0.5% Triton-X100 (Sigma-Aldrich) in DPBS (Life
Technologies) for 1 h at room temperature. Neurons were incubated
with primary antibodies overnight at 4.degree. C., washed 3.times.
with DPBS and incubated with secondary antibodies for 90 min at RT.
For antibody providers and concentrations used, see Table 3.
Coverslips were finally mounted using Prolong Gold Antifade Reagent
with DAPI (Life Technologies) and imaged on an Axio Scope A.1
(Zeiss) with an X-Cite 120Q light source (Lumen Dynamics). Image
were acquired using an AxioCam MRm camera and AxioVision 4.8.2.
[0383] For preparation of total protein lysates, primary cortical
neurons were harvested after light stimulation (see above) in
ice-cold lysis buffer (RIPA, Cell Signaling; 0.1% SDS,
Sigma-Aldrich; and cOmplete ultra protease inhibitor mix, Roche
Applied Science). Cell lysates were sonicated for 5 min at `M`
setting in a Bioruptor sonicator (Diagenode) and centrifuged at
21,000.times.g for 10 min at 4.degree. C. Protein concentration was
determined using the RC DC protein assay (Bio-Rad). 30-40 .mu.g of
total protein per lane was separated under non-reducing conditions
on 4-15% Tris-HCl gels (Bio-Rad) along with Precision Plus Protein
Dual Color Standard (Bio-Rad) After wet electrotransfer to
polyvinylidene difluoride membranes (Millipore) and membrane
blocking for 45 min in 5% BLOT-QuickBlocker (Millipore) in
Tris-buffered saline (TBS, Bio-Rad), western blots were probed with
anti-mGluR2 (Abcam, 1:1.000) and anti-.alpha.-tubulin
(Sigma-Aldrich 1:20,000) overnight at 4.degree. C., followed by
washing and anti-mouse-IgG HRP antibody incubation (Sigma-Aldrich,
1:5,000-1:10,000). For further antibody details see Table 3.
Detection was performed via ECL Western blot substrate (SuperSignal
West Femto Kit, Thermo Scientific). Blots were imaged with an
AlphaImager (Innotech) system, and quantified using ImageJ software
1.46r.
[0384] Production of concentrated and purified AAV for stereotactic
injection in-vivo was done using the same initial steps outlined
above for production of AAV1 supernatant. However, for
transfection, equal ratios of AAV1 and AAV2 serotype plasmids were
used instead of AAV1 alone. 5 plates were transfected per construct
and cells were harvested with a cell-scraper 48 h post
transfection. Purification of AAV1/2 particles was performed using
HiTrap heparin affinity columns (GE Healthcare).sup.42. Applicants
added a second concentration step down to a final volume of 100
.mu.l per construct using an Amicon 500 .mu.l concentration column
(100 kDa cutoff, Millipore) to achieve higher viral titers.
Titration of AAV was performed by qRT-PCR using a custom Taqman
probe for WPRE (Life Technologies). Prior to qRT-PCR, concentrated
AAV was treated with DNaseI (New England Biolabs) to achieve a
measurement of DNaseI-resistant particles only. Following DNaseI
heat-inactivation, the viral envelope was degraded by proteinase K
digestion (New England Biolabs). Viral titer was calculated based
on a standard curve with known WPRE copy numbers.
[0385] Adult (10-14 weeks old) male C57BL/6N mice were
anaesthetized by intraperitoneal (i.p.) injection of
Ketamine/Xylazine (100 mg/kg Ketamine and 10 mg/kg Xylazine) and
pre-emptive analgesia was given (Buprenex, 1 mg/kg, i.p.).
Craniotomy was performed according to approved procedures and 1
.mu.l of AAV1/2 was injected into ILC at 0.35/1.94/-2.94 (lateral,
anterior and inferior coordinates in mm relative to bregma). During
the same surgical procedure, an optical cannula with fiber (Doric
Lenses) was implanted into ILC unilaterally with the end of the
optical fiber located at 0.35/1.94/-2.64 relative to bregma. The
cannula was affixed to the skull using Metabond dental cement
(Parkell Inc) and Jet denture repair (Lang dental) to build a
stable cone around it. The incision was sutured and proper
post-operative analgesics were administered for three days
following surgery.
[0386] Mice were injected with a lethal dose of Ketamine/Xylazine
anaesthetic and transcardially perfused with PBS and 4%
paraformaldehyde (PFA). Brains were additionally fixed in 4% PFA at
4.degree. C. overnight and then transferred to 30% sucrose for
cryoprotection overnight at room temperature. Brains were then
transferred into Tissue-Tek Optimal Cutting Temperature (OCT)
Compound (Sakura Finetek) and frozen at -80.degree. C. 18 .mu.m
sections were cut on a cryostat (Leica Biosystems) and mounted on
Superfrost Plus glass slides (Thermo Fischer). Sections were
post-fixed with 4% PFA for 15 min, and immunohistochemistry was
performed as described for primary neurons above.
[0387] 8 days post-surgery, awake and freely moving mice were
stimulated using a 473 nm laser source (OEM Laser Systems)
connected to the optical implant via fiber patch cables and a
rotary joint. Stimulation parameters were the same as used on
primary neurons: 5 mW (total output), 0.8% duty cycle (500 ms light
pulses at 0.016 Hz) for a total of 12 h. Experimental conditions,
including transduced constructs and light stimulation are listed in
Table 4.
[0388] After the end of light stimulations, mice were euthanized
using CO2 and the prefrontal cortices (PFC) were quickly dissected
on ice and incubated in RNA later (Qiagen) at 4.degree. C.
overnight. 200 .mu.m sections were cut in RNA later at 4.degree. C.
on a vibratome (Leica Biosystems). Sections were then frozen on a
glass coverslide on dry ice and virally transduced ILC was
identified under a fluorescent stereomicroscope (Leica M165 FC). A
0.35 mm diameter punch of ILC, located directly ventrally to the
termination of the optical fiber tract, was extracted (Harris
uni-core, Ted Pella). The brain punch sample was then homogenized
using an RNase-free pellet-pestle grinder (Kimble Chase) in 50
.mu.l Cells-to-Ct RNA lysis buffer and RNA extraction, reverse
transcription and qRT-PCR was performed as described for primary
neuron samples.
[0389] All experiments were performed with a minimum of three
independent biological replicates. Statistical analysis was
performed with Prism (GraphPad) using student's t-test when
comparing two conditions, ANOVA with Tukey's post-hoc analysis when
comparing multiple samples with each other, ANOVA with Duncan's
post-hoc analysis when comparing multiple samples to the negative
control, and two-way ANOVA with Bonferroni post-hoc analysis to
compare multiple groups over time.
Example 7
Development of AAV1 Supernatant Process
[0390] Traditional AAV particle generation required laborious
production and purification processes, and made testing many
constructs in parallel impractical (4). In this study, a simple yet
highly effective process of AAV production using filtered
supernatant from transfected 293FT cells (FIG. 42). Recent reports
indicate that AAV particles produced in 293FT cells could be found
not only it the cytoplasm but also at considerable amounts in the
culture media (5). The ratio of viral particles between the
supernatant and cytosol of host cells varied depending on the AAV
serotype, and secretion was enhanced if polyethylenimine (PEI) was
used to transfect the viral packaging plasmids (5). In the current
study, it was found that 2.times.10.sup.5 293 FT cells transfected
with AAV vectors carrying TALEs (FIG. 37A) and packaged using AAV1
serotype were capable of producing 250 .mu.l of AAV1 at a
concentration of 5.6.+-.0.24.times.10.sup.10 DNAseI resistant
genome copies (gc) per mL. 250 .mu.l of filtered supernatant was
able to transduce 150,000 primary cortical neurons at efficiencies
of 80-90% (FIGS. 37B and 42B). This is a dramatic increase over the
1-2% transduction efficiency achieved using lentivirus supernatant
produced from the same number of 293FT cells (FIG. 42B).
TABLE-US-00004 TABLE 2 Product information for all Taqman probes
(Life Technologies) Target Species Probe # Ngn2 mouse Mm00437603_g1
Grm5 (mGluR5) mouse Mm00690332_m1 Grm2 (mGluR2) mouse Mm01235831_m1
Grin2a (NMDAR2A) mouse Mm00433802_m1 GAPD (GAPDH) mouse 4352932E
KLF4 human Hs00358836_m1 GAPD (GAPDH) human 4352934E WPRE
custom
TABLE-US-00005 TABLE 3 Clone, product numbers and concentrations
for antibodies used in this study Primary Antibodies Target Host
Clone # Manufacturer Product # IsoType Concentration mGluR2 mouse
mG2Na-s Abcam Ab15672 IgG 1:1000 .alpha.-tubulin mouse B-5-1-2
Sigma-Aldrich T5168 IgG1 1:20000 NeuN mouse A60 Millipore MAB377
IgG1 1:200 HA (Alexa Fluor mouse 6E2 Cell Signaling 3444 IgG1 1:100
594 conjugated) GFP chicken polyclonal Aves Labs GFP-1020 IgY 1:500
Secondary Antibodies Target Host Conjugate Manufacturer Product #
Concentration mouse IgG goat HRP Sigma-Aldrich A9917 1:5000-10000
mouse IgG goat Alexa Fluor 594 Life Technologies A11005 1:1000
chicken IgG Goat Alexa Fluor 488 Life Technologies A11039
1:1000
TABLE-US-00006 TABLE 4 Viral transduction and light stimulation
parameters for in vivo LITE-mediated activation of Grm2 in the
mouse infralimbic cortex (ILC). Grm2 mRNA levels in the ipsilateral
LITE-expressing hemisphere are compared with the contralateral
mCherry-expressing control hemisphere for all three experimental
conditions shown in FIG. 38J. ILC ILC Hemisphere (ipsilateral)
Hemisphere Experimental Light (contralateral) condition AAV vector
stimulation AAV vector GFP GFP yes mCherry LITEs/
TALE-CIB1::CRY2PHR- no mCherry no Light VP64 LITEs/
TALE-CIB1::CRY2PHR- yes mCherry +Light VP64
[0391] Sequences of constructs used in Neuro-2A cells (FIGS. 35,
36)
[0392] >TALE(Ngn2) (underlined)-NLS (in italics)-CRY2 (in
bold)
TABLE-US-00007 (SEQ ID NO: 168)
MSRTRLPSPPAPSPAFSADSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTEAATGEWDEVQSGLR
AADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDASPAAQVDLRTLGYSQQQQEKIKPKVRSTV
AQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEA
LLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGK
QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH
DGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVA
IASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPE
QVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHG
LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLC
QAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLL
PVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETV
QRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQA
LETVQRLLPVLCQAHGLTPEQVVAIASHDGGRPALESIVAQLSRPDPALAALTNDHLVALACLG
GRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQF
GMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFA
DSLERDLDAPSPMHEGDQTRASASPKKKRKVEASKMDKKTIVWFRRDLRIEDNPALAAAAHEGS
VFPVFIWCPEEEGQFYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGA
TKVVFNHLYDPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCL
DMSIESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLNE
FIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGEESADLF
LRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPLVDAGMRELWAT
GWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQYISGSIPDGHELDRLDN
PALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKASGVELGTNYAKPIVDIDTARE
LLAKAISRTREAQIMIGAAPDEIVADSFEALGANTIKEPGLCPSVSSNDQQVPSAVRYNGSKRV
KPEEEEERDMKKSRGFDERELFSTAESSSSSSVFFVSQSCSLASEGKNLEGIQDSSDQITTSLG
KNG
[0393] >TALE(Ngn2) (underlined)-NLS (in italics)-CRY2PHR (in
bold)
TABLE-US-00008 (SEQ ID NO: 169)
MSRTRLPSPPAPSPAFSADSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTEAATGEWDEVQSGLR
AADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDASPAAQVDLRTLGYSQQQQEKIKPKVRSTV
AQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEA
LLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGK
QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH
DGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVA
IASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPE
QVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHG
LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLC
QAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLL
PVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETV
QRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQA
LETVQRLLPVLCQAHGLTPEQVVAIASHDGGRPALESIVAQLSRPDPALAALTNDHLVALACLG
GRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQF
GMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFA
DSLERDLDAPSPMHEGDQTRASASPKKKRKVEASKMDKKTIVWFRRDLRIEDNPALAAAAHEGS
VFPVFIWCPEEEGQFYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGA
TKVVFNHLYDPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCL
DMSIESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLNE
FIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGEESADLF
LRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPLVDAGMRELWAT
GWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQYISGSIPDGHELDRLDN
PALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKASGVELGTNYAKPIVDIDTARE
LLAKAISRTREAQIMIGAAP
[0394] >CIB1 (in bold)-NLS (in italics)-VP64 (in bold,
underlined) .sub.--2A_ GFP (underlined)
TABLE-US-00009 (SEQ ID NO: 170)
MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYLSTAGLNLP
MMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCNEKKKKMTMNRDDLVEEGEEEKSKI
TEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKELEKTDYIHVRARRGQATDSHSIAERVRRE
KISERMKFLQDLVPGCDKITGKAGMLDEIINYVQSLQRQIEFLSMKLAIVNPRPDFDMDDIFAK
EVASTPMTVVPSPEMVLSGYSHEMVHSGYSSEMVNSGYLHVNPMQQVNTSSDPLSCFNNGEAPS
MWDSHVQNLYGNLGVASPKKKRKVEASGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDF
DLDMLGSDALDDFDLDMLINSRGSGEGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGD
VNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFK
SAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNE
KRDHMVLLEFVTAAGITLGMDELYK
[0395] >CIBN (in bold)-NLS (in italics)-VP64 (in bold,
underlined) .sub.--2A_ GFP (underlined)
TABLE-US-00010 (SEQ ID NO: 171)
MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYLSTAGLNLP
MMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCNEKKKKMTMNRDDLVEEGEEEKSKI
TEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKELEKTDYIASPKKKRKVEASGSGRADALDD
FDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINSRGSGEGRGSLLTCGDV
EENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWP
TLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNR
IELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNT
PIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
[0396] >CIB1 (in bold)-NLS (in italics)-VP16 (in bold,
underlined) .sub.--2A_ GFP (underlined)
TABLE-US-00011 (SEQ ID NO: 172)
MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYLSTAGLNLP
MMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCNEKKKKMTMNRDDLVEEGEEEKSKI
TEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKELEKTDYIHVRARRGQATDSHSIAERVRRE
KISERMKFLQDLVPGCDKITGKAGMLDEIINYVQSLQRQIEFLSMKLAIVNPRPDFDMDDIFAK
EVASTPMTVVPSPEMVLSGYSHEMVHSGYSSEMVNSGYLHVNPMQQVNTSSDPLSCFNNGEAPS
MWDSHVQNLYGNLGVASPKKKRKVEASAPPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGD
GDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGGEFPGIRRSRGSGEGRGSLLTCGD
VEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPW
PTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN
RIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQN
TPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
[0397] >CIB1 (in bold)-NLS (in italics)-p65 (in bold,
underlined) .sub.--2A_ GFP (underlined)
TABLE-US-00012 (SEQ ID NO: 173)
MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYLSTAGLNLP
MMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCNEKKKKMTMNRDDLVEEGEEEKSKI
TEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKELEKTDYIHVRARRGQATDSHSIAERVRRE
KISERMKFLQDLVPGCDKITGKAGMLDEIINYVQSLQRQIEFLSMKLAIVNPRPDFDMDDIFAK
EVASTPMTVVPSPEMVLSGYSHEMVHSGYSSEMVNSGYLHVNPMQQVNTSSDPLSCFNNGEAPS
MWDSHVQNLYGNLGVASPKKKRKVEASPSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPP
APAPVLTPGPPQSLSAPVPHSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVD
NSEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDEDFS
SIADMDFSALLSQISSSGQSRGSGEGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDV
NGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKS
AMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNV
YIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEK
RDHMVLLEFVTAAGITLGMDELYK
[0398] AAV constructs (constructs used in primary neurons and
in-vivo, FIGS. 37-38)
[0399] >HA-TALE(12mer) (in bold)-NLS (in italics)-VP64 (in bold,
underlined) .sub.--2A_ GFP (underlined)
TABLE-US-00013 (SEQ ID NO: 174)
MYPYDVPDYAVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTV
AVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVT
AVEAVHAWRNALTGAPLNLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASX
XGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVA
IASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPE
QVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHG
LTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLC
QAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLL
PVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGRPALESI
VAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAASP
KKKRKVEASGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML
INSRGSGEGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATY
GKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKI
RHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITL
GMDELYK
[0400] >HA-TALE(12mer) (in bold)-NLS (in italics)-SID4X (in
bold, underlined) .sub.--2A_ phiLOV2.1 (underlined)
TABLE-US-00014 (SEQ ID NO: 175)
MYPYDVPDYAVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTV
AVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVT
AVEAVHAWRNALTGAPLNLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASX
XGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVA
IASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPE
QVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHG
LTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLC
QAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLL
PVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGRPALESI
VAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAASP
KKKRKVEASPKKKRKVEASGSGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAA
DYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAA
DYLERREREAEHGYASMLPSRSRGSGEGRGSLLTCGDVEENPGPIEKSFVITDPRLPDYPIIFA
SDGFLELTEYSREEIMGRNARFLQGPETDQATVQKIRDAIRDQRETTVQLINYTKSGKKFWNLL
HLQPVRDRKGGLQYFIGVQLVGSDHV
[0401] >HA-TALE(12mer) (in bold)-NLS (in italics)-CIB1
(underlined)
TABLE-US-00015 (SEQ ID NO: 176)
MYPYDVPDYAVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTV
AVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVT
AVEAVHAWRNALTGAPLNLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASX
XGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVA
IASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPE
QVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHG
LTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLC
QAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLL
PVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGRPALESI
VAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAASP
KKKRKVEASNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYL
STAGLNLPMMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCNEKKKKMTMNRDDLVEE
GEEEKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKELEKTDYIHVRARRGQATDSHS
IAERVRREKISERMKFLQDLVPGCDKITGKAGMLDEIINYVQSLQRQIEFLSMKLAIVNPRPDF
DMDDIFAKEVASTPMTVVPSPEMVLSGYSHEMVHSGYSSEMVNSGYLHVNPMQQVNTSSDPLSC
FNNGEAPSMWDSHVQNLYGNLGV
[0402] >CRY2PHR(in bold)-NLS (in italics)-VP64 (in bold,
underlined) .sub.--2A_ GFP (underlined)
TABLE-US-00016 (SEQ ID NO: 177)
MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQFYPGRASRWWMKQSLAHLS
QSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLYDPVSLVRDHTVKEKLVERGISVQS
YNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSIESVMLPPPWRLMPITAAAEAIWACSIEEL
GLENEAEKPSNALLTRAWSPGWSNADKLLNEFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEI
SVRHVFQCARMKQIIWARDKNSEGEESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFP
WDADVDKFKAWRQGRTGYPLVDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDT
LLDADLECDILGWQYISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHP
WDAPLTVLKASGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAAPASPKKKRKVEASG
SGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINSRGSGEGR
GSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVK
FEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQ
LADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKV
[0403] >CRY2PHR (in bold)-NLS (in italics)-SID4X (in bold,
underlined) .sub.--2A_ phiLOV2.1 (underlined)
TABLE-US-00017 (SEQ ID NO: 178)
MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQFYPGRASRWWMKQSLAHLS
QSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLYDPVSLVRDHTVKEKLVERGISVQS
YNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSIESVMLPPPWRLMPITAAAEAIWACSIEEL
GLENEAEKPSNALLTRAWSPGWSNADKLLNEFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEI
SVRHVFQCARMKQIIWARDKNSEGEESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFP
WDADVDKFKAWRQGRTGYPLVDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDT
LLDADLECDILGWQYISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHP
WDAPLTVLKASGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAAPASPKKKRKVEASG
SGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERREREAEHGYASMLPG
SGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERREREAEHGYASMLPS
RSRGSGEGRGSLLTCGDVEENPGPIEKSFVITDPRLPDYPIIFASDGFLELTEYSREEIMGRNA
RFLQGPETDQATVQKIRDAIRDQRETTVQLINYTKSGKKFWNLLHLQPVRDRKGGLQYFIGVQL
VGSDHV
[0404] Sequences of FIGS. 39-48
[0405] >TALE(KLF4) (underlined)-NLS (in italics)-CRY2PHR (in
bold)
TABLE-US-00018 (SEQ ID NO: 179)
MSRTRLPSPPAPSPAFSADSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTEAATGEWDEVQSGLR
AADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDASPAAQVDLRTLGYSQQQQEKIKPKVRSTV
AQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEA
LLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNGGGK
QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN
GGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVA
IASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPE
QVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHG
LTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLC
QAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLL
PVLCQAHGLTPEQVVAIASHDGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAV
KKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLL
QLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLD
APSPMHEGDQTRASASPKKKRKVEASKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWC
PEEEGQFYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHL
YDPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSIESVM
LPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLNEFIEKQLID
YAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGEESADLFLRGIGLRE
YSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPLVDAGMRELWATGWMHNRIR
VIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQYISGSIPDGHELDRLDNPALQGAKY
DPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKASGVELGTNYAKPIVDIDTARELLAKAISR
TREAQIMIGAAP_
[0406] >HA-NLS (in italics)-TALE(p11, N136) (in bold)-SID
(underlined)
TABLE-US-00019 (SEQ ID NO: 180)
MYPYDVPDYASPKKKRKVEASVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVA
LSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQ
LLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGL
TPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQ
AHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLP
VLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQ
RLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQAL
ETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGG
KQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS
NNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVV
AIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP
EQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAH
GLTPEQVVAIASHDGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHA
PALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVG
VTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPMHE
GDQTRASASGSGMNIQMLLEAADYLERREREAEHGYASMLP.
[0407] >HA-NLS (in italics)-TALE(p11, N136) (in bold)-SID4X
(underlined)
TABLE-US-00020 (SEQ ID NO: 181)
MYPYDVPDYASPKKKRKVEASVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVA
LSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQ
LLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGL
TPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQ
AHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLP
VLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQ
RLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQAL
ETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGG
KQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS
NNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVV
AIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP
EQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAH
GLTPEQVVAIASHDGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHA
PALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVG
VTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPMHE
GDQTRASASGSGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERREREA
EHGYASMLPGSGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERREREA
EHGYASMLPSR
[0408] The following Arduino script was used to enable the
individual control of each 4-well column of a light-stimulated
24-well plate:
TABLE-US-00021 //Basic control code for LITE LED array using
Arduino UNO //LED column address initialization to PWM-ready
Arduino outputs int led1_pin = 3; int led2_pin = 5; int led3_pin =
6; int led4_pin = 9; int led5_pin = 10; int led6_pin = 11;
//Maximum setting for Arduino PWM int uniform_brightness = 255;
//PWM settings for individual LED columns int led1_brightness =
uniform_brightness/2; int led2_brightness = uniform_brightness/2;
int led3_brightness = uniform_brightness/2; int led4_brightness =
uniform_brightness/2; int led5_brightness = uniform_brightness/2;
int led6_brightness = uniform_brightness/2; //`on` time in msec
unsigned long uniform_stim_time = 1000; / //individual `on` time
settings for LED columns unsigned long_led1_stim_time =
uniform_stim_time; unsigned long_led2_stim_time =
uniform_stim_time; unsigned long_led3_stim_time =
uniform_stim_time; unsigned long_led4_stim_time =
uniform_stim_time; unsigned long_led5_stim_time =
uniform_stim_time; unsigned long_led6_stim_time =
uniform_stim_time; //`off` time in msec unsigned long
uniform_off_time = 14000; //individual `off` time settings for LED
columns unsigned long led1_off_time = uniform_off_time; unsigned
long led2_off_time = uniform_off_time; unsigned long led3_off_time
= uniform_off_time; unsigned long led4_off_time = uniform_off_time;
unsigned long led5_off_time = uniform_off_time; unsigned long
led6_off_time = uniform_off_time; unsigned long currentMillis = 0;
//initialize timing and state variables unsigned long
led1_last_change = 0; unsigned long led2_last_change = 0; unsigned
long led3_last_change = 0; unsigned long led4_last_change = 0;
unsigned long led5_last_change = 0; unsigned long led6_last_change
= 0; int led1_state = HIGH; int led2_state = HIGH; int led3_state =
HIGH; int led4_state = HIGH; int led5_state = HIGH; int led6_state
= HIGH; unsigned long led1_timer = 0; unsigned long led2_timer = 0;
unsigned long led3_timer = 0; unsigned long led4_timer = 0;
unsigned long led5_timer = 0; unsigned long led6_timer = 0; void
setup( ) { // setup PWM pins for output pinMode(led1_pin, OUTPUT);
pinMode(led2_pin, OUTPUT); pinMode(led3_pin, OUTPUT);
pinMode(led4_pin, OUTPUT); pinMode(led5_pin, OUTPUT);
pinMode(led6_pin, OUTPUT); //LED starting state
analogWrite(led1_pin, led1_brightness); analogWrite(led2_pin,
led2_brightness); analogWrite(led3_pin, led3_brightness);
analogWrite(led4_pin, led4_brightness); analogWrite(led5_pin,
led5_brightness); analogWrite(led6_pin, led6_brightness); } void
loop( ) { currentMillis = millis( ); //identical timing loops for
the 6 PWM output pins led1_timer = currentMillis -
led1_last_change; if (led1_state == HIGH){ //led state is on if
(led1_timer >= led1_stim_time){ //TRUE if stim time is complete
analogWrite(led1_pin, 0); //turn LED off led1_state = LOW; //change
LED state variable led1_last_change = currentMillis; //mark time of
most recent change } } else{ //led1 state is off if (led1_timer
>= led1_off_time){ //TRUE if off time is complete
analogWrite(led1_pin, led1_brightness); //turn LED on led1_state =
HIGH; //change LED state variable led1_last_change = currentMillis;
//mark time of most recent change } } led2_timer = currentMillis -
led2_last_change; if (led2_state == HIGH){ if (led2_timer >=
led2_stim_time){ analogWrite(led2_pin, 0); led2_state = LOW;
led2_last_change = currentMillis; } } else{ //led2 state is off if
(led2_timer >= led2_off_time){ analogWrite(led2_pin,
led2_brightness); led2_state = HIGH; led2_last_change =
currentMillis; } } led3_timer = currentMillis - led3_last_change;
if (led3_state == HIGH){ if (led3_timer >= led3_stim_time){
analogWrite(led3_pin, 0); led3_state = LOW; led3_last_change =
currentMillis; } } else{ //led3 state is off if (led3_timer >=
led3_off_time){ analogWrite(led3_pin, led3_brightness); led3_state
= HIGH; led3_last_change = currentMillis; } } led4_timer =
currentMillis - led4_last_change; if (led4_state == HIGH){ if
(led4_timer >= led4_stim_time){ analogWrite(led4_pin, 0);
led4_state = LOW; led4_last_change = currentMillis; } } else{
//led4 state is off if (led4_timer >= led4_off_time){
analogWrite(led4_pin, led4_brightness); led4_state = HIGH;
led4_last_change = currentMillis; } } led5_timer = currentMillis -
led5_last_change; if (led5_state == HIGH){ if (led5_timer >=
led5_stim_time){ analogWrite(led5_pin, 0); led5_state = LOW;
led5_last_change = currentMillis; } } else{ //led5 state is off if
(led5_timer >= led5_off_time){ analogWrite(led5_pin,
led5_brightness); led5_state = HIGH; led5_last_change =
currentMillis; } } led6_timer = currentMillis - led6_last_change;
if (led6_state == HIGH){ if (led6_timer >= led6_stim_time){
analogWrite(led6_pin, 0); led6_state = LOW; led6_last_change =
currentMillis; } } else{ //led6 state is off if (led6_timer >=
led6_off_time){ analogWrite(led6_pin, led6_brightness); led6_state
= HIGH; led6_last_change = currentMillis; } } }
REFERENCES
[0409] 1 Banerjee, R. et al. The Signaling State of Arabidopsis
Cryptochrome 2 Contains Flavin Semiquinone. Journal of Biological
Chemistry 282, 14916-14922, doi:10.1074/jbc.M700616200 (2007).
[0410] 2 McClure, C., Cole, K. L., Wulff, P., Klugmann, M. &
Murray, A. J. Production and titering of recombinant
adeno-associated viral vectors. J Vis Exp, e3348, doi:10.3791/3348
(2011). [0411] 3 Witten, Ilana B. et al. Recombinase-Driver Rat
Lines: Tools, Techniques, and Optogenetic Application to
Dopamine-Mediated Reinforcement. Neuron 72, 721-733, doi: at the
website dx.doi.org/10.1016/j.neuron.2011.10.028 (2011). [0412] 4
Grieger, J. C., Choi, V. W. & Samulski, R. J. Production and
characterization of adeno-associated viral vectors. Nat Protoc 1,
1412-1428, doi:10.1038/nprot.2006.207 (2006). [0413] 5 Lock M, A.
M., Vandenberghe L H, Samanta A, Toelen J, Debyser Z, Wilson J M.
Rapid, Simple, and Versatile Manufacturing of Recombinant
Adeno-Associated Viral Vectors at Scale. Human Gene Therapy 21,
1259-1271, doi:10.1089/hum.2010.055 (2010).
Example 8
Cloning (Construction) of AAV Constructs
[0414] Construction of AAV-Promoter-TALE-Effector Backbone
[0415] For construction of AAV-promoter-TALE-effector a backbone
was cloned by standard subcloning methods. Specifically, the vector
contained an antibiotics resistance gene, such as ampicillin
resistance and two AAV inverted terminal repeats (itr's) flanking
the promoter-TALE-effector insert (sequences, see below). The
promoter (hSyn), the effector domain (VP64, SID4X or CIB1 in this
example)/the N- and C-terminal portion of the TALE gene containing
a spacer with two typeIIS restriction sites (BsaI in this instance)
were subcloned into this vector. To achieve subcloning, each DNA
component was amplified using polymerase-chain reaction and then
digested with specific restriction enzymes to create matching DNA
sticky ends. The vector was similarly digested with DNA restriction
enzymes. All DNA fragments were subsequently allowed to anneal at
matching ends and fused together using a ligase enzyme.
[0416] Assembly of Individual TALEs into AAV-Promoter-TALE-Effector
Backbone
[0417] For incorporating different TALE monomer sequences into the
AAV-promoter-TALE-effector backbone described above, a strategy
based on restriction of individual monomers with type IIS
restriction enzymes and ligation of their unique overhangs to form
an assembly of 12 to 16 monomers to form the final TALE and ligate
it into the AAV-promoter-TALE-effector backbone by using the type
IIS sites present in the spacer between the N- and C-term (termed
golden gate assembly). This method of TALE monomer assembly has
previously been described by us (NE Sanjana, L Cong, Y Zhou, M M
Cunniff, G Feng & F Zhang A transcription activator-like
effector toolbox for genome engineering Nature Protocols 7, 171-192
(2012) doi:10.1038/nprot.2011.431)
[0418] By using the general cloning strategy outlined above, AAV
vectors containing different promoters, effector domains and TALE
monomer sequences can be easily constructed.
Nucleotide Sequences:
TABLE-US-00022 [0419] Left AAV ITR (SEQ ID NO: 182)
Cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggc
gacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatc
actaggggttcct_ Right AAV ITR (SEQ ID NO: 183)
Aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgg
gcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgc
agctgcctgcagg_ hSyn promoter (SEQ ID NO: 184)
gtgtctagactgcagagggccctgcgtatgagtgcaagtgggttttaggaccaggatgaggcgg
ggtgggggtgcctacctgacgaccgaccccgacccactggacaagcacccaacccccattcccc
aaattgcgcatcccctatcagagagggggaggggaaacaggatgcggcgaggcgcgtgcgcact
gccagcttcagcaccgcggacagtgccttcgcccccgcctggcggcgcgcgccaccgccgcctc
agcactgaaggcgcgctgacgtcactcgccggtcccccgcaaactccccttcccggccaccttg
gtcgcgtccgcgccgccgccggcccagccggaccgcaccacgcgaggcgcgagataggggggca
cgggcgcgaccatctgcgctgcggcgccggcgactcagcgctgcctcagtctgcggtgggcagc
ggaggagtcgtgtcgtgcctgagagcgcagtcgagaa_ TALE N-term (+136 AA
truncation) (SEQ ID NO: 185)
GTAGATTTGAGAACTTTGGGATATTCACAGCAGCAGCAGGAAAAGATCAAGCCCAAAGTGAGGT
CGACAGTCGCGCAGCATCACGAAGCGCTGGTGGGTCATGGGTTTACACATGCCCACATCGTAGC
CTTGTCGCAGCACCCTGCAGCCCTTGGCACGGTCGCCGTCAAGTACCAGGACATGATTGCGGCG
TTGCCGGAAGCCACACATGAGGCGATCGTCGGTGTGGGGAAACAGTGGAGCGGAGCCCGAGCGC
TTGAGGCCCTGTTGACGGTCGCGGGAGAGCTGAGAGGGCCTCCCCTTCAGCTGGACACGGGCCA
GTTGCTGAAGATCGCGAAGCGGGGAGGAGTCACGGCGGTCGAGGCGGTGCACGCGTGGCGCAAT
GCGCTCACGGGAGCACCCCTCAAC_ TALE C-term (+63 AA truncation) (SEQ ID
NO: 186)
CGGACCCCGCGCTGGCCGCACTCACTAATGATCATCTTGTAGCGCTGGCCTGCCTCGGCGGACG
ACCCGCCTTGGATGCGGTGAAGAAGGGGCTCCCGCACGCGCCTGCATTGATTAAGCGGACCAAC
AGAAGGATTCCCGAGAGGACATCACATCGAGTGGCA_ Ampicillin resistance gene
(SEQ ID NO: 187)
atgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgttt
ttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtggg
ttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgtttt
ccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggc
aagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcac
agaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagt
gataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttt
tgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccat
accaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactatta
actggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaag
ttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagc
cggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatc
gtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgaga
taggtgcctcactgattaagcattgg_
Example 9
DNA Ratios
[0420] In this application, Applicants provide for varying plasmid
ratios. The ratios of vector of interest plasmid: AAV serotype
plasmid: pHelper plasmid may be varied. Specific values used in
examples above are: 1:1.7:2 for AAV supernatant production down to
24-well scale. Values that may be used for production in 96-well
format are: 1:2:1. Values may be varied in a wider range (e.g. up
to fivefold excess of one plasmid) if desired.
[0421] Scalability
[0422] The present invention also comprehends AAV supernatant
production as described herein being easily scaled up into higher
throughput formats. The examples listed describe scaling from 15 cm
dishes to 96-well plates for production. Through the same principle
of scaling it may be possible to produce AAV in more dense well
plate formats (e.g. 384-well, 1536-well etc.). The invention
further comprehends using this process in even smaller volume units
as would be possible with e.g. a microfluidic device capable of
maintaining cell cultures in individual chambers. Hence, the
present invention allows for an unprecedented throughput of
production of different AAV viral particles. Applicants submit that
one further important advantage of the invention described is that
due to the highly efficient recovery of functional viral particles
(due to minimal loss compared to extensive purification procedures
traditionally used) AAV supernatant can be produced at the same
scale as it will be applied. This is especially relevant for
automated processing as it provides not only a simplified
production and application process but also reduces the possibility
for variability. In a preferred embodiment, the invention
comprehends the automated production of 96 different AAV particles
in 96-well plate format and application of the harvested
supernatant to 3 replicate plates of cells to be transduced. This
requires minimal pipetting steps, no necessary rearrangement
(entire plates of virus can be applied to cells with a 96-channel
pipette head) and minimal chance of pipetting error.
[0423] Filtering/Purification
[0424] Multiple methods may be used to purify the cell supernatant
containing AAV particles after harvest and before application to
cells for transduction. For a basic purification which mostly
serves to remove any potential 293FT cells and large cell debris
from the supernatant, filtration with a 22 micron or 45 micron pore
size low protein binding filter or centrifugation for pelleting
cells and cell debris may be employed. In the case of filtration,
the flow-through will be harvested and used subsequently and in the
case of centrifugation (at speeds in a range of e.g. 200 g for 10
min to 6000 g for 1-10 min) the supernatant will be used. In cases
where more stringent purification is desired (e.g. for particularly
sensitive cell types such as human ES cells or in a clinical
application) it may be possible to follow up with subsequent
purification steps. In an aspect of the invention, a sequence of
molecular weight cutoff filters may be used (e.g. Amicon filters,
millipore).
[0425] FBS Substitutes
[0426] The use of fetal bovine serum in the production of
supernatant AA V may prove problematic for certain downstream
applications. For example, the application of FBS-containing AAV
supernatant to embryonic stem cells would result in uncontrolled
differentiation of the pluripotent cultures. Also, the use of
undefined FBS is incompatible with human clinical applications. In
order to mitigate the issues arising from the use of FBS, the
invention comprehends the culture medium used to support the AAV
producing 293FT cells being replaced with a chemically-defined
serum-free medium. For example, Pro293a from Lanza Biologics is a
chemically-defined, serum-free medium designed to support the
growth and protein production of adherent 293 lineage cells. With
regards to the AAV supernatant production protocol details in the
examples herein, all media components would simply be replaced with
Pro293a or another suitable medium substitute.
[0427] Reasons to Use AAV
[0428] Non-integration: A major motivation for the use of AAV in
the field of gene therapy is the relative lack of random genomic
integration compared to lentivirus, retrovirus, and other
integrating viral vectors. The majority of transduced recombinant
AAV genetic material exists in the host cell as episomes, rather
than at randomly integrated chromosomal locations. In human cells,
if the appropriate helper genes are provided, the AAV genome can
integrate at the well-characterized safe harbor locus AAVS 1. These
characteristics reduce the chance for oncogenic integration, making
AAV the current preferred viral system for human gene therapy. The
non-integration of AAV also provides advantages for functional
genomic studies. By providing trans genes or expression modulation
systems via AAV, rather than an integrating virus, one can be
assured that the cell population being used maintains an otherwise
isogenic background.
[0429] Functional Genomics: Cell Type Addressability
[0430] The generation of large libraries of RNAi, ORFs, targeted
nucleases (ZFNs, TALENs, CRISPR/Cas9), transcriptional modulators
(TALE-TFs, CRISPR/dCAS9 effectors), and other gene expression tools
has enabled large-scale arrayed functional genomics. These types of
experiments, however, are limited to cell types to which such gene
expression tools can be delivered in high-throughput. The
high-throughput scalability of Applicants' AAV supernatant
production protocol allows for the application of functional
genomics techniques to cell types for which AAV is the ideal
delivery mechanism. For example, AAV may be used to transduce
primary cortical neurons with higher efficiency than lentiviral
transduction or plasmid transfection, with lower toxicity than
lentiviral delivery.
[0431] Pooling
[0432] The herein described AAV supernatant production method may
be used to generate functional, pooled AAV supernatant. In an
embodiment of the invention, several genes of interest, encoded on
separate AAV backbone plasmids can be pooled at the plasmid stage
to produce a final supernatant containing a mixture of the desired
AAV vectors. Several types of gene delivery applications may
benefit from a pooling approach. First, some experiments in which a
large number of viral vectors must be functionally tested could be
performed in a hierarchical pooled fashion. For example, groups of
multiple RNAi or ORFs could be delivered in pooled AAV format to
reduce the size of the initial search space, saving experimental
time and cost. Second, complicated multicomponent gene expression
systems may be produced via a pooled AAV format. For example, the
differentiation of embryonic stem cells or reprogramming of one
cell type to another often requires the delivery of numerous
transcription factors simultaneously. Methods of the invention
encompassing pooled AAV supernatant production could rapidly
provide many different transcription factor combinations, simply by
altering the mixtures of AAV backbone plasmids, which may be
automated by liquid handling robotics. Third, artificial
transcription factors, such as TALE-TFs and CRISPR/Cas9 activators,
have been shown to have synergistic effects when provided in
combination to target cells. Pooled AAV supernatant production
could rapidly provide many different TALE-TF, CRISPR/Cas9, or other
engineered gene expression modulators, simply by altering the
mixtures of AAV backbone plasmids. This approach has been validated
for pooled TALE-TFs designed to activate gene expression in mouse
primary cortical neurons. Ten separate TALE-VP64 activators
designed to target the Drd2 locus were produced by Applicants'
standard AAV supernatant production method. Simultaneously, an
equimolar mixture of all10 Drd2 targeting TALE-VP64 plasmids was
made, referred to as the "10 TALE mixture". The identical AAV
supernatant production protocol was used produce the pooled AAV
mixture, with the exception that the gene of interest backbone
plasmid was replaced by an equal mass of "10 TALE mixture"
plasmids. All AAV supernatants were harvested and applied to mouse
primary neuron cultures as previously described. Six days after
transduction, cell lysis, reverse transcription and qPCR were
performed on the neuron cultures to determine the expression levels
of Drd2. Gene expression levels were elevated for several of the
TALE-VP64 transduced cultures. The culture transduced with
supernatant from the "10 TALE mixture" was found to activate
expression from the Drd2 locus at a level equivalent to the most
potent individual TALE-VP64.
[0433] Multiple Harvests
[0434] Multiple supernatant AAV batches may be harvested from a
single AAV producing 293FT culture. Specifically, following the 48
hour post-transfection harvested described in Applicants' standard
AAV supernatant protocol, the culture medium may be replenished and
harvested again 24 hours later (72 hours post-transfection). Both
harvests contain functional AAV particles. In this presently
described multiple harvest protocol, the value of producing twice
as much AAV supernatant as Applicants' standard protocol saves time
and resources when producing many AAV cultures in an arrayed
format. This approach offers an advantage over current large-scale
AAV production methods. In current methods, the amount of AAV that
can be produced is limited by the mass of 293 cells producing the
viral particles, as these methods typically require lysing the
producer cells to harvest the AAV particles. By stably expressing
the AAV expression plasmids in a 293 producer cell line, one could
continually harvest AAV supernatant batches simply by maintaining
the cell cultures, periodically collecting the supernatant, and
replenishing the culture medium.
[0435] In additional embodiments, the invention comprises a method
for obtaining and optionally storing a sample containing a set
amount of a Dependovirus-based vector comprising or consisting
essentially of: (a) creating infected or transfected cells by a
process comprising or consisting essentially of one or more methods
selected from: (i) transfecting plasmid(s) containing or consisting
essentially of exogenous DNA including DNA for expression into
Dependovirus-based vector-infected cells along with another helper
plasmid that provides Dependovirus rep and/or cap genes which are
obligatory for replication and packaging of the Dependovirus-based
vector; or (ii) infecting susceptible cells with a
Dependovirus-based vector containing or consisting essentially of
exogenous DNA including DNA for expression, and helper virus
wherein the Dependovirus-based vector lacks functioning cap and/or
rep and the helper virus provides the cap and/or rev function that
the Dependovirus-based vector lacks; or (iii) infecting susceptible
cells with a Dependovirus-based vector containing or consisting
essentially of exogenous DNA including DNA for expression, wherein
the recombinant construct lacks functioning cap and/or rep, and
transfecting said cells with a plasmid supplying cap and/or rep
function that the Dependovirus-based vector lacks; or (iv)
infecting susceptible cells with a Dependovirus-based vector
containing or consisting essentially of exogenous DNA including DNA
for expression, wherein the recombinant construct lacks functioning
cap and/or rep, wherein said cells supply cap and/or rep function
that the recombinant construct lacks; or (v) transfecting the
susceptible cells with a Dependovirus-based vector lacking
functioning cap and/or rep and plasmids for inserting exogenous DNA
into the recombinant construct so that the exogenous DNA is
expressed by the recombinant construct and for supplying rep and/or
cap functions whereby transfection results in a Dependovirus-based
vector containing or consisting essentially of the exogenous DNA
including DNA for expression that lacks functioning cap and/or rep;
and (b) incubating the infected or transfected cells, whereby there
results infected or transfected cells and supernatant containing
the Dependovirus-based vector lacking functioning cap and/or rep;
(c) after incubating, extracting an aliquot from the supernatant;
(d) filtering the aliquot, whereby the filtered aliquot contains
and the method obtains a sample containing set amount of the
Dependovirus-based vector relative to the type and amount of
susceptible cells infected or transfected; and (e) optionally
freezing the filtered aliquot, whereby the method optionally
includes storing a sample containing set amount of the
Dependovirus-based vector relative to the type and amount of
susceptible cells infected or transfected.
[0436] In one aspect, the Dependovirus-based vector of the
invention is derived from one or more Dependoviruses selected from
one or more of: adeno associated virus (AAV), Adenovirus,
parvovirus, Erythrovirus, Bocavirus and the like. In one aspect,
the Dependovirus-based vector of the invention is derived from a
recombinant adeno associated virus (rAAV).
[0437] The invention is further described by the following numbered
paragraphs:
[0438] 1. A method for obtaining and optionally storing a sample
containing a set amount of rAAV comprising or consisting
essentially of:
[0439] (a) creating infected or transfected cells by a process
comprising or consisting essentially of one or more methods
selected from:
[0440] (i) transfecting plasmid(s) containing or consisting
essentially of exogenous DNA including DNA for expression into
AAV-infected cells along with another helper plasmid that provides
AAV rep and/or cap genes which are obligatory for replication and
packaging of the rAAV; or
[0441] (ii) infecting susceptible cells with a rAAV containing or
consisting essentially of exogenous DNA including DNA for
expression, and helper virus wherein the rAAV lacks functioning cap
and/or rep and the helper virus provides the cap and/or rev
function that the rAAV lacks; or
[0442] (iii) infecting susceptible cells with a rAAV containing or
consisting essentially of exogenous DNA including DNA for
expression, wherein the recombinant construct lacks functioning cap
and/or rep, and transfecting said cells with a plasmid supplying
cap and/or rep function that the rAAV lacks; or
[0443] (iv) infecting susceptible cells with a rAAV containing or
consisting essentially of exogenous DNA including DNA for
expression, wherein the recombinant construct lacks functioning cap
and/or rep, wherein said cells supply cap and/or rep function that
the recombinant construct lacks; or
[0444] (v) transfecting the susceptible cells with an AAV lacking
functioning cap and/or rep and plasmids for inserting exogenous DNA
into the recombinant construct so that the exogenous DNA is
expressed by the recombinant construct and for supplying rep and/or
cap functions whereby transfection results in an rAAV containing or
consisting essentially of the exogenous DNA including DNA for
expression that lacks functioning cap and/or rep; and
[0445] (b) incubating the infected or transfected cells, whereby
there results infected or transfected cells and supernatant
containing the rAAV lacking functioning cap and/or rep;
[0446] (c) after incubating, extracting an aliquot from the
supernatant;
[0447] (d) filtering the aliquot, whereby the filtered aliquot
contains and the method obtains a sample containing set amount of
the rAAV relative to the type and amount of susceptible cells
infected or transfected; and
[0448] (e) optionally freezing the filtered aliquot,
whereby the method optionally includes storing a sample containing
set amount of the rAAV relative to the type and amount of
susceptible cells infected or transfected.
[0449] 2. A method for screening rAAV comprising or consisting
essentially of,
[0450] preparing the filtered aliquot or the stored filtered
aliquot of paragraph 1,
[0451] if necessary, thawing the stored filtered aliquot,
[0452] contacting the filtered aliquot with cells, and
[0453] determining whether the exogenous DNA is expressed in an
amount and/or duration sufficient for an intended use.
[0454] 3. The method of paragraph 2 wherein the contacting of the
filtered aliquot with cells comprises or consists essentially of
transducing said cells.
[0455] 4. The method of paragraph 3 wherein the contacting is for
5-6 days.
[0456] 5. The method of paragraph 2 wherein the rAAV expresses a
TALE and the contacting includes or consists essentially of
detecting nuclease, activator or repressor activity.
[0457] 6. The method of paragraph 2 wherein the rAAV expresses a
LITE, and the contacting includes or consists essentially of
inducing gene expression or subjecting the contacted cells to a
suitable stimulus, and detecting whether a transcriptional effector
has been induced.
[0458] 7. The method of paragraph 6 wherein detecting whether a
transcriptional effector has been induced includes or consists
essentially of detecting a color change.
[0459] 8. The method of paragraph 2 wherein the rAAV expresses a
CRISPR system, and the contacting includes or consists essentially
of detecting gene knockdown or other effects of the CRISPR
system.
[0460] 9. The method of paragraph 1 or 2 wherein the AAV is AAV1,
AAV2, AAV5 or an AAV having a hybrid or mosaic AAV1, AAV2 and/or
AAV5 capsid.
[0461] 10. The method of paragraph 1 or 2 wherein the susceptible
cells are 293FT cells.
[0462] 11. The method of paragraph 10 wherein 2.times.10.sup.5
cells are transfected or infected.
[0463] 12. The method of paragraph 11 wherein a 250 .mu.L filtered
aliquot contains the recombinant AAV at a concentration of about
5.6+/-0.24.times.10.sup.5.
[0464] 13. The method of any one of paragraphs 1 or 2 including
freezing the filtered aliquot.
[0465] 14. The method of paragraph 13 wherein the filtered aliquot
is frozen at about -80 C.
[0466] 15. The method of any one of paragraphs 1 or 2 including
adding a secretion enhancer to the cells before, during or after
and within the incubating.
[0467] 16. The method of paragraph 15 wherein the secretion
enhancer is polyethylenimine (PEI).
[0468] 17. A method of high-throughput screening of a sample
comprising or consisting essentially of contacting the supernatant
containing the rAAV lacking functioning cap and/or rep of any one
of paragraphs 1-16 with the sample and determining whether the
exogenous DNA of paragraph 1 is present in the sample.
[0469] 18. The method of paragraph 17, wherein the supernatant is
thawed from the filtered aliquot.
[0470] Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention
defined by the above paragraphs is not to be limited to particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope of the present invention.
Sequence CWU 1
1
350134PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser
Asn Gly Gly Gly Lys 1 5 10 15 Gln Ala Leu Glu Thr Val Gln Arg Leu
Leu Pro Val Leu Cys Gln Ala 20 25 30 His Gly 214DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 2tttattccct gacc 14332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
3Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly 20 25 30 432PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 4Leu Thr Pro Glu Gln Val Val Ala Ile
Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu
Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30 532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
5Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly 20 25 30 632PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 6Leu Thr Pro Ala Gln Val Val Ala Ile
Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu
Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30 732PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
7Leu Thr Pro Ala Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly 20 25 30 832PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 8Leu Thr Pro Asp Gln Val Val Ala Ile
Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu
Leu Pro Val Leu Cys Glu Gln His Gly 20 25 30 932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
9Leu Thr Pro Asp Gln Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly 20 25 30 1032PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 10Leu Thr Leu Asp Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30 1132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
11Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly 20 25 30 1232PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 12Leu Thr Pro Asp Gln Val Val Ala
Ile Ala Asn Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30 1332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
13Leu Thr Pro Asn Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly 20 25 30 1432PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 14Leu Thr Leu Asp Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30 1532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
15Leu Thr Pro Ala Gln Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly 20 25 30 1632PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 16Leu Thr Pro Glu Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Ala
Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30 1732PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
17Leu Thr Leu Asp Gln Val Val Ala Ile Ala Ser Gly Ser Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly 20 25 30 1832PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 18Leu Thr Gln Asp Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30 1932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
19Leu Ser Pro Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly 20 25 30 2032PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 20Leu Thr Pro Asp Gln Val Val Ala
Ile Ala Asn Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Leu Gln Arg
Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30 2132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
21Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Leu Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly 20 25 30 2232PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 22Leu Thr Pro Asp Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Arg Gln Ala His Gly 20 25 30 2332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
23Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Gly Gly Asn Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly 20 25 30 2432PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 24Leu Thr Pro Asp Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Ala Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30 2532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
25Leu Thr Pro Ala Gln Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly 20 25 30 2632PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 26Leu Thr Leu Ala Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30 2732PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
27Leu Thr Pro Glu Gln Val Val Ala Ile Ala Cys Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly 20 25 30 2832PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 28Leu Thr Pro Ala Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Gln
Leu Leu Pro Val Leu Cys Glu Gln His Gly 20 25 30 2932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
29Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Gly Gly Arg Pro Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly 20 25 30 3032PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 30Leu Thr Pro Asp Gln Val Val Ala
Ile Ala Ser Gly Ser Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30 3132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
31Leu Thr Pro Asn Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly 20 25 30 3232PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 32Leu Thr Pro Asp Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Gly Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30 3332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
33Leu Thr Leu Ala Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly 20 25 30 3432PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 34Leu Thr Pro Ala Gln Ala Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30 3532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
35Leu Thr Pro Ala Gln Val Val Ala Ile Ala Ser Gly Gly Asn Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly 20 25 30 3632PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 36Leu Thr Pro Asp Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Leu Gln Arg
Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30 3732PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
37Leu Thr Pro Asp Gln Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Leu Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly 20 25 30 3832PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 38Leu Thr Pro Asp Gln Val Val Thr
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30 3932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
39Leu Thr Pro Ala Gln Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Arg Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly 20 25 30 4032PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 40Leu Thr Pro Asp Gln Val Val Ala
Ile Ala Ser Gly Gly Asn Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30 4132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
41Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Thr His
Gly 20 25 30 4232PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 42Leu Pro Pro Asp Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30 4332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
43Leu Thr Ser Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly 20 25 30 4432PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 44Leu Thr Pro Ala Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Glu Gln His Gly 20 25 30 4532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
45Leu Ile Pro Ala Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly 20 25 30 4632PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 46Leu Thr Pro Ala Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Met Gln Arg
Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30 4732PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
47Leu Thr Arg Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly 20 25 30 4832PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 48Leu Thr Pro Asp Gln Val Val Ala
Thr Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30 4932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
49Leu Ile Pro Asp Gln Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly 20 25 30 5032PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 50Leu Thr Pro Asp Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Asn His Gly 20 25 30 5132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
51Leu Thr Leu Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Lys Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly 20
25 30 5232PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 52Leu Thr Pro Asp Gln Leu Val Ala Ile Ala Asn
Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro
Val Leu Cys Gln Asp His Gly 20 25 30 5332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
53Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Gly His
Gly 20 25 30 5432PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 54Leu Thr Pro Asp Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Glu His Gly 20 25 30 5532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
55Leu Thr Leu Asp Lys Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly 20 25 30 5632PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 56Leu Thr Pro Ala Gln Val Val Ala
Ile Ala Ser Gly Ser Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30 5732PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
57Leu Thr Pro Asp Lys Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly 20 25 30 5832PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 58Leu Thr Gln Asp Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Tyr Gln Asp His Gly 20 25 30 5932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
59Leu Thr Pro Ala Gln Val Val Ala Ile Val Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly 20 25 30 6032PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 60Leu Thr Pro Asp Lys Val Val Ala
Ile Ala Asn Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30 6132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
61Leu Thr Gln Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly 20 25 30 6232PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 62Leu Thr Pro Asp Gln Val Met Ala
Ile Ala Asn Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30 6332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
63Leu Thr Thr Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly 20 25 30 6432PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 64Leu Thr Pro Asp Gln Val Val Ala
Ile Ala Ser Gly Ser Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30 6532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
65Leu Thr Pro Asp Gln Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Leu Val Leu Cys Gln Ala His
Gly 20 25 30 6632PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 66Leu Thr Gln Glu Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30 6732PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
67Leu Thr Pro Asp Gln Val Val Thr Ile Ala Asn Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly 20 25 30 6832PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 68Leu Ser Pro Ala Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys His Asp His Gly 20 25 30 6932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
69Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Met Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly 20 25 30 7032PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 70Leu Ile Pro Asp Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30 7132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
71Leu Thr Pro Val Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
Gly 20 25 30 7232PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 72Leu Thr Pro Asp Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Lys Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30 7332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
73Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Met Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly 20 25 30 7432PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 74Leu Thr Pro Ala Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Phe Pro Val Leu Cys Gln Asp His Gly 20 25 30 7514DNAHomo
sapiens 75tttattccct gaca 147618DNAHomo sapiens 76tcggcccctg
ccggccca 187720DNAMus musculus 77tgcctgccct ccaggctcct
207840DNAHomo sapiens 78aaacggaagg gcctgagtcc gagcagaaga agaagtttta
407940DNAHomo sapiens 79aggaggaagg gcctgagtcc gagcagaaga agaagggctc
408040DNAHomo sapiens 80gagcccttct tcttctgctc ggactcaggc ccttcctcct
408126RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 81gaguccgagc agaagaagaa guuuua
268239DNAHomo sapiens 82aggaggaagg gcctgagtcc gagcagaaga gaagggctc
398339DNAHomo sapiens 83ggaggaaggg cctgagtccg agcagaagaa gaagggctc
398438DNAHomo sapiens 84ggaggaaggg cctgagtccg agcagaagag aagggctc
388540DNAHomo sapiens 85ggaggaaggg cctgagtccg agcagaagaa agaagggctc
408637DNAHomo sapiens 86ggaggaaggg cctgagtccg agcagaagga agggctc
378736DNAHomo sapiens 87ggaggaaggg cctgagtccg agcagaagaa gggctc
368833DNAHomo sapiens 88ggaggaaggg cctgagtccg agcagaaggg ctc
338933DNAHomo sapiens 89ggaggaaggg cctgagcccg agcagaaggg ctc
3390122DNAHomo sapiens 90ggaggaaggg cctgagtccg agcagaagaa
gaagggctcc catcacatca accggtggcg 60cattgccacg aagcaggcca atggggagga
catcgatgtc acctccaatg actagggtgg 120gc 12291122DNAHomo sapiens
91gcccacccta gtcattggag gtgacatcga tgtcctcccc attggcctgc ttcgtggcaa
60tgcgccaccg gttgatgtga tgggagccct tcttcttctg ctcggactca ggcccttcct
120cc 1229248RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 92acnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnguuuuaga gcuaugcu 489367DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 93agcauagcaa guuaaaauaa ggctaguccg uuaucaacuu
gaaaaagugg caccgagucg 60gugcuuu 679462RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 94nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc
aaguuaaaau aaggcuaguc 60cg 629548DNAHomo sapiens 95ctggaggagg
aagggcctga gtccgagcag aagaagaagg gctcccat 489648DNAHomo sapiens
96atgggagccc ttcttcttct gctcggactc aggcccttcc tcctccag
489730RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 97gaguccgagc agaagaagaa guuuuagagc
309820RNAHomo sapiens 98gaguccgagc agaagaagau 209920RNAHomo sapiens
99gaguccgagc agaagaagua 2010020RNAHomo sapiens 100gaguccgagc
agaagaacaa 2010120RNAHomo sapiens 101gaguccgagc agaagaugaa
2010220RNAHomo sapiens 102gaguccgagc agaaguagaa 2010320RNAHomo
sapiens 103gaguccgagc agaugaagaa 2010420RNAHomo sapiens
104gaguccgagc acaagaagaa 2010520RNAHomo sapiens 105gaguccgagg
agaagaagaa 2010620RNAHomo sapiens 106gaguccgugc agaagaagaa
2010720RNAHomo sapiens 107gagucggagc agaagaagaa 2010820RNAHomo
sapiens 108gagaccgagc agaagaagaa 2010924DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 109aatgacaagc ttgctagcgg tggg 2411039DNAHomo
sapiens 110aaaacggaag ggcctgagtc cgagcagaag aagaagttt
3911139DNAHomo sapiens 111aaacaggggc cgagattggg tgttcagggc
agaggtttt 3911238DNAHomo sapiens 112aaaacggaag ggcctgagtc
cgagcagaag aagaagtt 3811340DNAHomo sapiens 113aacggaggga ggggcacaga
tgagaaactc agggttttag 4011438DNAHomo sapiens 114agcccttctt
cttctgctcg gactcaggcc cttcctcc 3811540DNAHomo sapiens 115cagggaggga
ggggcacaga tgagaaactc aggaggcccc 4011638DNAHomo sapiens
116ggaggaaggg cctgagtccg agcagaagaa gaagggct 3811740DNAHomo sapiens
117ggggcctcct gagtttctca tctgtgcccc tccctccctg 4011880DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 118ggcaatgcgc caccggttga tgtgatggga gcccttctag
gaggccccca gagcagccac 60tggggcctca acactcaggc 8011998DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 119ggacgaaaca ccggaaccat tcaaaacagc atagcaagtt
aaaataaggc tagtccgtta 60tcaacttgaa aaagtggcac cgagtcggtg cttttttt
98120186DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 120ggacgaaaca ccggtagtat taagtattgt
tttatggctg ataaatttct ttgaatttct 60ccttgattat ttgttataaa agttataaaa
taatcttgtt ggaaccattc aaaacagcat 120agcaagttaa aataaggcta
gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 180tttttt
18612173DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 121tgaatggtcc caaaacggaa gggcctgagt
ccgagcagaa gaagaagttt tagagctatg 60ctgttttgaa tgg
7312295DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 122gggttttaga gctatgctgt tttgaatggt
cccaaaacgg gtcttcgaga agacgtttta 60gagctatgct gttttgaatg gtcccaaaac
ttttt 9512395DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 123aaaaagtttt gggaccattc
aaaacagcat agctctaaaa cgtcttctcg aagacccgtt 60ttgggaccat tcaaaacagc
atagctctaa aaccc 9512436DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 124aaacnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnngt 3612536DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 125taaaacnnnn nnnnnnnnnn nnnnnnnnnn nnnnnn
3612684DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 126gtggaaagga cgaaacaccg ggtcttcgag
aagacctgtt ttagagctag aaatagcaag 60ttaaaataag gctagtccgt tttt
8412784DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 127aaaaacggac tagccttatt ttaacttgct
atttctagct ctaaaacagg tcttctcgaa 60gacccggtgt ttcgtccttt ccac
8412824DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 128caccgnnnnn nnnnnnnnnn nnnn
2412924DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 129aaacnnnnnn nnnnnnnnnn nnnc
2413069DNAHomo sapiens 130ctggtcttcc acctctctgc cctgaacacc
caatctcggc ccctctcgcc accctcctgc 60atttctgtt 6913169DNAHomo sapiens
131aacagaaatg caggagggtg gcgagagggg ccgagattgg gtgttcaggg
cagagaggtg 60gaagaccag 69132138DNAMus musculus 132acccaagcac
tgagtgccat tagctaaatg catagggtac cacccacagg tgccaggggc 60ctttcccaaa
gttcccagcc ccttctccaa cctttcctgg cccagaggct ttcccatgtg
120tgtggctgga ccctttga 138133138DNAMus musculus 133tcaaagggtc
cagccacaca catgggaaag cctctgggcc aggaaaggtt ggagaagggg 60ctgggaactt
tgggaaaggc ccctggcacc tgtgggtggt accctatgca tttagctaat
120ggcactcagt gcttgggt 13813446RNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 134nnnnnnnnnn
nnnnnnnnng uuauuguacu cucaagauuu auuuuu 4613591RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 135guuacuuaaa ucuugcagaa gcuacaaaga uaaggcuuca
ugccgaaauc aacacccugu 60cauuuuaugg caggguguuu ucguuauuua a
9113670DNAHomo sapiens 136ttttctagtg ctgagtttct gtgactcctc
tacattctac ttctctgtgt ttctgtatac 60tacctcctcc 7013770DNAHomo
sapiens 137ggaggaggta gtatacagaa acacagagaa gtagaatgta gaggagtcac
agaaactcag 60cactagaaaa 701387DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 138nnagaaw
713934PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 139Leu Thr Pro Glu Gln Val Val Ala Ile Ala
Ser Xaa Xaa Gly Gly Lys 1 5 10
15 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30 His Gly 14014DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 140tgcaagagta ggag
1414114DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 141tctgcaagag tagg 1414214DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 142ttggaggagc acca 1414314DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 143tgcactccac cttg 1414414DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 144tcaagcagct tctc 1414514DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 145tcagagctgt cctc 1414620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 146tgcctgccct ccaggctcct 20147288PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
147Met Asp Pro Ile Arg Ser Arg Thr Pro Ser Pro Ala Arg Glu Leu Leu
1 5 10 15 Ser Gly Pro Gln Pro Asp Gly Val Gln Pro Thr Ala Asp Arg
Gly Val 20 25 30 Ser Pro Pro Ala Gly Gly Pro Leu Asp Gly Leu Pro
Ala Arg Arg Thr 35 40 45 Met Ser Arg Thr Arg Leu Pro Ser Pro Pro
Ala Pro Ser Pro Ala Phe 50 55 60 Ser Ala Asp Ser Phe Ser Asp Leu
Leu Arg Gln Phe Asp Pro Ser Leu 65 70 75 80 Phe Asn Thr Ser Leu Phe
Asp Ser Leu Pro Pro Phe Gly Ala His His 85 90 95 Thr Glu Ala Ala
Thr Gly Glu Trp Asp Glu Val Gln Ser Gly Leu Arg 100 105 110 Ala Ala
Asp Ala Pro Pro Pro Thr Met Arg Val Ala Val Thr Ala Ala 115 120 125
Arg Pro Pro Arg Ala Lys Pro Ala Pro Arg Arg Arg Ala Ala Gln Pro 130
135 140 Ser Asp Ala Ser Pro Ala Ala Gln Val Asp Leu Arg Thr Leu Gly
Tyr 145 150 155 160 Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val
Arg Ser Thr Val 165 170 175 Ala Gln His His Glu Ala Leu Val Gly His
Gly Phe Thr His Ala His 180 185 190 Ile Val Ala Leu Ser Gln His Pro
Ala Ala Leu Gly Thr Val Ala Val 195 200 205 Lys Tyr Gln Asp Met Ile
Ala Ala Leu Pro Glu Ala Thr His Glu Ala 210 215 220 Ile Val Gly Val
Gly Lys Gln Trp Ser Gly Ala Arg Ala Leu Glu Ala 225 230 235 240 Leu
Leu Thr Val Ala Gly Glu Leu Arg Gly Pro Pro Leu Gln Leu Asp 245 250
255 Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly Gly Val Thr Ala Val
260 265 270 Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr Gly Ala Pro
Leu Asn 275 280 285 148183PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 148Arg Pro Ala Leu Glu
Ser Ile Val Ala Gln Leu Ser Arg Pro Asp Pro 1 5 10 15 Ala Leu Ala
Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu 20 25 30 Gly
Gly Arg Pro Ala Leu Asp Ala Val Lys Lys Gly Leu Pro His Ala 35 40
45 Pro Ala Leu Ile Lys Arg Thr Asn Arg Arg Ile Pro Glu Arg Thr Ser
50 55 60 His Arg Val Ala Asp His Ala Gln Val Val Arg Val Leu Gly
Phe Phe 65 70 75 80 Gln Cys His Ser His Pro Ala Gln Ala Phe Asp Asp
Ala Met Thr Gln 85 90 95 Phe Gly Met Ser Arg His Gly Leu Leu Gln
Leu Phe Arg Arg Val Gly 100 105 110 Val Thr Glu Leu Glu Ala Arg Ser
Gly Thr Leu Pro Pro Ala Ser Gln 115 120 125 Arg Trp Asp Arg Ile Leu
Gln Ala Ser Gly Met Lys Arg Ala Lys Pro 130 135 140 Ser Pro Thr Ser
Thr Gln Thr Pro Asp Gln Ala Ser Leu His Ala Phe 145 150 155 160 Ala
Asp Ser Leu Glu Arg Asp Leu Asp Ala Pro Ser Pro Met His Glu 165 170
175 Gly Asp Gln Thr Arg Ala Ser 180 14918DNAMus musculus
149tgaatgatga taatacga 1815011PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 150Ser Pro Lys Lys Lys Arg
Lys Val Glu Ala Ser 1 5 10 1517PRTSimian virus 40 151Pro Lys Lys
Lys Arg Lys Val 1 5 15216PRTUnknownDescription of Unknown
Nucleoplasmin bipartite NLS sequence 152Lys Arg Pro Ala Ala Thr Lys
Lys Ala Gly Gln Ala Lys Lys Lys Lys 1 5 10 15
1539PRTUnknownDescription of Unknown C-myc NLS sequence 153Pro Ala
Ala Lys Arg Val Lys Leu Asp 1 5 15411PRTUnknownDescription of
Unknown C-myc NLS sequence 154Arg Gln Arg Arg Asn Glu Leu Lys Arg
Ser Pro 1 5 10 15538PRTHomo sapiens 155Asn Gln Ser Ser Asn Phe Gly
Pro Met Lys Gly Gly Asn Phe Gly Gly 1 5 10 15 Arg Ser Ser Gly Pro
Tyr Gly Gly Gly Gly Gln Tyr Phe Ala Lys Pro 20 25 30 Arg Asn Gln
Gly Gly Tyr 35 15642PRTUnknownDescription of Unknown IBB domain
from importin-alpha sequence 156Arg Met Arg Ile Glx Phe Lys Asn Lys
Gly Lys Asp Thr Ala Glu Leu 1 5 10 15 Arg Arg Arg Arg Val Glu Val
Ser Val Glu Leu Arg Lys Ala Lys Lys 20 25 30 Asp Glu Gln Ile Leu
Lys Arg Arg Asn Val 35 40 1578PRTUnknownDescription of Unknown
Myoma T protein sequence 157Val Ser Arg Lys Arg Pro Arg Pro 1 5
1588PRTUnknownDescription of Unknown Myoma T protein sequence
158Pro Pro Lys Lys Ala Arg Glu Asp 1 5 1598PRTHomo sapiens 159Pro
Gln Pro Lys Lys Lys Pro Leu 1 5 16012PRTMus musculus 160Ser Ala Leu
Ile Lys Lys Lys Lys Lys Met Ala Pro 1 5 10 1615PRTInfluenza virus
161Asp Arg Leu Arg Arg 1 5 1627PRTInfluenza virus 162Pro Lys Gln
Lys Lys Arg Lys 1 5 16310PRTHepatitus delta virus 163Arg Lys Leu
Lys Lys Lys Ile Lys Lys Leu 1 5 10 16410PRTMus musculus 164Arg Glu
Lys Lys Lys Phe Leu Lys Arg Arg 1 5 10 16520PRTHomo sapiens 165Lys
Arg Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val Ala Lys Lys 1 5 10
15 Lys Ser Lys Lys 20 16617PRTHomo sapiens 166Arg Lys Cys Leu Gln
Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys 1 5 10 15 Lys
16714DNAHomo sapiens 167ttcttactta taac 141681603PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
168Met Ser Arg Thr Arg Leu Pro Ser Pro Pro Ala Pro Ser Pro Ala Phe
1 5 10 15 Ser Ala Asp Ser Phe Ser Asp Leu Leu Arg Gln Phe Asp Pro
Ser Leu 20 25 30 Phe Asn Thr Ser Leu Phe Asp Ser Leu Pro Pro Phe
Gly Ala His His 35 40 45 Thr Glu Ala Ala Thr Gly Glu Trp Asp Glu
Val Gln Ser Gly Leu Arg 50 55 60 Ala Ala Asp Ala Pro Pro Pro Thr
Met Arg Val Ala Val Thr Ala Ala 65 70 75 80 Arg Pro Pro Arg Ala Lys
Pro Ala Pro Arg Arg Arg Ala Ala Gln Pro 85 90 95 Ser Asp Ala Ser
Pro Ala Ala Gln Val Asp Leu Arg Thr Leu Gly Tyr 100 105 110 Ser Gln
Gln Gln Gln Glu Lys Ile Lys Pro Lys Val Arg Ser Thr Val 115 120 125
Ala Gln His His Glu Ala Leu Val Gly His Gly Phe Thr His Ala His 130
135 140 Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly Thr Val Ala
Val 145 150 155 160 Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu Ala
Thr His Glu Ala 165 170 175 Ile Val Gly Val Gly Lys Gln Trp Ser Gly
Ala Arg Ala Leu Glu Ala 180 185 190 Leu Leu Thr Val Ala Gly Glu Leu
Arg Gly Pro Pro Leu Gln Leu Asp 195 200 205 Thr Gly Gln Leu Leu Lys
Ile Ala Lys Arg Gly Gly Val Thr Ala Val 210 215 220 Glu Ala Val His
Ala Trp Arg Asn Ala Leu Thr Gly Ala Pro Leu Asn 225 230 235 240 Leu
Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys 245 250
255 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
260 265 270 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
Asp Gly 275 280 285 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys 290 295 300 Gln Ala His Gly Leu Thr Pro Glu Gln Val
Val Ala Ile Ala Ser His 305 310 315 320 Asp Gly Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu Pro Val 325 330 335 Leu Cys Gln Ala His
Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 340 345 350 Ser Asn Ile
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 355 360 365 Pro
Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 370 375
380 Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
385 390 395 400 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
Glu Gln Val 405 410 415 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
Ala Leu Glu Thr Val 420 425 430 Gln Arg Leu Leu Pro Val Leu Cys Gln
Ala His Gly Leu Thr Pro Glu 435 440 445 Gln Val Val Ala Ile Ala Ser
His Asp Gly Gly Lys Gln Ala Leu Glu 450 455 460 Thr Val Gln Arg Leu
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 465 470 475 480 Pro Glu
Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala 485 490 495
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 500
505 510 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly
Lys 515 520 525 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
Cys Gln Ala 530 535 540 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile
Ala Ser Asn Asn Gly 545 550 555 560 Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg Leu Leu Pro Val Leu Cys 565 570 575 Gln Ala His Gly Leu Thr
Pro Glu Gln Val Val Ala Ile Ala Ser Asn 580 585 590 Gly Gly Gly Lys
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 595 600 605 Leu Cys
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 610 615 620
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 625
630 635 640 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
Val Ala 645 650 655 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg 660 665 670 Leu Leu Pro Val Leu Cys Gln Ala His Gly
Leu Thr Pro Glu Gln Val 675 680 685 Val Ala Ile Ala Ser Asn Asn Gly
Gly Lys Gln Ala Leu Glu Thr Val 690 695 700 Gln Arg Leu Leu Pro Val
Leu Cys Gln Ala His Gly Leu Thr Pro Glu 705 710 715 720 Gln Val Val
Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu 725 730 735 Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 740 745
750 Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala
755 760 765 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 770 775 780 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
Asp Gly Gly Arg 785 790 795 800 Pro Ala Leu Glu Ser Ile Val Ala Gln
Leu Ser Arg Pro Asp Pro Ala 805 810 815 Leu Ala Ala Leu Thr Asn Asp
His Leu Val Ala Leu Ala Cys Leu Gly 820 825 830 Gly Arg Pro Ala Leu
Asp Ala Val Lys Lys Gly Leu Pro His Ala Pro 835 840 845 Ala Leu Ile
Lys Arg Thr Asn Arg Arg Ile Pro Glu Arg Thr Ser His 850 855 860 Arg
Val Ala Asp His Ala Gln Val Val Arg Val Leu Gly Phe Phe Gln 865 870
875 880 Cys His Ser His Pro Ala Gln Ala Phe Asp Asp Ala Met Thr Gln
Phe 885 890 895 Gly Met Ser Arg His Gly Leu Leu Gln Leu Phe Arg Arg
Val Gly Val 900 905 910 Thr Glu Leu Glu Ala Arg Ser Gly Thr Leu Pro
Pro Ala Ser Gln Arg 915 920 925 Trp Asp Arg Ile Leu Gln Ala Ser Gly
Met Lys Arg Ala Lys Pro Ser 930 935 940 Pro Thr Ser Thr Gln Thr Pro
Asp Gln Ala Ser Leu His Ala Phe Ala 945 950 955 960 Asp Ser Leu Glu
Arg Asp Leu Asp Ala Pro Ser Pro Met His Glu Gly 965 970 975 Asp Gln
Thr Arg Ala Ser Ala Ser Pro Lys Lys Lys Arg Lys Val Glu 980 985 990
Ala Ser Lys Met Asp Lys Lys Thr Ile Val Trp Phe Arg Arg Asp Leu 995
1000 1005 Arg Ile Glu Asp Asn Pro Ala Leu Ala Ala Ala Ala His Glu
Gly 1010 1015 1020 Ser Val Phe Pro Val Phe Ile Trp Cys Pro Glu Glu
Glu Gly Gln 1025 1030 1035 Phe Tyr Pro Gly Arg Ala Ser Arg Trp Trp
Met Lys Gln Ser Leu 1040 1045 1050 Ala His Leu Ser Gln Ser Leu Lys
Ala Leu Gly Ser Asp Leu Thr 1055 1060 1065 Leu Ile Lys Thr His Asn
Thr Ile Ser Ala Ile Leu Asp Cys Ile 1070 1075 1080 Arg Val Thr Gly
Ala Thr Lys Val Val Phe Asn His Leu Tyr Asp 1085 1090 1095 Pro Val
Ser Leu Val Arg Asp His Thr Val Lys Glu Lys Leu Val 1100 1105 1110
Glu Arg Gly Ile Ser Val Gln Ser Tyr Asn Gly Asp Leu Leu Tyr 1115
1120 1125 Glu Pro Trp Glu Ile Tyr Cys Glu Lys Gly Lys Pro Phe Thr
Ser 1130 1135 1140 Phe Asn Ser Tyr Trp Lys Lys Cys Leu Asp Met Ser
Ile Glu Ser 1145 1150 1155 Val Met Leu Pro Pro Pro Trp Arg Leu Met
Pro Ile Thr Ala Ala 1160 1165 1170 Ala Glu Ala Ile Trp Ala Cys Ser
Ile Glu Glu Leu Gly Leu Glu 1175 1180 1185 Asn Glu Ala Glu Lys Pro
Ser Asn Ala Leu Leu Thr Arg Ala Trp 1190 1195 1200 Ser Pro Gly Trp
Ser Asn Ala Asp Lys Leu Leu Asn Glu Phe Ile 1205 1210 1215 Glu Lys
Gln Leu Ile Asp Tyr Ala Lys Asn Ser Lys Lys Val Val 1220 1225 1230
Gly Asn Ser Thr Ser Leu Leu Ser Pro Tyr Leu His Phe Gly Glu 1235
1240 1245
Ile Ser Val Arg His Val Phe Gln Cys Ala Arg Met Lys Gln Ile 1250
1255 1260 Ile Trp Ala Arg Asp Lys Asn Ser Glu Gly Glu Glu Ser Ala
Asp 1265 1270 1275 Leu Phe Leu Arg Gly Ile Gly Leu Arg Glu Tyr Ser
Arg Tyr Ile 1280 1285 1290 Cys Phe Asn Phe Pro Phe Thr His Glu Gln
Ser Leu Leu Ser His 1295 1300 1305 Leu Arg Phe Phe Pro Trp Asp Ala
Asp Val Asp Lys Phe Lys Ala 1310 1315 1320 Trp Arg Gln Gly Arg Thr
Gly Tyr Pro Leu Val Asp Ala Gly Met 1325 1330 1335 Arg Glu Leu Trp
Ala Thr Gly Trp Met His Asn Arg Ile Arg Val 1340 1345 1350 Ile Val
Ser Ser Phe Ala Val Lys Phe Leu Leu Leu Pro Trp Lys 1355 1360 1365
Trp Gly Met Lys Tyr Phe Trp Asp Thr Leu Leu Asp Ala Asp Leu 1370
1375 1380 Glu Cys Asp Ile Leu Gly Trp Gln Tyr Ile Ser Gly Ser Ile
Pro 1385 1390 1395 Asp Gly His Glu Leu Asp Arg Leu Asp Asn Pro Ala
Leu Gln Gly 1400 1405 1410 Ala Lys Tyr Asp Pro Glu Gly Glu Tyr Ile
Arg Gln Trp Leu Pro 1415 1420 1425 Glu Leu Ala Arg Leu Pro Thr Glu
Trp Ile His His Pro Trp Asp 1430 1435 1440 Ala Pro Leu Thr Val Leu
Lys Ala Ser Gly Val Glu Leu Gly Thr 1445 1450 1455 Asn Tyr Ala Lys
Pro Ile Val Asp Ile Asp Thr Ala Arg Glu Leu 1460 1465 1470 Leu Ala
Lys Ala Ile Ser Arg Thr Arg Glu Ala Gln Ile Met Ile 1475 1480 1485
Gly Ala Ala Pro Asp Glu Ile Val Ala Asp Ser Phe Glu Ala Leu 1490
1495 1500 Gly Ala Asn Thr Ile Lys Glu Pro Gly Leu Cys Pro Ser Val
Ser 1505 1510 1515 Ser Asn Asp Gln Gln Val Pro Ser Ala Val Arg Tyr
Asn Gly Ser 1520 1525 1530 Lys Arg Val Lys Pro Glu Glu Glu Glu Glu
Arg Asp Met Lys Lys 1535 1540 1545 Ser Arg Gly Phe Asp Glu Arg Glu
Leu Phe Ser Thr Ala Glu Ser 1550 1555 1560 Ser Ser Ser Ser Ser Val
Phe Phe Val Ser Gln Ser Cys Ser Leu 1565 1570 1575 Ala Ser Glu Gly
Lys Asn Leu Glu Gly Ile Gln Asp Ser Ser Asp 1580 1585 1590 Gln Ile
Thr Thr Ser Leu Gly Lys Asn Gly 1595 1600 1691492PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
169Met Ser Arg Thr Arg Leu Pro Ser Pro Pro Ala Pro Ser Pro Ala Phe
1 5 10 15 Ser Ala Asp Ser Phe Ser Asp Leu Leu Arg Gln Phe Asp Pro
Ser Leu 20 25 30 Phe Asn Thr Ser Leu Phe Asp Ser Leu Pro Pro Phe
Gly Ala His His 35 40 45 Thr Glu Ala Ala Thr Gly Glu Trp Asp Glu
Val Gln Ser Gly Leu Arg 50 55 60 Ala Ala Asp Ala Pro Pro Pro Thr
Met Arg Val Ala Val Thr Ala Ala 65 70 75 80 Arg Pro Pro Arg Ala Lys
Pro Ala Pro Arg Arg Arg Ala Ala Gln Pro 85 90 95 Ser Asp Ala Ser
Pro Ala Ala Gln Val Asp Leu Arg Thr Leu Gly Tyr 100 105 110 Ser Gln
Gln Gln Gln Glu Lys Ile Lys Pro Lys Val Arg Ser Thr Val 115 120 125
Ala Gln His His Glu Ala Leu Val Gly His Gly Phe Thr His Ala His 130
135 140 Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly Thr Val Ala
Val 145 150 155 160 Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu Ala
Thr His Glu Ala 165 170 175 Ile Val Gly Val Gly Lys Gln Trp Ser Gly
Ala Arg Ala Leu Glu Ala 180 185 190 Leu Leu Thr Val Ala Gly Glu Leu
Arg Gly Pro Pro Leu Gln Leu Asp 195 200 205 Thr Gly Gln Leu Leu Lys
Ile Ala Lys Arg Gly Gly Val Thr Ala Val 210 215 220 Glu Ala Val His
Ala Trp Arg Asn Ala Leu Thr Gly Ala Pro Leu Asn 225 230 235 240 Leu
Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys 245 250
255 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
260 265 270 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
Asp Gly 275 280 285 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys 290 295 300 Gln Ala His Gly Leu Thr Pro Glu Gln Val
Val Ala Ile Ala Ser His 305 310 315 320 Asp Gly Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu Pro Val 325 330 335 Leu Cys Gln Ala His
Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 340 345 350 Ser Asn Ile
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 355 360 365 Pro
Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 370 375
380 Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
385 390 395 400 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
Glu Gln Val 405 410 415 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
Ala Leu Glu Thr Val 420 425 430 Gln Arg Leu Leu Pro Val Leu Cys Gln
Ala His Gly Leu Thr Pro Glu 435 440 445 Gln Val Val Ala Ile Ala Ser
His Asp Gly Gly Lys Gln Ala Leu Glu 450 455 460 Thr Val Gln Arg Leu
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 465 470 475 480 Pro Glu
Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala 485 490 495
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 500
505 510 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly
Lys 515 520 525 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
Cys Gln Ala 530 535 540 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile
Ala Ser Asn Asn Gly 545 550 555 560 Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg Leu Leu Pro Val Leu Cys 565 570 575 Gln Ala His Gly Leu Thr
Pro Glu Gln Val Val Ala Ile Ala Ser Asn 580 585 590 Gly Gly Gly Lys
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 595 600 605 Leu Cys
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 610 615 620
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 625
630 635 640 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
Val Ala 645 650 655 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg 660 665 670 Leu Leu Pro Val Leu Cys Gln Ala His Gly
Leu Thr Pro Glu Gln Val 675 680 685 Val Ala Ile Ala Ser Asn Asn Gly
Gly Lys Gln Ala Leu Glu Thr Val 690 695 700 Gln Arg Leu Leu Pro Val
Leu Cys Gln Ala His Gly Leu Thr Pro Glu 705 710 715 720 Gln Val Val
Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu 725 730 735 Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 740 745
750 Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala
755 760 765 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 770 775 780 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
Asp Gly Gly Arg 785 790 795 800 Pro Ala Leu Glu Ser Ile Val Ala Gln
Leu Ser Arg Pro Asp Pro Ala 805 810 815 Leu Ala Ala Leu Thr Asn Asp
His Leu Val Ala Leu Ala Cys Leu Gly 820 825 830 Gly Arg Pro Ala Leu
Asp Ala Val Lys Lys Gly Leu Pro His Ala Pro 835 840 845 Ala Leu Ile
Lys Arg Thr Asn Arg Arg Ile Pro Glu Arg Thr Ser His 850 855 860 Arg
Val Ala Asp His Ala Gln Val Val Arg Val Leu Gly Phe Phe Gln 865 870
875 880 Cys His Ser His Pro Ala Gln Ala Phe Asp Asp Ala Met Thr Gln
Phe 885 890 895 Gly Met Ser Arg His Gly Leu Leu Gln Leu Phe Arg Arg
Val Gly Val 900 905 910 Thr Glu Leu Glu Ala Arg Ser Gly Thr Leu Pro
Pro Ala Ser Gln Arg 915 920 925 Trp Asp Arg Ile Leu Gln Ala Ser Gly
Met Lys Arg Ala Lys Pro Ser 930 935 940 Pro Thr Ser Thr Gln Thr Pro
Asp Gln Ala Ser Leu His Ala Phe Ala 945 950 955 960 Asp Ser Leu Glu
Arg Asp Leu Asp Ala Pro Ser Pro Met His Glu Gly 965 970 975 Asp Gln
Thr Arg Ala Ser Ala Ser Pro Lys Lys Lys Arg Lys Val Glu 980 985 990
Ala Ser Lys Met Asp Lys Lys Thr Ile Val Trp Phe Arg Arg Asp Leu 995
1000 1005 Arg Ile Glu Asp Asn Pro Ala Leu Ala Ala Ala Ala His Glu
Gly 1010 1015 1020 Ser Val Phe Pro Val Phe Ile Trp Cys Pro Glu Glu
Glu Gly Gln 1025 1030 1035 Phe Tyr Pro Gly Arg Ala Ser Arg Trp Trp
Met Lys Gln Ser Leu 1040 1045 1050 Ala His Leu Ser Gln Ser Leu Lys
Ala Leu Gly Ser Asp Leu Thr 1055 1060 1065 Leu Ile Lys Thr His Asn
Thr Ile Ser Ala Ile Leu Asp Cys Ile 1070 1075 1080 Arg Val Thr Gly
Ala Thr Lys Val Val Phe Asn His Leu Tyr Asp 1085 1090 1095 Pro Val
Ser Leu Val Arg Asp His Thr Val Lys Glu Lys Leu Val 1100 1105 1110
Glu Arg Gly Ile Ser Val Gln Ser Tyr Asn Gly Asp Leu Leu Tyr 1115
1120 1125 Glu Pro Trp Glu Ile Tyr Cys Glu Lys Gly Lys Pro Phe Thr
Ser 1130 1135 1140 Phe Asn Ser Tyr Trp Lys Lys Cys Leu Asp Met Ser
Ile Glu Ser 1145 1150 1155 Val Met Leu Pro Pro Pro Trp Arg Leu Met
Pro Ile Thr Ala Ala 1160 1165 1170 Ala Glu Ala Ile Trp Ala Cys Ser
Ile Glu Glu Leu Gly Leu Glu 1175 1180 1185 Asn Glu Ala Glu Lys Pro
Ser Asn Ala Leu Leu Thr Arg Ala Trp 1190 1195 1200 Ser Pro Gly Trp
Ser Asn Ala Asp Lys Leu Leu Asn Glu Phe Ile 1205 1210 1215 Glu Lys
Gln Leu Ile Asp Tyr Ala Lys Asn Ser Lys Lys Val Val 1220 1225 1230
Gly Asn Ser Thr Ser Leu Leu Ser Pro Tyr Leu His Phe Gly Glu 1235
1240 1245 Ile Ser Val Arg His Val Phe Gln Cys Ala Arg Met Lys Gln
Ile 1250 1255 1260 Ile Trp Ala Arg Asp Lys Asn Ser Glu Gly Glu Glu
Ser Ala Asp 1265 1270 1275 Leu Phe Leu Arg Gly Ile Gly Leu Arg Glu
Tyr Ser Arg Tyr Ile 1280 1285 1290 Cys Phe Asn Phe Pro Phe Thr His
Glu Gln Ser Leu Leu Ser His 1295 1300 1305 Leu Arg Phe Phe Pro Trp
Asp Ala Asp Val Asp Lys Phe Lys Ala 1310 1315 1320 Trp Arg Gln Gly
Arg Thr Gly Tyr Pro Leu Val Asp Ala Gly Met 1325 1330 1335 Arg Glu
Leu Trp Ala Thr Gly Trp Met His Asn Arg Ile Arg Val 1340 1345 1350
Ile Val Ser Ser Phe Ala Val Lys Phe Leu Leu Leu Pro Trp Lys 1355
1360 1365 Trp Gly Met Lys Tyr Phe Trp Asp Thr Leu Leu Asp Ala Asp
Leu 1370 1375 1380 Glu Cys Asp Ile Leu Gly Trp Gln Tyr Ile Ser Gly
Ser Ile Pro 1385 1390 1395 Asp Gly His Glu Leu Asp Arg Leu Asp Asn
Pro Ala Leu Gln Gly 1400 1405 1410 Ala Lys Tyr Asp Pro Glu Gly Glu
Tyr Ile Arg Gln Trp Leu Pro 1415 1420 1425 Glu Leu Ala Arg Leu Pro
Thr Glu Trp Ile His His Pro Trp Asp 1430 1435 1440 Ala Pro Leu Thr
Val Leu Lys Ala Ser Gly Val Glu Leu Gly Thr 1445 1450 1455 Asn Tyr
Ala Lys Pro Ile Val Asp Ile Asp Thr Ala Arg Glu Leu 1460 1465 1470
Leu Ala Lys Ala Ile Ser Arg Thr Arg Glu Ala Gln Ile Met Ile 1475
1480 1485 Gly Ala Ala Pro 1490 170665PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
170Met Asn Gly Ala Ile Gly Gly Asp Leu Leu Leu Asn Phe Pro Asp Met
1 5 10 15 Ser Val Leu Glu Arg Gln Arg Ala His Leu Lys Tyr Leu Asn
Pro Thr 20 25 30 Phe Asp Ser Pro Leu Ala Gly Phe Phe Ala Asp Ser
Ser Met Ile Thr 35 40 45 Gly Gly Glu Met Asp Ser Tyr Leu Ser Thr
Ala Gly Leu Asn Leu Pro 50 55 60 Met Met Tyr Gly Glu Thr Thr Val
Glu Gly Asp Ser Arg Leu Ser Ile 65 70 75 80 Ser Pro Glu Thr Thr Leu
Gly Thr Gly Asn Phe Lys Lys Arg Lys Phe 85 90 95 Asp Thr Glu Thr
Lys Asp Cys Asn Glu Lys Lys Lys Lys Met Thr Met 100 105 110 Asn Arg
Asp Asp Leu Val Glu Glu Gly Glu Glu Glu Lys Ser Lys Ile 115 120 125
Thr Glu Gln Asn Asn Gly Ser Thr Lys Ser Ile Lys Lys Met Lys His 130
135 140 Lys Ala Lys Lys Glu Glu Asn Asn Phe Ser Asn Asp Ser Ser Lys
Val 145 150 155 160 Thr Lys Glu Leu Glu Lys Thr Asp Tyr Ile His Val
Arg Ala Arg Arg 165 170 175 Gly Gln Ala Thr Asp Ser His Ser Ile Ala
Glu Arg Val Arg Arg Glu 180 185 190 Lys Ile Ser Glu Arg Met Lys Phe
Leu Gln Asp Leu Val Pro Gly Cys 195 200 205 Asp Lys Ile Thr Gly Lys
Ala Gly Met Leu Asp Glu Ile Ile Asn Tyr 210 215 220 Val Gln Ser Leu
Gln Arg Gln Ile Glu Phe Leu Ser Met Lys Leu Ala 225 230 235 240 Ile
Val Asn Pro Arg Pro Asp Phe Asp Met Asp Asp Ile Phe Ala Lys 245 250
255 Glu Val Ala Ser Thr Pro Met Thr Val Val Pro Ser Pro Glu Met Val
260 265 270 Leu Ser Gly Tyr Ser His Glu Met Val His Ser Gly Tyr Ser
Ser Glu 275 280 285 Met Val Asn Ser Gly Tyr Leu His Val Asn Pro Met
Gln Gln Val Asn 290 295 300 Thr Ser Ser Asp Pro Leu Ser Cys Phe Asn
Asn Gly Glu Ala Pro Ser 305 310 315 320 Met Trp Asp Ser His Val Gln
Asn Leu Tyr Gly Asn Leu Gly Val Ala 325 330 335 Ser Pro Lys Lys Lys
Arg Lys Val Glu Ala Ser Gly Ser Gly Arg Ala 340 345 350 Asp Ala Leu
Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu 355 360 365 Asp
Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe 370 375
380 Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp
Asp Phe Asp Leu Asp 385 390 395 400 Met Leu Ile Asn Ser Arg Gly Ser
Gly Glu Gly Arg Gly Ser Leu Leu 405 410 415 Thr Cys Gly Asp Val Glu
Glu Asn Pro Gly Pro Val Ser Lys Gly Glu 420 425 430 Glu Leu Phe Thr
Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp 435 440 445 Val Asn
Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala 450 455 460
Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu 465
470 475 480 Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly
Val Gln 485 490 495 Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His
Asp Phe Phe Lys 500 505 510 Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
Arg Thr Ile Phe Phe Lys 515 520 525 Asp Asp Gly Asn Tyr Lys Thr Arg
Ala Glu Val Lys Phe Glu Gly Asp 530 535 540 Thr Leu Val Asn Arg Ile
Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp 545 550 555 560 Gly Asn Ile
Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn 565 570 575 Val
Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe 580 585
590 Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His
595 600 605 Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu
Pro Asp 610 615 620 Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys
Asp Pro Asn Glu 625 630 635 640 Lys Arg Asp His Met Val Leu Leu Glu
Phe Val Thr Ala Ala Gly Ile 645 650 655 Thr Leu Gly Met Asp Glu Leu
Tyr Lys 660 665 171500PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 171Met Asn Gly Ala Ile
Gly Gly Asp Leu Leu Leu Asn Phe Pro Asp Met 1 5 10 15 Ser Val Leu
Glu Arg Gln Arg Ala His Leu Lys Tyr Leu Asn Pro Thr 20 25 30 Phe
Asp Ser Pro Leu Ala Gly Phe Phe Ala Asp Ser Ser Met Ile Thr 35 40
45 Gly Gly Glu Met Asp Ser Tyr Leu Ser Thr Ala Gly Leu Asn Leu Pro
50 55 60 Met Met Tyr Gly Glu Thr Thr Val Glu Gly Asp Ser Arg Leu
Ser Ile 65 70 75 80 Ser Pro Glu Thr Thr Leu Gly Thr Gly Asn Phe Lys
Lys Arg Lys Phe 85 90 95 Asp Thr Glu Thr Lys Asp Cys Asn Glu Lys
Lys Lys Lys Met Thr Met 100 105 110 Asn Arg Asp Asp Leu Val Glu Glu
Gly Glu Glu Glu Lys Ser Lys Ile 115 120 125 Thr Glu Gln Asn Asn Gly
Ser Thr Lys Ser Ile Lys Lys Met Lys His 130 135 140 Lys Ala Lys Lys
Glu Glu Asn Asn Phe Ser Asn Asp Ser Ser Lys Val 145 150 155 160 Thr
Lys Glu Leu Glu Lys Thr Asp Tyr Ile Ala Ser Pro Lys Lys Lys 165 170
175 Arg Lys Val Glu Ala Ser Gly Ser Gly Arg Ala Asp Ala Leu Asp Asp
180 185 190 Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe
Asp Leu 195 200 205 Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp
Leu Asp Met Leu 210 215 220 Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu
Asp Met Leu Ile Asn Ser 225 230 235 240 Arg Gly Ser Gly Glu Gly Arg
Gly Ser Leu Leu Thr Cys Gly Asp Val 245 250 255 Glu Glu Asn Pro Gly
Pro Val Ser Lys Gly Glu Glu Leu Phe Thr Gly 260 265 270 Val Val Pro
Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys 275 280 285 Phe
Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu 290 295
300 Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro
305 310 315 320 Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe
Ser Arg Tyr 325 330 335 Pro Asp His Met Lys Gln His Asp Phe Phe Lys
Ser Ala Met Pro Glu 340 345 350 Gly Tyr Val Gln Glu Arg Thr Ile Phe
Phe Lys Asp Asp Gly Asn Tyr 355 360 365 Lys Thr Arg Ala Glu Val Lys
Phe Glu Gly Asp Thr Leu Val Asn Arg 370 375 380 Ile Glu Leu Lys Gly
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly 385 390 395 400 His Lys
Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala 405 410 415
Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn 420
425 430 Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn
Thr 435 440 445 Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His
Tyr Leu Ser 450 455 460 Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu
Lys Arg Asp His Met 465 470 475 480 Val Leu Leu Glu Phe Val Thr Ala
Ala Gly Ile Thr Leu Gly Met Asp 485 490 495 Glu Leu Tyr Lys 500
172693PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 172Met Asn Gly Ala Ile Gly Gly Asp Leu Leu
Leu Asn Phe Pro Asp Met 1 5 10 15 Ser Val Leu Glu Arg Gln Arg Ala
His Leu Lys Tyr Leu Asn Pro Thr 20 25 30 Phe Asp Ser Pro Leu Ala
Gly Phe Phe Ala Asp Ser Ser Met Ile Thr 35 40 45 Gly Gly Glu Met
Asp Ser Tyr Leu Ser Thr Ala Gly Leu Asn Leu Pro 50 55 60 Met Met
Tyr Gly Glu Thr Thr Val Glu Gly Asp Ser Arg Leu Ser Ile 65 70 75 80
Ser Pro Glu Thr Thr Leu Gly Thr Gly Asn Phe Lys Lys Arg Lys Phe 85
90 95 Asp Thr Glu Thr Lys Asp Cys Asn Glu Lys Lys Lys Lys Met Thr
Met 100 105 110 Asn Arg Asp Asp Leu Val Glu Glu Gly Glu Glu Glu Lys
Ser Lys Ile 115 120 125 Thr Glu Gln Asn Asn Gly Ser Thr Lys Ser Ile
Lys Lys Met Lys His 130 135 140 Lys Ala Lys Lys Glu Glu Asn Asn Phe
Ser Asn Asp Ser Ser Lys Val 145 150 155 160 Thr Lys Glu Leu Glu Lys
Thr Asp Tyr Ile His Val Arg Ala Arg Arg 165 170 175 Gly Gln Ala Thr
Asp Ser His Ser Ile Ala Glu Arg Val Arg Arg Glu 180 185 190 Lys Ile
Ser Glu Arg Met Lys Phe Leu Gln Asp Leu Val Pro Gly Cys 195 200 205
Asp Lys Ile Thr Gly Lys Ala Gly Met Leu Asp Glu Ile Ile Asn Tyr 210
215 220 Val Gln Ser Leu Gln Arg Gln Ile Glu Phe Leu Ser Met Lys Leu
Ala 225 230 235 240 Ile Val Asn Pro Arg Pro Asp Phe Asp Met Asp Asp
Ile Phe Ala Lys 245 250 255 Glu Val Ala Ser Thr Pro Met Thr Val Val
Pro Ser Pro Glu Met Val 260 265 270 Leu Ser Gly Tyr Ser His Glu Met
Val His Ser Gly Tyr Ser Ser Glu 275 280 285 Met Val Asn Ser Gly Tyr
Leu His Val Asn Pro Met Gln Gln Val Asn 290 295 300 Thr Ser Ser Asp
Pro Leu Ser Cys Phe Asn Asn Gly Glu Ala Pro Ser 305 310 315 320 Met
Trp Asp Ser His Val Gln Asn Leu Tyr Gly Asn Leu Gly Val Ala 325 330
335 Ser Pro Lys Lys Lys Arg Lys Val Glu Ala Ser Ala Pro Pro Thr Asp
340 345 350 Val Ser Leu Gly Asp Glu Leu His Leu Asp Gly Glu Asp Val
Ala Met 355 360 365 Ala His Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp
Met Leu Gly Asp 370 375 380 Gly Asp Ser Pro Gly Pro Gly Phe Thr Pro
His Asp Ser Ala Pro Tyr 385 390 395 400 Gly Ala Leu Asp Met Ala Asp
Phe Glu Phe Glu Gln Met Phe Thr Asp 405 410 415 Ala Leu Gly Ile Asp
Glu Tyr Gly Gly Glu Phe Pro Gly Ile Arg Arg 420 425 430 Ser Arg Gly
Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp 435 440 445 Val
Glu Glu Asn Pro Gly Pro Val Ser Lys Gly Glu Glu Leu Phe Thr 450 455
460 Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His
465 470 475 480 Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr
Tyr Gly Lys 485 490 495 Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys
Leu Pro Val Pro Trp 500 505 510 Pro Thr Leu Val Thr Thr Leu Thr Tyr
Gly Val Gln Cys Phe Ser Arg 515 520 525 Tyr Pro Asp His Met Lys Gln
His Asp Phe Phe Lys Ser Ala Met Pro 530 535 540 Glu Gly Tyr Val Gln
Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn 545 550 555 560 Tyr Lys
Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn 565 570 575
Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu 580
585 590 Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile
Met 595 600 605 Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys
Ile Arg His 610 615 620 Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp
His Tyr Gln Gln Asn 625 630 635 640 Thr Pro Ile Gly Asp Gly Pro Val
Leu Leu Pro Asp Asn His Tyr Leu 645 650 655 Ser Thr Gln Ser Ala Leu
Ser Lys Asp Pro Asn Glu Lys Arg Asp His 660 665 670 Met Val Leu Leu
Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met 675 680 685 Asp Glu
Leu Tyr Lys 690 173792PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 173Met Asn Gly Ala Ile
Gly Gly Asp Leu Leu Leu Asn Phe Pro Asp Met 1 5 10 15 Ser Val Leu
Glu Arg Gln Arg Ala His Leu Lys Tyr Leu Asn Pro Thr 20 25 30 Phe
Asp Ser Pro Leu Ala Gly Phe Phe Ala Asp Ser Ser Met Ile Thr 35 40
45 Gly Gly Glu Met Asp Ser Tyr Leu Ser Thr Ala Gly Leu Asn Leu Pro
50 55 60 Met Met Tyr Gly Glu Thr Thr Val Glu Gly Asp Ser Arg Leu
Ser Ile 65 70 75 80 Ser Pro Glu Thr Thr Leu Gly Thr Gly Asn Phe Lys
Lys Arg Lys Phe 85 90 95 Asp Thr Glu Thr Lys Asp Cys Asn Glu Lys
Lys Lys Lys Met Thr Met 100 105 110 Asn Arg Asp Asp Leu Val Glu Glu
Gly Glu Glu Glu Lys Ser Lys Ile 115 120 125 Thr Glu Gln Asn Asn Gly
Ser Thr Lys Ser Ile Lys Lys Met Lys His 130 135 140 Lys Ala Lys Lys
Glu Glu Asn Asn Phe Ser Asn Asp Ser Ser Lys Val 145 150 155 160 Thr
Lys Glu Leu Glu Lys Thr Asp Tyr Ile His Val Arg Ala Arg Arg 165 170
175 Gly Gln Ala Thr Asp Ser His Ser Ile Ala Glu Arg Val Arg Arg Glu
180 185 190 Lys Ile Ser Glu Arg Met Lys Phe Leu Gln Asp Leu Val Pro
Gly Cys 195 200 205 Asp Lys Ile Thr Gly Lys Ala Gly Met Leu Asp Glu
Ile Ile Asn Tyr 210 215 220 Val Gln Ser Leu Gln Arg Gln Ile Glu Phe
Leu Ser Met Lys Leu Ala 225 230 235 240 Ile Val Asn Pro Arg Pro Asp
Phe Asp Met Asp Asp Ile Phe Ala Lys 245 250 255 Glu Val Ala Ser Thr
Pro Met Thr Val Val Pro Ser Pro Glu Met Val 260 265 270 Leu Ser Gly
Tyr Ser His Glu Met Val His Ser Gly Tyr Ser Ser Glu 275 280 285 Met
Val Asn Ser Gly Tyr Leu His Val Asn Pro Met Gln Gln Val Asn 290 295
300 Thr Ser Ser Asp Pro Leu Ser Cys Phe Asn Asn Gly Glu Ala Pro Ser
305 310 315 320 Met Trp Asp Ser His Val Gln Asn Leu Tyr Gly Asn Leu
Gly Val Ala 325 330 335 Ser Pro Lys Lys Lys Arg Lys Val Glu Ala Ser
Pro Ser Gly Gln Ile 340 345 350 Ser Asn Gln Ala Leu Ala Leu Ala Pro
Ser Ser Ala Pro Val Leu Ala 355 360 365 Gln Thr Met Val Pro Ser Ser
Ala Met Val Pro Leu Ala Gln Pro Pro 370 375 380 Ala Pro Ala Pro Val
Leu Thr Pro Gly Pro Pro Gln Ser Leu Ser Ala 385 390 395 400 Pro Val
Pro Lys Ser Thr Gln Ala Gly Glu Gly Thr Leu Ser Glu Ala 405 410 415
Leu Leu His Leu Gln Phe Asp Ala Asp Glu Asp Leu Gly Ala Leu Leu 420
425 430 Gly Asn Ser Thr Asp Pro Gly Val Phe Thr Asp Leu Ala Ser Val
Asp 435 440 445 Asn Ser Glu Phe Gln Gln Leu Leu Asn Gln Gly Val Ser
Met Ser His 450 455 460 Ser Thr Ala Glu Pro Met Leu Met Glu Tyr Pro
Glu Ala Ile Thr Arg 465 470 475 480 Leu Val Thr Gly Ser Gln Arg Pro
Pro Asp Pro Ala Pro Thr Pro Leu 485 490 495 Gly Thr Ser Gly Leu Pro
Asn Gly Leu Ser Gly Asp Glu Asp Phe Ser 500 505 510 Ser Ile Ala Asp
Met Asp Phe Ser Ala Leu Leu Ser Gln Ile Ser Ser 515 520 525 Ser Gly
Gln Ser Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr 530 535 540
Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Val Ser Lys Gly Glu Glu 545
550 555 560 Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly
Asp Val 565 570 575 Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu
Gly Asp Ala Thr 580 585 590 Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys
Thr Thr Gly Lys Leu Pro 595 600 605 Val Pro Trp Pro Thr Leu Val Thr
Thr Leu Thr Tyr Gly Val Gln Cys 610 615 620 Phe Ser Arg Tyr Pro Asp
His Met Lys Gln His Asp Phe Phe Lys Ser 625 630 635 640 Ala Met Pro
Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp 645 650 655 Asp
Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr 660 665
670 Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly
675 680 685 Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His
Asn Val 690 695 700 Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys
Val Asn Phe Lys 705 710 715 720 Ile Arg His Asn Ile Glu Asp Gly Ser
Val Gln Leu Ala Asp His Tyr 725 730 735 Gln Gln Asn Thr Pro Ile Gly
Asp Gly Pro Val Leu Leu Pro Asp Asn 740 745 750 His Tyr Leu Ser Thr
Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys 755 760 765 Arg Asp His
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr 770 775 780 Leu
Gly Met Asp Glu Leu Tyr Lys 785 790 174967PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptide 174Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Val Asp Leu
Arg Thr Leu 1 5 10 15 Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys
Pro Lys Val Arg Ser 20 25 30 Thr Val Ala Gln His His Glu Ala Leu
Val Gly His Gly Phe Thr His 35 40 45 Ala His Ile Val Ala Leu Ser
Gln His Pro Ala Ala Leu Gly Thr Val 50 55 60 Ala Val Lys Tyr Gln
Asp Met Ile Ala Ala Leu Pro Glu Ala Thr His 65 70 75 80 Glu Ala Ile
Val Gly Val Gly Lys Gln Trp Ser Gly Ala Arg Ala Leu 85 90 95 Glu
Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro Pro Leu Gln 100 105
110 Leu Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly Gly Val Thr
115 120 125 Ala Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr Gly
Ala Pro 130 135 140 Leu Asn Leu Thr Pro Glu Gln Val Val Ala Ile Ala
Ser Xaa Xaa Gly 145 150 155 160 Gly Lys Gln Ala Leu Glu Thr Val Gln
Arg Leu Leu Pro Val Leu Cys 165 170 175 Gln Ala His Gly Leu Thr Pro
Glu Gln Val Val Ala Ile Ala Ser Xaa 180 185 190 Xaa Gly Gly Lys Gln
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 195 200 205 Leu Cys Gln
Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 210 215 220 Ser
Xaa Xaa Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 225 230
235 240 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val
Ala 245 250 255 Ile Ala Ser Xaa Xaa Gly Gly Lys Gln Ala Leu Glu Thr
Val Gln Arg 260 265 270 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
Thr Pro Glu Gln Val 275 280 285 Val Ala Ile Ala Ser Xaa Xaa Gly Gly
Lys Gln Ala Leu Glu Thr Val 290 295 300 Gln Arg Leu Leu Pro Val Leu
Cys Gln Ala His Gly Leu Thr Pro Glu 305 310 315 320 Gln Val Val Ala
Ile Ala Ser Xaa Xaa Gly Gly Lys Gln Ala Leu Glu 325 330 335 Thr Val
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 340 345 350
Pro Glu Gln Val Val Ala Ile Ala Ser Xaa Xaa Gly Gly Lys Gln Ala 355
360 365 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly 370 375 380 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Xaa Xaa
Gly Gly Lys 385 390 395 400 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Ala 405 410 415 His Gly Leu Thr Pro Glu Gln Val
Val Ala Ile Ala Ser Xaa Xaa Gly 420 425 430 Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg Leu Leu Pro Val Leu Cys 435 440 445 Gln Ala His Gly
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Xaa 450 455 460 Xaa Gly
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 465 470 475
480 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
485 490 495 Ser Xaa Xaa Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu 500 505 510 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
Gln Val Val Ala 515 520 525 Ile Ala Ser Xaa Xaa Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg 530 535 540 Leu Leu Pro Val Leu Cys Gln Ala
His Gly Leu Thr Pro Glu Gln Val 545 550 555 560 Val Ala Ile Ala Ser
Xaa Xaa Gly Gly Arg Pro Ala Leu Glu Ser Ile 565 570 575 Val Ala Gln
Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn 580 585 590 Asp
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Leu Asp 595 600
605 Ala Val Lys Lys Gly Leu Pro His Ala Pro Ala Leu Ile Lys Arg Thr
610 615 620 Asn Arg Arg Ile Pro Glu Arg Thr Ser His Arg Val Ala Ala
Ser Pro 625 630 635 640 Lys Lys Lys Arg Lys Val Glu Ala Ser Gly Ser
Gly Arg Ala Asp Ala 645 650 655 Leu Asp Asp Phe Asp Leu Asp Met Leu
Gly Ser Asp Ala Leu Asp Asp 660 665 670 Phe Asp Leu Asp Met Leu Gly
Ser Asp Ala Leu Asp Asp Phe Asp Leu 675 680 685 Asp Met Leu Gly Ser
Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu 690 695 700 Ile Asn Ser
Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys 705 710 715 720
Gly Asp Val Glu Glu Asn Pro Gly Pro Val Ser Lys Gly Glu Glu Leu 725
730 735 Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val
Asn 740 745 750 Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp
Ala Thr Tyr 755 760 765 Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr
Gly Lys Leu Pro Val 770 775 780 Pro Trp Pro Thr Leu Val Thr Thr Leu
Thr Tyr Gly Val Gln Cys Phe 785 790 795 800 Ser Arg Tyr Pro Asp His
Met Lys Gln His Asp Phe Phe Lys Ser Ala 805 810 815 Met Pro Glu Gly
Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp 820 825 830 Gly Asn
Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu 835 840 845
Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn 850
855 860 Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val
Tyr 865 870 875 880 Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val
Asn Phe Lys Ile 885 890 895 Arg His Asn Ile Glu Asp Gly Ser Val Gln
Leu Ala Asp His Tyr Gln 900 905 910 Gln Asn Thr Pro Ile Gly Asp Gly
Pro Val Leu Leu Pro Asp Asn His 915 920 925 Tyr Leu Ser Thr Gln Ser
Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg 930 935 940 Asp His Met Val
Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu 945 950 955 960 Gly
Met Asp Glu Leu Tyr Lys 965 175922PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 175Met Tyr Pro Tyr Asp
Val Pro Asp Tyr Ala Val Asp Leu Arg Thr Leu 1 5 10 15 Gly Tyr Ser
Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val Arg Ser 20 25 30 Thr
Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe Thr His 35 40
45 Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly Thr Val
50 55 60 Ala Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu Ala
Thr His 65 70 75 80 Glu Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly
Ala Arg Ala Leu 85 90 95 Glu Ala Leu Leu Thr Val Ala Gly Glu Leu
Arg Gly Pro Pro Leu Gln 100 105 110 Leu Asp Thr Gly Gln Leu Leu Lys
Ile Ala Lys Arg Gly Gly Val Thr 115 120 125 Ala Val Glu Ala Val His
Ala Trp Arg Asn Ala Leu Thr Gly Ala Pro 130 135 140 Leu Asn Leu Thr
Pro Glu Gln Val Val Ala Ile Ala Ser Xaa Xaa Gly 145 150 155 160 Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 165 170
175 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Xaa
180 185 190 Xaa Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val 195 200 205 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
Val Ala Ile Ala 210 215 220 Ser Xaa Xaa Gly Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg Leu Leu 225 230 235 240 Pro Val Leu Cys Gln Ala His
Gly Leu Thr Pro Glu Gln Val Val Ala 245 250 255 Ile Ala Ser Xaa Xaa
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 260 265 270 Leu Leu Pro
Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 275 280 285 Val
Ala Ile Ala Ser Xaa Xaa Gly Gly Lys Gln Ala Leu Glu Thr Val 290 295
300 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
305 310 315 320 Gln Val Val Ala Ile Ala Ser Xaa Xaa Gly Gly Lys Gln
Ala Leu Glu 325 330 335 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
Ala His Gly Leu Thr 340 345 350 Pro Glu Gln Val Val Ala Ile Ala Ser
Xaa Xaa Gly Gly Lys Gln Ala 355 360 365 Leu Glu Thr Val Gln Arg Leu
Leu Pro Val Leu Cys Gln Ala His Gly 370 375 380 Leu Thr Pro Glu Gln
Val Val Ala Ile Ala Ser Xaa Xaa Gly Gly Lys 385 390 395 400 Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 405 410 415
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Xaa Xaa Gly 420
425 430 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
Cys 435 440 445 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile
Ala Ser Xaa 450 455 460 Xaa Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
Arg Leu Leu Pro Val 465 470 475 480 Leu Cys Gln Ala His Gly Leu Thr
Pro Glu Gln Val Val Ala Ile Ala 485 490 495 Ser Xaa Xaa Gly Gly Lys
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 500 505 510 Pro Val Leu Cys
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 515 520 525 Ile Ala
Ser Xaa Xaa Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 530 535 540
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 545
550 555 560 Val Ala Ile Ala Ser Xaa Xaa Gly Gly Arg Pro Ala Leu Glu
Ser Ile 565 570 575 Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala
Ala Leu Thr Asn 580 585 590 Asp His Leu Val Ala Leu Ala Cys Leu Gly
Gly Arg Pro Ala Leu Asp 595 600 605 Ala Val Lys Lys Gly Leu Pro His
Ala Pro Ala Leu Ile Lys Arg Thr 610 615 620 Asn Arg Arg Ile Pro Glu
Arg Thr Ser His Arg Val Ala Ala Ser Pro 625 630 635 640 Lys Lys Lys
Arg Lys Val Glu Ala Ser Pro Lys Lys Lys Arg Lys Val 645 650 655 Glu
Ala Ser Gly Ser Gly Met Asn Ile Gln Met Leu Leu Glu Ala Ala 660 665
670 Asp Tyr Leu Glu Arg Arg Glu Arg Glu Ala Glu His Gly Tyr Ala Ser
675 680 685 Met Leu Pro Gly Ser Gly Met Asn Ile Gln Met Leu Leu Glu
Ala Ala 690 695 700 Asp Tyr Leu Glu Arg Arg Glu Arg Glu Ala Glu His
Gly Tyr Ala Ser 705 710 715 720 Met Leu Pro Gly Ser Gly Met Asn Ile
Gln Met Leu Leu Glu Ala Ala 725 730 735 Asp Tyr Leu Glu Arg Arg Glu
Arg Glu Ala Glu His Gly Tyr Ala Ser 740 745 750 Met Leu Pro Gly Ser
Gly Met Asn Ile Gln Met Leu Leu Glu Ala Ala 755 760 765 Asp Tyr Leu
Glu Arg Arg Glu Arg Glu Ala Glu His Gly Tyr Ala Ser 770 775 780 Met
Leu Pro Ser Arg Ser Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu 785 790
795 800 Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Ile Glu Lys
Ser 805 810 815 Phe Val Ile Thr Asp Pro Arg Leu Pro Asp Tyr Pro Ile
Ile Phe Ala 820 825 830 Ser Asp Gly Phe Leu Glu Leu Thr Glu Tyr Ser
Arg Glu Glu Ile Met 835 840 845 Gly Arg Asn Ala Arg Phe Leu Gln Gly
Pro Glu Thr Asp Gln Ala Thr 850 855 860 Val Gln Lys Ile Arg Asp Ala
Ile Arg Asp Gln Arg Glu Thr Thr Val 865 870 875 880 Gln Leu Ile Asn
Tyr Thr Lys Ser Gly Lys Lys Phe Trp Asn Leu Leu 885 890 895 His Leu
Gln Pro Val Arg Asp Arg Lys Gly Gly Leu Gln Tyr Phe Ile 900 905 910
Gly Val Gln Leu Val Gly Ser Asp His Val 915 920 176983PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
176Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Val Asp Leu Arg Thr Leu
1 5 10 15 Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val
Arg Ser 20 25 30 Thr Val Ala Gln His His Glu Ala Leu Val Gly His
Gly Phe Thr His 35 40 45 Ala His Ile Val Ala Leu Ser Gln His Pro
Ala Ala Leu Gly Thr Val 50 55 60 Ala Val Lys Tyr Gln Asp Met Ile
Ala Ala Leu Pro Glu Ala Thr His 65 70 75 80 Glu Ala Ile Val Gly Val
Gly Lys Gln Trp Ser Gly Ala Arg Ala Leu 85 90 95 Glu Ala Leu Leu
Thr Val Ala Gly Glu Leu Arg Gly Pro Pro Leu Gln 100 105 110 Leu Asp
Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly Gly Val Thr 115 120 125
Ala Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr Gly Ala Pro 130
135 140 Leu Asn Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Xaa Xaa
Gly 145 150 155 160 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys 165 170 175 Gln Ala His Gly Leu Thr Pro Glu Gln Val
Val Ala Ile Ala Ser Xaa 180 185 190 Xaa Gly Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg Leu Leu Pro Val 195 200 205 Leu Cys Gln Ala His Gly
Leu Thr Pro Glu Gln Val Val Ala Ile Ala 210 215 220 Ser Xaa Xaa Gly
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 225 230 235 240 Pro
Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 245 250
255 Ile Ala Ser Xaa Xaa Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
260 265 270 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
Gln Val 275 280 285 Val Ala Ile Ala Ser Xaa Xaa Gly Gly Lys Gln Ala
Leu Glu Thr Val 290 295 300 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly Leu Thr Pro Glu 305 310 315 320 Gln Val Val Ala Ile Ala Ser
Xaa Xaa Gly Gly Lys Gln Ala Leu Glu 325 330 335 Thr Val Gln Arg Leu
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 340 345 350 Pro Glu Gln
Val Val Ala Ile Ala Ser Xaa Xaa Gly Gly Lys Gln Ala 355 360 365 Leu
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 370 375
380 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Xaa
Xaa Gly Gly Lys 385 390 395 400 Gln Ala Leu Glu Thr Val Gln Arg Leu
Leu Pro Val Leu Cys Gln Ala 405 410 415 His Gly Leu Thr Pro Glu Gln
Val Val Ala Ile Ala Ser Xaa Xaa Gly 420 425 430 Gly Lys Gln Ala Leu
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 435 440 445 Gln Ala His
Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Xaa 450 455 460 Xaa
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 465 470
475 480 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile
Ala 485 490 495 Ser Xaa Xaa Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
Arg Leu Leu 500 505 510 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
Glu Gln Val Val Ala 515 520 525 Ile Ala Ser Xaa Xaa Gly Gly Lys Gln
Ala Leu Glu Thr Val Gln Arg 530 535 540 Leu Leu Pro Val Leu Cys Gln
Ala His Gly Leu Thr Pro Glu Gln Val 545 550 555 560 Val Ala Ile Ala
Ser Xaa Xaa Gly Gly Arg Pro Ala Leu Glu Ser Ile 565 570 575 Val Ala
Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn 580 585 590
Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Leu Asp 595
600 605 Ala Val Lys Lys Gly Leu Pro His Ala Pro Ala Leu Ile Lys Arg
Thr 610 615 620 Asn Arg Arg Ile Pro Glu Arg Thr Ser His Arg Val Ala
Ala Ser Pro 625 630 635 640 Lys Lys Lys Arg Lys Val Glu Ala Ser Asn
Gly Ala Ile Gly Gly Asp 645 650 655 Leu Leu Leu Asn Phe Pro Asp Met
Ser Val Leu Glu Arg Gln Arg Ala 660 665 670 His Leu Lys Tyr Leu Asn
Pro Thr Phe Asp Ser Pro Leu Ala Gly Phe 675 680 685 Phe Ala Asp Ser
Ser Met Ile Thr Gly Gly Glu Met Asp Ser Tyr Leu 690 695 700 Ser Thr
Ala Gly Leu Asn Leu Pro Met Met Tyr Gly Glu Thr Thr Val 705 710 715
720 Glu Gly Asp Ser Arg Leu Ser Ile Ser Pro Glu Thr Thr Leu Gly Thr
725 730 735 Gly Asn Phe Lys Lys Arg Lys Phe Asp Thr Glu Thr Lys Asp
Cys Asn 740 745 750 Glu Lys Lys Lys Lys Met Thr Met Asn Arg Asp Asp
Leu Val Glu Glu 755 760 765 Gly Glu Glu Glu Lys Ser Lys Ile Thr Glu
Gln Asn Asn Gly Ser Thr 770 775 780 Lys Ser Ile Lys Lys Met Lys His
Lys Ala Lys Lys Glu Glu Asn Asn 785 790 795 800 Phe Ser Asn Asp Ser
Ser Lys Val Thr Lys Glu Leu Glu Lys Thr Asp 805 810 815 Tyr Ile His
Val Arg Ala Arg Arg Gly Gln Ala Thr Asp Ser His Ser 820 825 830 Ile
Ala Glu Arg Val Arg Arg Glu Lys Ile Ser Glu Arg Met Lys Phe 835 840
845 Leu Gln Asp Leu Val Pro Gly Cys Asp Lys Ile Thr Gly Lys Ala Gly
850 855 860 Met Leu Asp Glu Ile Ile Asn Tyr Val Gln Ser Leu Gln Arg
Gln Ile 865 870 875 880 Glu Phe Leu Ser Met Lys Leu Ala Ile Val Asn
Pro Arg Pro Asp Phe 885 890 895 Asp Met Asp Asp Ile Phe Ala Lys Glu
Val Ala Ser Thr Pro Met Thr 900 905 910 Val Val Pro Ser Pro Glu Met
Val Leu Ser Gly Tyr Ser His Glu Met 915 920 925 Val His Ser Gly Tyr
Ser Ser Glu Met Val Asn Ser Gly Tyr Leu His 930 935 940 Val Asn Pro
Met Gln Gln Val Asn Thr Ser Ser Asp Pro Leu Ser Cys 945 950 955 960
Phe Asn Asn Gly Glu Ala Pro Ser Met Trp Asp Ser His Val Gln Asn 965
970 975 Leu Tyr Gly Asn Leu Gly Val 980 177830PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
177Met Lys Met Asp Lys Lys Thr Ile Val Trp Phe Arg Arg Asp Leu Arg
1 5 10 15 Ile Glu Asp Asn Pro Ala Leu Ala Ala Ala Ala His Glu Gly
Ser Val 20 25 30 Phe Pro Val Phe Ile Trp Cys Pro Glu Glu Glu Gly
Gln Phe Tyr Pro 35 40 45 Gly Arg Ala Ser Arg Trp Trp Met Lys Gln
Ser Leu Ala His Leu Ser 50 55 60 Gln Ser Leu Lys Ala Leu Gly Ser
Asp Leu Thr Leu Ile Lys Thr His 65 70 75 80 Asn Thr Ile Ser Ala Ile
Leu Asp Cys Ile Arg Val Thr Gly Ala Thr 85 90 95 Lys Val Val Phe
Asn His Leu Tyr Asp Pro Val Ser Leu Val Arg Asp 100 105 110 His Thr
Val Lys Glu Lys Leu Val Glu Arg Gly Ile Ser Val Gln Ser 115 120 125
Tyr Asn Gly Asp Leu Leu Tyr Glu Pro Trp Glu Ile Tyr Cys Glu Lys 130
135 140 Gly Lys Pro Phe Thr Ser Phe Asn Ser Tyr Trp Lys Lys Cys Leu
Asp 145 150 155 160 Met Ser Ile Glu Ser Val Met Leu Pro Pro Pro Trp
Arg Leu Met Pro 165 170 175 Ile Thr Ala Ala Ala Glu Ala Ile Trp Ala
Cys Ser Ile Glu Glu Leu 180 185 190 Gly Leu Glu Asn Glu Ala Glu Lys
Pro Ser Asn Ala Leu Leu Thr Arg 195 200 205 Ala Trp Ser Pro Gly Trp
Ser Asn Ala Asp Lys Leu Leu Asn Glu Phe 210 215 220 Ile Glu Lys Gln
Leu Ile Asp Tyr Ala Lys Asn Ser Lys Lys Val Val 225 230 235 240 Gly
Asn Ser Thr Ser Leu Leu Ser Pro Tyr Leu His Phe Gly Glu Ile 245 250
255 Ser Val Arg His Val Phe Gln Cys Ala Arg Met Lys Gln Ile Ile Trp
260 265 270 Ala Arg Asp Lys Asn Ser Glu Gly Glu Glu Ser Ala Asp Leu
Phe Leu 275 280 285 Arg Gly Ile Gly Leu Arg Glu Tyr Ser Arg Tyr Ile
Cys Phe Asn Phe 290 295 300 Pro Phe Thr His Glu Gln Ser Leu Leu Ser
His Leu Arg Phe Phe Pro 305 310 315 320 Trp Asp Ala Asp Val Asp Lys
Phe Lys Ala Trp Arg Gln Gly Arg Thr 325 330 335 Gly Tyr Pro Leu Val
Asp Ala Gly Met Arg Glu Leu Trp Ala Thr Gly 340 345 350 Trp Met His
Asn Arg Ile Arg Val Ile Val Ser Ser Phe Ala Val Lys 355 360 365 Phe
Leu Leu Leu Pro Trp Lys Trp Gly Met Lys Tyr Phe Trp Asp Thr 370 375
380 Leu Leu Asp Ala Asp Leu Glu Cys Asp Ile Leu Gly Trp Gln Tyr Ile
385 390 395 400 Ser Gly Ser Ile Pro Asp Gly His Glu Leu Asp Arg Leu
Asp Asn Pro 405 410 415 Ala Leu Gln Gly Ala Lys Tyr Asp Pro Glu Gly
Glu Tyr Ile Arg Gln 420 425 430 Trp Leu Pro Glu Leu Ala Arg Leu Pro
Thr Glu Trp Ile His His Pro 435 440 445 Trp Asp Ala Pro Leu Thr Val
Leu Lys Ala Ser Gly Val Glu Leu Gly 450 455 460 Thr Asn Tyr Ala Lys
Pro Ile Val Asp Ile Asp Thr Ala Arg Glu Leu 465 470 475 480 Leu Ala
Lys Ala Ile Ser Arg Thr Arg Glu Ala Gln Ile Met Ile Gly 485 490 495
Ala Ala Pro Ala Ser Pro Lys Lys Lys Arg Lys Val Glu Ala Ser Gly 500
505 510 Ser Gly Arg Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu
Gly 515 520 525 Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly
Ser Asp Ala 530 535 540 Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser
Asp Ala Leu Asp Asp 545 550 555 560 Phe Asp Leu Asp Met Leu Ile Asn
Ser Arg Gly Ser Gly Glu Gly Arg 565 570 575 Gly Ser Leu Leu Thr Cys
Gly Asp Val Glu Glu Asn Pro Gly Pro Val 580 585 590 Ser Lys Gly Glu
Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu 595 600 605 Leu Asp
Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly 610 615 620
Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr 625
630 635 640 Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
Leu Thr 645 650 655 Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His
Met Lys Gln His 660 665 670 Asp Phe Phe Lys Ser Ala Met Pro Glu Gly
Tyr Val Gln Glu Arg Thr 675 680 685 Ile Phe Phe Lys Asp Asp Gly Asn
Tyr Lys Thr Arg Ala Glu Val Lys 690 695 700 Phe Glu Gly Asp Thr Leu
Val Asn Arg Ile Glu Leu Lys Gly Ile Asp 705 710 715 720 Phe Lys Glu
Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr 725 730 735 Asn
Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile 740 745
750 Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln
755 760 765 Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
Pro Val 770 775 780 Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser
Ala Leu Ser Lys 785 790 795 800 Asp Pro Asn Glu Lys Arg Asp His Met
Val Leu Leu Glu Phe Val Thr 805 810 815 Ala Ala Gly Ile Thr Leu Gly
Met Asp Glu Leu Tyr Lys Val 820 825 830 178774PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
178Met Lys Met Asp Lys Lys Thr Ile Val Trp Phe Arg Arg Asp Leu Arg
1 5 10 15 Ile Glu Asp Asn Pro Ala Leu Ala Ala Ala Ala His Glu Gly
Ser Val 20 25 30 Phe Pro Val Phe Ile Trp Cys Pro Glu Glu Glu Gly
Gln Phe Tyr Pro 35 40 45 Gly Arg Ala Ser Arg Trp Trp Met Lys Gln
Ser Leu Ala His Leu Ser 50 55 60 Gln Ser Leu Lys Ala Leu Gly Ser
Asp Leu Thr Leu Ile Lys Thr His 65 70 75 80 Asn Thr Ile Ser Ala Ile
Leu Asp Cys Ile Arg Val Thr Gly Ala Thr 85 90 95 Lys Val Val Phe
Asn His Leu Tyr Asp Pro Val Ser Leu Val Arg Asp 100 105 110 His Thr
Val Lys Glu Lys Leu Val Glu Arg Gly Ile Ser Val Gln Ser 115 120 125
Tyr Asn Gly Asp Leu Leu Tyr Glu Pro Trp Glu Ile Tyr Cys Glu Lys 130
135 140 Gly Lys Pro Phe Thr Ser Phe Asn Ser Tyr Trp Lys Lys Cys Leu
Asp 145 150 155 160 Met Ser Ile Glu Ser Val Met Leu Pro Pro Pro Trp
Arg Leu Met Pro 165 170 175 Ile Thr Ala Ala Ala Glu Ala Ile Trp Ala
Cys Ser Ile Glu Glu Leu 180 185 190 Gly Leu Glu Asn Glu Ala Glu Lys
Pro Ser Asn Ala Leu Leu Thr Arg 195 200 205 Ala Trp Ser Pro Gly Trp
Ser Asn Ala Asp Lys Leu Leu Asn Glu Phe 210 215 220 Ile Glu Lys Gln
Leu Ile Asp Tyr Ala Lys Asn Ser Lys Lys Val Val 225 230 235 240 Gly
Asn Ser Thr Ser Leu Leu Ser Pro Tyr Leu His Phe Gly Glu Ile 245 250
255 Ser Val Arg His Val Phe Gln Cys Ala Arg Met Lys Gln Ile Ile Trp
260 265 270 Ala Arg Asp Lys Asn Ser Glu Gly Glu Glu Ser Ala Asp Leu
Phe Leu 275 280 285 Arg Gly Ile Gly Leu Arg Glu Tyr Ser Arg Tyr Ile
Cys Phe Asn Phe 290 295 300 Pro Phe Thr His Glu Gln Ser Leu Leu Ser
His Leu Arg Phe Phe Pro 305 310 315 320 Trp Asp Ala Asp Val Asp Lys
Phe Lys Ala Trp Arg Gln Gly Arg Thr 325 330 335 Gly Tyr Pro Leu Val
Asp Ala Gly Met Arg Glu Leu Trp Ala Thr Gly 340 345 350 Trp Met His
Asn Arg Ile Arg Val Ile Val Ser Ser Phe Ala Val Lys 355 360 365 Phe
Leu Leu Leu Pro Trp Lys Trp Gly Met Lys Tyr Phe Trp Asp Thr 370 375
380 Leu Leu Asp Ala Asp Leu Glu Cys Asp Ile Leu Gly Trp Gln Tyr Ile
385 390 395 400 Ser Gly Ser Ile Pro Asp Gly His Glu Leu Asp Arg Leu
Asp Asn Pro 405 410 415 Ala Leu Gln Gly Ala Lys Tyr Asp Pro Glu Gly
Glu Tyr Ile Arg Gln 420 425 430 Trp Leu Pro Glu Leu Ala Arg Leu Pro
Thr Glu Trp Ile His His Pro 435 440 445 Trp Asp Ala Pro Leu Thr Val
Leu Lys Ala Ser Gly Val Glu Leu Gly 450 455 460 Thr Asn Tyr Ala Lys
Pro Ile Val Asp Ile Asp Thr Ala Arg Glu Leu 465 470 475 480 Leu Ala
Lys Ala Ile Ser Arg Thr Arg Glu Ala Gln Ile Met Ile Gly 485 490 495
Ala Ala Pro Ala Ser Pro Lys Lys Lys Arg Lys Val Glu Ala Ser Gly 500
505 510 Ser Gly Met Asn Ile Gln Met Leu Leu Glu Ala Ala Asp Tyr Leu
Glu 515 520 525 Arg Arg Glu Arg Glu Ala Glu His Gly Tyr Ala Ser Met
Leu Pro Gly 530 535 540 Ser Gly Met Asn Ile Gln Met Leu Leu Glu Ala
Ala Asp Tyr Leu Glu 545 550 555 560 Arg Arg Glu Arg Glu Ala Glu His
Gly Tyr Ala Ser Met Leu Pro Gly 565 570 575 Ser Gly Met Asn Ile Gln
Met Leu Leu Glu Ala Ala Asp Tyr Leu Glu 580 585 590 Arg Arg Glu Arg
Glu Ala Glu His Gly Tyr Ala Ser Met Leu Pro Gly 595 600 605 Ser Gly
Met Asn Ile Gln Met Leu Leu Glu Ala Ala Asp Tyr Leu Glu 610 615 620
Arg Arg Glu Arg Glu Ala Glu His Gly Tyr Ala Ser Met Leu Pro Ser 625
630 635 640 Arg Ser Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr
Cys Gly 645 650 655 Asp Val Glu Glu Asn Pro Gly Pro Ile Glu Lys Ser
Phe Val Ile Thr 660 665 670 Asp Pro Arg Leu Pro Asp Tyr Pro Ile Ile
Phe Ala Ser Asp Gly Phe 675 680 685 Leu Glu Leu Thr Glu Tyr Ser Arg
Glu Glu Ile Met Gly Arg Asn Ala 690 695 700 Arg Phe Leu Gln Gly Pro
Glu Thr Asp Gln Ala Thr Val Gln Lys Ile 705 710 715 720 Arg Asp Ala
Ile Arg Asp Gln Arg Glu Thr Thr Val Gln Leu Ile Asn 725 730 735 Tyr
Thr Lys Ser Gly Lys Lys Phe Trp Asn Leu Leu His Leu Gln Pro 740 745
750 Val Arg Asp Arg Lys Gly Gly Leu Gln Tyr Phe Ile Gly Val Gln Leu
755 760 765 Val Gly Ser Asp His Val 770 1791356PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
179Met Ser Arg Thr Arg Leu Pro Ser Pro Pro Ala Pro Ser Pro Ala Phe
1 5 10 15 Ser Ala Asp Ser Phe Ser Asp Leu Leu Arg Gln Phe Asp Pro
Ser Leu 20 25 30 Phe Asn Thr Ser Leu Phe Asp Ser Leu Pro Pro Phe
Gly Ala His His 35 40 45 Thr Glu Ala Ala Thr Gly Glu Trp Asp Glu
Val Gln Ser Gly Leu Arg 50 55 60 Ala Ala Asp Ala Pro Pro Pro Thr
Met Arg Val Ala Val
Thr Ala Ala 65 70 75 80 Arg Pro Pro Arg Ala Lys Pro Ala Pro Arg Arg
Arg Ala Ala Gln Pro 85 90 95 Ser Asp Ala Ser Pro Ala Ala Gln Val
Asp Leu Arg Thr Leu Gly Tyr 100 105 110 Ser Gln Gln Gln Gln Glu Lys
Ile Lys Pro Lys Val Arg Ser Thr Val 115 120 125 Ala Gln His His Glu
Ala Leu Val Gly His Gly Phe Thr His Ala His 130 135 140 Ile Val Ala
Leu Ser Gln His Pro Ala Ala Leu Gly Thr Val Ala Val 145 150 155 160
Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu Ala Thr His Glu Ala 165
170 175 Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala Arg Ala Leu Glu
Ala 180 185 190 Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro Pro Leu
Gln Leu Asp 195 200 205 Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly
Gly Val Thr Ala Val 210 215 220 Glu Ala Val His Ala Trp Arg Asn Ala
Leu Thr Gly Ala Pro Leu Asn 225 230 235 240 Leu Thr Pro Glu Gln Val
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys 245 250 255 Gln Ala Leu Glu
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 260 265 270 His Gly
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly 275 280 285
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 290
295 300 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser
Asn 305 310 315 320 Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val 325 330 335 Leu Cys Gln Ala His Gly Leu Thr Pro Glu
Gln Val Val Ala Ile Ala 340 345 350 Ser Asn Gly Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu 355 360 365 Pro Val Leu Cys Gln Ala
His Gly Leu Thr Pro Glu Gln Val Val Ala 370 375 380 Ile Ala Ser Asn
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 385 390 395 400 Leu
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 405 410
415 Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
420 425 430 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
Pro Glu 435 440 445 Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
Gln Ala Leu Glu 450 455 460 Thr Val Gln Arg Leu Leu Pro Val Leu Cys
Gln Ala His Gly Leu Thr 465 470 475 480 Pro Glu Gln Val Val Ala Ile
Ala Ser Asn Gly Gly Gly Lys Gln Ala 485 490 495 Leu Glu Thr Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 500 505 510 Leu Thr Pro
Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 515 520 525 Gln
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 530 535
540 His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly
545 550 555 560 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
Val Leu Cys 565 570 575 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val
Ala Ile Ala Ser Asn 580 585 590 Ile Gly Gly Lys Gln Ala Leu Glu Thr
Val Gln Arg Leu Leu Pro Val 595 600 605 Leu Cys Gln Ala His Gly Leu
Thr Pro Glu Gln Val Val Ala Ile Ala 610 615 620 Ser Asn Ile Gly Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 625 630 635 640 Pro Val
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 645 650 655
Ile Ala Ser His Asp Gly Gly Arg Pro Ala Leu Glu Ser Ile Val Ala 660
665 670 Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp
His 675 680 685 Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Leu
Asp Ala Val 690 695 700 Lys Lys Gly Leu Pro His Ala Pro Ala Leu Ile
Lys Arg Thr Asn Arg 705 710 715 720 Arg Ile Pro Glu Arg Thr Ser His
Arg Val Ala Asp His Ala Gln Val 725 730 735 Val Arg Val Leu Gly Phe
Phe Gln Cys His Ser His Pro Ala Gln Ala 740 745 750 Phe Asp Asp Ala
Met Thr Gln Phe Gly Met Ser Arg His Gly Leu Leu 755 760 765 Gln Leu
Phe Arg Arg Val Gly Val Thr Glu Leu Glu Ala Arg Ser Gly 770 775 780
Thr Leu Pro Pro Ala Ser Gln Arg Trp Asp Arg Ile Leu Gln Ala Ser 785
790 795 800 Gly Met Lys Arg Ala Lys Pro Ser Pro Thr Ser Thr Gln Thr
Pro Asp 805 810 815 Gln Ala Ser Leu His Ala Phe Ala Asp Ser Leu Glu
Arg Asp Leu Asp 820 825 830 Ala Pro Ser Pro Met His Glu Gly Asp Gln
Thr Arg Ala Ser Ala Ser 835 840 845 Pro Lys Lys Lys Arg Lys Val Glu
Ala Ser Lys Met Asp Lys Lys Thr 850 855 860 Ile Val Trp Phe Arg Arg
Asp Leu Arg Ile Glu Asp Asn Pro Ala Leu 865 870 875 880 Ala Ala Ala
Ala His Glu Gly Ser Val Phe Pro Val Phe Ile Trp Cys 885 890 895 Pro
Glu Glu Glu Gly Gln Phe Tyr Pro Gly Arg Ala Ser Arg Trp Trp 900 905
910 Met Lys Gln Ser Leu Ala His Leu Ser Gln Ser Leu Lys Ala Leu Gly
915 920 925 Ser Asp Leu Thr Leu Ile Lys Thr His Asn Thr Ile Ser Ala
Ile Leu 930 935 940 Asp Cys Ile Arg Val Thr Gly Ala Thr Lys Val Val
Phe Asn His Leu 945 950 955 960 Tyr Asp Pro Val Ser Leu Val Arg Asp
His Thr Val Lys Glu Lys Leu 965 970 975 Val Glu Arg Gly Ile Ser Val
Gln Ser Tyr Asn Gly Asp Leu Leu Tyr 980 985 990 Glu Pro Trp Glu Ile
Tyr Cys Glu Lys Gly Lys Pro Phe Thr Ser Phe 995 1000 1005 Asn Ser
Tyr Trp Lys Lys Cys Leu Asp Met Ser Ile Glu Ser Val 1010 1015 1020
Met Leu Pro Pro Pro Trp Arg Leu Met Pro Ile Thr Ala Ala Ala 1025
1030 1035 Glu Ala Ile Trp Ala Cys Ser Ile Glu Glu Leu Gly Leu Glu
Asn 1040 1045 1050 Glu Ala Glu Lys Pro Ser Asn Ala Leu Leu Thr Arg
Ala Trp Ser 1055 1060 1065 Pro Gly Trp Ser Asn Ala Asp Lys Leu Leu
Asn Glu Phe Ile Glu 1070 1075 1080 Lys Gln Leu Ile Asp Tyr Ala Lys
Asn Ser Lys Lys Val Val Gly 1085 1090 1095 Asn Ser Thr Ser Leu Leu
Ser Pro Tyr Leu His Phe Gly Glu Ile 1100 1105 1110 Ser Val Arg His
Val Phe Gln Cys Ala Arg Met Lys Gln Ile Ile 1115 1120 1125 Trp Ala
Arg Asp Lys Asn Ser Glu Gly Glu Glu Ser Ala Asp Leu 1130 1135 1140
Phe Leu Arg Gly Ile Gly Leu Arg Glu Tyr Ser Arg Tyr Ile Cys 1145
1150 1155 Phe Asn Phe Pro Phe Thr His Glu Gln Ser Leu Leu Ser His
Leu 1160 1165 1170 Arg Phe Phe Pro Trp Asp Ala Asp Val Asp Lys Phe
Lys Ala Trp 1175 1180 1185 Arg Gln Gly Arg Thr Gly Tyr Pro Leu Val
Asp Ala Gly Met Arg 1190 1195 1200 Glu Leu Trp Ala Thr Gly Trp Met
His Asn Arg Ile Arg Val Ile 1205 1210 1215 Val Ser Ser Phe Ala Val
Lys Phe Leu Leu Leu Pro Trp Lys Trp 1220 1225 1230 Gly Met Lys Tyr
Phe Trp Asp Thr Leu Leu Asp Ala Asp Leu Glu 1235 1240 1245 Cys Asp
Ile Leu Gly Trp Gln Tyr Ile Ser Gly Ser Ile Pro Asp 1250 1255 1260
Gly His Glu Leu Asp Arg Leu Asp Asn Pro Ala Leu Gln Gly Ala 1265
1270 1275 Lys Tyr Asp Pro Glu Gly Glu Tyr Ile Arg Gln Trp Leu Pro
Glu 1280 1285 1290 Leu Ala Arg Leu Pro Thr Glu Trp Ile His His Pro
Trp Asp Ala 1295 1300 1305 Pro Leu Thr Val Leu Lys Ala Ser Gly Val
Glu Leu Gly Thr Asn 1310 1315 1320 Tyr Ala Lys Pro Ile Val Asp Ile
Asp Thr Ala Arg Glu Leu Leu 1325 1330 1335 Ala Lys Ala Ile Ser Arg
Thr Arg Glu Ala Gln Ile Met Ile Gly 1340 1345 1350 Ala Ala Pro 1355
1801001PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 180Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
Ser Pro Lys Lys Lys Arg 1 5 10 15 Lys Val Glu Ala Ser Val Asp Leu
Arg Thr Leu Gly Tyr Ser Gln Gln 20 25 30 Gln Gln Glu Lys Ile Lys
Pro Lys Val Arg Ser Thr Val Ala Gln His 35 40 45 His Glu Ala Leu
Val Gly His Gly Phe Thr His Ala His Ile Val Ala 50 55 60 Leu Ser
Gln His Pro Ala Ala Leu Gly Thr Val Ala Val Lys Tyr Gln 65 70 75 80
Asp Met Ile Ala Ala Leu Pro Glu Ala Thr His Glu Ala Ile Val Gly 85
90 95 Val Gly Lys Gln Trp Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu
Thr 100 105 110 Val Ala Gly Glu Leu Arg Gly Pro Pro Leu Gln Leu Asp
Thr Gly Gln 115 120 125 Leu Leu Lys Ile Ala Lys Arg Gly Gly Val Thr
Ala Val Glu Ala Val 130 135 140 His Ala Trp Arg Asn Ala Leu Thr Gly
Ala Pro Leu Asn Leu Thr Pro 145 150 155 160 Glu Gln Val Val Ala Ile
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu 165 170 175 Glu Thr Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu 180 185 190 Thr Pro
Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln 195 200 205
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His 210
215 220 Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
Gly 225 230 235 240 Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
Val Leu Cys Gln 245 250 255 Ala His Gly Leu Thr Pro Glu Gln Val Val
Ala Ile Ala Ser Asn Gly 260 265 270 Gly Gly Lys Gln Ala Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu 275 280 285 Cys Gln Ala His Gly Leu
Thr Pro Glu Gln Val Val Ala Ile Ala Ser 290 295 300 Asn Asn Gly Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 305 310 315 320 Val
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile 325 330
335 Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
340 345 350 Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln
Val Val 355 360 365 Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
Glu Thr Val Gln 370 375 380 Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly Leu Thr Pro Glu Gln 385 390 395 400 Val Val Ala Ile Ala Ser His
Asp Gly Gly Lys Gln Ala Leu Glu Thr 405 410 415 Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro 420 425 430 Glu Gln Val
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu 435 440 445 Glu
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu 450 455
460 Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
465 470 475 480 Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
Gln Ala His 485 490 495 Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
Ser His Asp Gly Gly 500 505 510 Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln 515 520 525 Ala His Gly Leu Thr Pro Glu
Gln Val Val Ala Ile Ala Ser Asn Ile 530 535 540 Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu 545 550 555 560 Cys Gln
Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser 565 570 575
Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 580
585 590 Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
Ile 595 600 605 Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg Leu 610 615 620 Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
Pro Glu Gln Val Val 625 630 635 640 Ala Ile Ala Ser His Asp Gly Gly
Lys Gln Ala Leu Glu Thr Val Gln 645 650 655 Arg Leu Leu Pro Val Leu
Cys Gln Ala His Gly Leu Thr Pro Glu Gln 660 665 670 Val Val Ala Ile
Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr 675 680 685 Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro 690 695 700
Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu 705
710 715 720 Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly Leu 725 730 735 Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp
Gly Gly Lys Gln 740 745 750 Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
Val Leu Cys Gln Ala His 755 760 765 Gly Leu Thr Pro Glu Gln Val Val
Ala Ile Ala Ser His Asp Gly Gly 770 775 780 Arg Pro Ala Leu Glu Ser
Ile Val Ala Gln Leu Ser Arg Pro Asp Pro 785 790 795 800 Ala Leu Ala
Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu 805 810 815 Gly
Gly Arg Pro Ala Leu Asp Ala Val Lys Lys Gly Leu Pro His Ala 820 825
830 Pro Ala Leu Ile Lys Arg Thr Asn Arg Arg Ile Pro Glu Arg Thr Ser
835 840 845 His Arg Val Ala Asp His Ala Gln Val Val Arg Val Leu Gly
Phe Phe 850 855 860 Gln Cys His Ser His Pro Ala Gln Ala Phe Asp Asp
Ala Met Thr Gln 865 870 875 880 Phe Gly Met Ser Arg His Gly Leu Leu
Gln Leu Phe Arg Arg Val Gly 885 890 895 Val Thr Glu Leu Glu Ala Arg
Ser Gly Thr Leu Pro Pro Ala Ser Gln 900 905 910 Arg Trp Asp Arg Ile
Leu Gln Ala Ser Gly Met Lys Arg Ala Lys Pro 915 920 925 Ser Pro Thr
Ser Thr Gln Thr Pro Asp Gln Ala Ser Leu His Ala Phe 930 935 940 Ala
Asp Ser Leu Glu Arg Asp Leu Asp Ala Pro Ser Pro Met His Glu 945 950
955 960 Gly Asp Gln Thr Arg Ala Ser Ala Ser Gly Ser Gly Met Asn Ile
Gln 965 970 975 Met Leu Leu Glu Ala Ala Asp Tyr Leu Glu Arg Arg Glu
Arg Glu Ala 980 985
990 Glu His Gly Tyr Ala Ser Met Leu Pro 995 1000
1811099PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 181Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
Ser Pro Lys Lys Lys Arg 1 5 10 15 Lys Val Glu Ala Ser Val Asp Leu
Arg Thr Leu Gly Tyr Ser Gln Gln 20 25 30 Gln Gln Glu Lys Ile Lys
Pro Lys Val Arg Ser Thr Val Ala Gln His 35 40 45 His Glu Ala Leu
Val Gly His Gly Phe Thr His Ala His Ile Val Ala 50 55 60 Leu Ser
Gln His Pro Ala Ala Leu Gly Thr Val Ala Val Lys Tyr Gln 65 70 75 80
Asp Met Ile Ala Ala Leu Pro Glu Ala Thr His Glu Ala Ile Val Gly 85
90 95 Val Gly Lys Gln Trp Ser Gly Ala Arg Ala Leu Glu Ala Leu Leu
Thr 100 105 110 Val Ala Gly Glu Leu Arg Gly Pro Pro Leu Gln Leu Asp
Thr Gly Gln 115 120 125 Leu Leu Lys Ile Ala Lys Arg Gly Gly Val Thr
Ala Val Glu Ala Val 130 135 140 His Ala Trp Arg Asn Ala Leu Thr Gly
Ala Pro Leu Asn Leu Thr Pro 145 150 155 160 Glu Gln Val Val Ala Ile
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu 165 170 175 Glu Thr Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu 180 185 190 Thr Pro
Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln 195 200 205
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His 210
215 220 Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
Gly 225 230 235 240 Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
Val Leu Cys Gln 245 250 255 Ala His Gly Leu Thr Pro Glu Gln Val Val
Ala Ile Ala Ser Asn Gly 260 265 270 Gly Gly Lys Gln Ala Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu 275 280 285 Cys Gln Ala His Gly Leu
Thr Pro Glu Gln Val Val Ala Ile Ala Ser 290 295 300 Asn Asn Gly Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 305 310 315 320 Val
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile 325 330
335 Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
340 345 350 Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln
Val Val 355 360 365 Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
Glu Thr Val Gln 370 375 380 Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly Leu Thr Pro Glu Gln 385 390 395 400 Val Val Ala Ile Ala Ser His
Asp Gly Gly Lys Gln Ala Leu Glu Thr 405 410 415 Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro 420 425 430 Glu Gln Val
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu 435 440 445 Glu
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu 450 455
460 Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
465 470 475 480 Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
Gln Ala His 485 490 495 Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
Ser His Asp Gly Gly 500 505 510 Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln 515 520 525 Ala His Gly Leu Thr Pro Glu
Gln Val Val Ala Ile Ala Ser Asn Ile 530 535 540 Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu 545 550 555 560 Cys Gln
Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser 565 570 575
Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 580
585 590 Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
Ile 595 600 605 Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Arg Leu 610 615 620 Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
Pro Glu Gln Val Val 625 630 635 640 Ala Ile Ala Ser His Asp Gly Gly
Lys Gln Ala Leu Glu Thr Val Gln 645 650 655 Arg Leu Leu Pro Val Leu
Cys Gln Ala His Gly Leu Thr Pro Glu Gln 660 665 670 Val Val Ala Ile
Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr 675 680 685 Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro 690 695 700
Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu 705
710 715 720 Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly Leu 725 730 735 Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp
Gly Gly Lys Gln 740 745 750 Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
Val Leu Cys Gln Ala His 755 760 765 Gly Leu Thr Pro Glu Gln Val Val
Ala Ile Ala Ser His Asp Gly Gly 770 775 780 Arg Pro Ala Leu Glu Ser
Ile Val Ala Gln Leu Ser Arg Pro Asp Pro 785 790 795 800 Ala Leu Ala
Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu 805 810 815 Gly
Gly Arg Pro Ala Leu Asp Ala Val Lys Lys Gly Leu Pro His Ala 820 825
830 Pro Ala Leu Ile Lys Arg Thr Asn Arg Arg Ile Pro Glu Arg Thr Ser
835 840 845 His Arg Val Ala Asp His Ala Gln Val Val Arg Val Leu Gly
Phe Phe 850 855 860 Gln Cys His Ser His Pro Ala Gln Ala Phe Asp Asp
Ala Met Thr Gln 865 870 875 880 Phe Gly Met Ser Arg His Gly Leu Leu
Gln Leu Phe Arg Arg Val Gly 885 890 895 Val Thr Glu Leu Glu Ala Arg
Ser Gly Thr Leu Pro Pro Ala Ser Gln 900 905 910 Arg Trp Asp Arg Ile
Leu Gln Ala Ser Gly Met Lys Arg Ala Lys Pro 915 920 925 Ser Pro Thr
Ser Thr Gln Thr Pro Asp Gln Ala Ser Leu His Ala Phe 930 935 940 Ala
Asp Ser Leu Glu Arg Asp Leu Asp Ala Pro Ser Pro Met His Glu 945 950
955 960 Gly Asp Gln Thr Arg Ala Ser Ala Ser Gly Ser Gly Met Asn Ile
Gln 965 970 975 Met Leu Leu Glu Ala Ala Asp Tyr Leu Glu Arg Arg Glu
Arg Glu Ala 980 985 990 Glu His Gly Tyr Ala Ser Met Leu Pro Gly Ser
Gly Met Asn Ile Gln 995 1000 1005 Met Leu Leu Glu Ala Ala Asp Tyr
Leu Glu Arg Arg Glu Arg Glu 1010 1015 1020 Ala Glu His Gly Tyr Ala
Ser Met Leu Pro Gly Ser Gly Met Asn 1025 1030 1035 Ile Gln Met Leu
Leu Glu Ala Ala Asp Tyr Leu Glu Arg Arg Glu 1040 1045 1050 Arg Glu
Ala Glu His Gly Tyr Ala Ser Met Leu Pro Gly Ser Gly 1055 1060 1065
Met Asn Ile Gln Met Leu Leu Glu Ala Ala Asp Tyr Leu Glu Arg 1070
1075 1080 Arg Glu Arg Glu Ala Glu His Gly Tyr Ala Ser Met Leu Pro
Ser 1085 1090 1095 Arg 182141DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 182cctgcaggca
gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60gggcgacctt
tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
120actccatcac taggggttcc t 141183141DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
183aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
ctcactgagg 60ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
gtgagcgagc 120gagcgcgcag ctgcctgcag g 141184485DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
184gtgtctagac tgcagagggc cctgcgtatg agtgcaagtg ggttttagga
ccaggatgag 60gcggggtggg ggtgcctacc tgacgaccga ccccgaccca ctggacaagc
acccaacccc 120cattccccaa attgcgcatc ccctatcaga gagggggagg
ggaaacagga tgcggcgagg 180cgcgtgcgca ctgccagctt cagcaccgcg
gacagtgcct tcgcccccgc ctggcggcgc 240gcgccaccgc cgcctcagca
ctgaaggcgc gctgacgtca ctcgccggtc ccccgcaaac 300tccccttccc
ggccaccttg gtcgcgtccg cgccgccgcc ggcccagccg gaccgcacca
360cgcgaggcgc gagatagggg ggcacgggcg cgaccatctg cgctgcggcg
ccggcgactc 420agcgctgcct cagtctgcgg tgggcagcgg aggagtcgtg
tcgtgcctga gagcgcagtc 480gagaa 485185408DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
185gtagatttga gaactttggg atattcacag cagcagcagg aaaagatcaa
gcccaaagtg 60aggtcgacag tcgcgcagca tcacgaagcg ctggtgggtc atgggtttac
acatgcccac 120atcgtagcct tgtcgcagca ccctgcagcc cttggcacgg
tcgccgtcaa gtaccaggac 180atgattgcgg cgttgccgga agccacacat
gaggcgatcg tcggtgtggg gaaacagtgg 240agcggagccc gagcgcttga
ggccctgttg acggtcgcgg gagagctgag agggcctccc 300cttcagctgg
acacgggcca gttgctgaag atcgcgaagc ggggaggagt cacggcggtc
360gaggcggtgc acgcgtggcg caatgcgctc acgggagcac ccctcaac
408186164DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 186cggaccccgc gctggccgca ctcactaatg
atcatcttgt agcgctggcc tgcctcggcg 60gacgacccgc cttggatgcg gtgaagaagg
ggctcccgca cgcgcctgca ttgattaagc 120ggaccaacag aaggattccc
gagaggacat cacatcgagt ggca 164187858DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
187atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
ttgccttcct 60gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
gttgggtgca 120cgagtgggtt acatcgaact ggatctcaac agcggtaaga
tccttgagag ttttcgcccc 180gaagaacgtt ttccaatgat gagcactttt
aaagttctgc tatgtggcgc ggtattatcc 240cgtattgacg ccgggcaaga
gcaactcggt cgccgcatac actattctca gaatgacttg 300gttgagtact
caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
360tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
gacaacgatc 420ggaggaccga aggagctaac cgcttttttg cacaacatgg
gggatcatgt aactcgcctt 480gatcgttggg aaccggagct gaatgaagcc
ataccaaacg acgagcgtga caccacgatg 540cctgtagcaa tggcaacaac
gttgcgcaaa ctattaactg gcgaactact tactctagct 600tcccggcaac
aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
660tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
gcgtgggtct 720cgcggtatca ttgcagcact ggggccagat ggtaagccct
cccgtatcgt agttatctac 780acgacgggga gtcaggcaac tatggatgaa
cgaaatagac agatcgctga gataggtgcc 840tcactgatta agcattgg
8581888PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 188Leu Asp Leu Ala Ser Leu Ile Leu 1 5
18932PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 189Leu Thr Pro Ala Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Gln Leu Leu
Pro Val Leu Cys Gln Ala His Gly 20 25 30 19032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
190Leu Thr Pro Ala Gln Val Val Ala Leu Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 19132PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 191Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Gly Gly Arg Pro Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Glu Gln His Gly 20 25 30
19232PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 192Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Ala Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30 19332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
193Leu Thr Gln Val Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 20 25 30 19432PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 194Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Arg Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30
19532PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 195Leu Pro Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Ala His Gly 20 25 30 19632PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
196Leu Thr Leu Asp Gln Val Val Ala Ile Ala Ser Gly Ser Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 20 25 30 19732PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 197Leu Ser Pro Asp Gln
Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Leu Gln Arg Leu Leu Pro Val Leu Cys Gln Thr His Ala 20 25 30
19832PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 198Leu Asn Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30 19932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
199Leu Thr Pro Asp Gln Val Met Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 20032PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 200Leu Thr Pro Ala Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Arg Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30
20132PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 201Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Thr 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30 20232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
202Leu Thr Pro Asp Gln Val Met Thr Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 20332PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 203Leu Thr Pro Ala Gln
Val Val Thr Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
20432PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 204Leu Thr Pro Ala Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1
5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Arg Ala His
Gly 20 25 30 20532PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 205Leu Ser Pro Asp Gln Val Val Ala
Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30 20632PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
206Leu Thr Pro Asp Gln Val Val Gly Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 20732PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 207Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala Asn Gly 20 25 30
20832PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 208Leu Thr Pro Ala Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Thr His Gly 20 25 30 20932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
209Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Met Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 21032PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 210Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Met Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
21132PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 211Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Asn Gly Gly Lys Gln Ala 1 5 10 15 Leu Ala Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30 21232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
212Leu Thr Pro Asp Gln Val Val Thr Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 20 25 30 21332PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 213Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Thr Val Leu Cys Gln Asp His Gly 20 25 30
21432PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 214Met Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30 21532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
215Leu Ala Pro Asp Gln Val Val Ala Val Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 21632PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 216Leu Thr Pro Ala Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Lys Thr
Val Gln Gln Leu Leu Pro Val Leu Cys Glu Gln His Gly 20 25 30
21732PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 217Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Arg Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30 21832PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
218Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Gln Leu Leu Pro Val Leu Cys Gln Ala
His Gly 20 25 30 21932PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 219Leu Thr Pro Asp Gln
Val Leu Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Leu Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
22032PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 220Leu Thr Pro Glu Gln Val Val Ala Ile Ala
Arg Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Ala His Gly 20 25 30 22132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
221Leu Thr Pro Ala Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Met Gln Arg Leu Leu Pro Val Leu Cys Arg Ala
His Gly 20 25 30 22232PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 222Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Met
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
22332PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 223Leu Thr Thr Asp Gln Val Val Thr Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30 22432PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
224Leu Thr Pro Thr Gln Val Met Ala Ile Ala Asn Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 22532PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 225Leu Thr Pro Gln Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30
22632PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 226Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Met Leu Cys Gln Asp His Gly 20 25 30 22732PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
227Leu Thr Ser Ala Gln Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 22832PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 228Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Gln Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
22932PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 229Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Asn Gly Gly Lys Gln Ala 1 5 10 15 Leu Ala Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Ala His Gly 20 25 30 23032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
230Leu Thr Pro Ala Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Met Leu Cys Gln Ala
His Gly 20 25 30 23132PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 231Leu Thr Leu Asp Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala Arg Gly 20 25 30
23232PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 232Leu Thr Pro Ala Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Leu Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30 23332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
233Leu Thr Pro Asp Gln Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asn
His Gly 20 25 30 23432PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 234Leu Thr Pro Asp Gln
Val Val Thr Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Met
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
23532PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 235Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Arg Val Gln Arg Leu Leu
Pro Val Leu Cys Glu Gln His Gly 20 25 30 23632PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
236Leu Thr Pro Glu Gln Val Val Ala Ile Ala Cys Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Arg Gln Ala
His Gly 20 25 30 23732PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 237Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Arg Asp His Gly 20 25 30
23832PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 238Leu Thr Pro Glu Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Met Leu Cys Gln Ala His Gly 20 25 30 23932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
239Leu Thr Pro Glu Gln Val Val Ala Ile Ala Cys Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Arg His Ala
His Gly 20 25 30 24032PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 240Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln His His Gly 20 25 30
24132PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 241Leu Ile Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln His His Gly 20 25 30 24232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
242Leu Thr Arg Ala Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Glu Gln
His Gly 20 25 30 24332PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 243Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala 1 5 10 15 Val Gly Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30
24432PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 244Leu Thr Leu Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Glu Gln His Gly 20 25 30 24532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
245Leu Thr Pro Ala Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Met Leu Cys Gln Asp
His Gly 20 25 30 24632PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 246Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Gly Ser Lys Gln Ala 1 5 10 15 Leu Glu Thr
Met Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
24732PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 247Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Lys Gln His Gly 20 25 30 24832PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
248Leu Thr Leu Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Thr
His Gly 20 25 30 24932PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 249Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Ala
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
25032PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 250Leu Thr Pro Ala Gln Val Val Thr Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Glu Gln His Gly 20 25 30 25132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
251Leu Thr Pro Ala Gln Val Met Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 25232PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 252Leu Thr Arg Glu Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Arg Gln Ala His Gly 20 25 30
25332PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 253Leu Thr Leu Ala Gln Val Val Ala Ile Ala
Asn Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Ala His Gly 20 25 30 25432PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
254Leu Thr Leu Glu Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30
25532PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 255Leu Thr Pro Gln Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Glu Gln His Gly 20 25 30 25632PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
256Leu Ser Pro Asp Gln Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 25732PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 257Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln His His Gly 20 25 30
25832PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 258Leu Thr Pro Glu Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Ala Leu Leu
Pro Val Leu Arg Gln Ala His Gly 20 25 30 25932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
259Leu Ser Gln Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 26032PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 260Leu Pro Pro Glu Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30
26132PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 261Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Ala Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Ala His Gly 20 25 30 26232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
262Leu Thr Pro Asp Gln Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Glu
His Gly 20 25 30 26332PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 263Leu Thr Leu Asp Gln
Val Ala Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30
26432PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 264Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Val Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30 26532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
265Leu Ile Pro Ala Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 20 25 30 26632PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 266Leu Thr Pro Ala Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Arg Gln Ala His Gly 20 25 30
26732PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 267Leu Thr Pro Ala Gln Val Val Ala Ile Ala
Ser Gly Ser Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Thr His Gly 20 25 30 26832PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
268Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 26932PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 269Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala 1 5 10 15 Val Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30
27032PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 270Leu Ser Pro Asp Gln Val Val Thr Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Leu Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30 27132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
271Leu Thr Pro Val Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 20 25 30 27232PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 272Leu Thr Leu Asp Gln
Val Val Ala Ile Ala Ser Gly Ser Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Thr His Gly 20 25 30
27332PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 273Leu Thr Pro Ala Gln Val Val Ala Ile Ala
Cys Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Arg Arg Leu Leu
Pro Val Leu Cys Gln Ala His Gly 20 25 30 27432PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
274Leu Thr Pro Ala Gln Val Val Ala Ile Ala Ser Gly Ser Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Phe Pro Val Leu Cys Gln Ala
His Gly 20 25 30 27532PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 275Leu Pro Pro Ala Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30
27632PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 276Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Phe Gln Glu His Gly 20 25 30 27732PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
277Leu Thr Pro Ala Lys Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 27832PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 278Leu Thr Pro Val Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Ala Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
27932PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 279Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Gly Leu Cys Gln Asp His Gly 20 25 30 28032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
280Leu Thr Leu Ala Gln Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 28132PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 281Leu Thr Pro Ala Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Thr Val Leu Cys Gln Asp His Gly 20 25 30
28232PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 282Leu Pro Pro Ala Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30 28332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
283Leu Thr Pro Ala Gln Ala Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 20 25 30 28432PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 284Leu Thr Pro Ala Gln
Val Val Ala Ile Val Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Thr His Gly 20 25 30
28532PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 285Leu Thr Pro Asp Gln Val Val Ala Val Ala
Gly Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30 28632PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
286Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Gly Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 20 25 30 28732PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 287Leu Pro Pro Ala Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Glu Ala His Gly 20 25 30
28832PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 288Leu Thr Thr Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30 28932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
289Leu Thr Pro Asp Gln Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Val Pro Val Leu Cys Gln Asp
His Gly 20 25 30 29032PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 290Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Thr His Ala 20 25 30
29132PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 291Leu Thr Leu Ala Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Thr His Gly 20 25 30 29232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
292Leu Thr Pro Asn Gln Leu Val Ala Ile Ala Asn Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 29332PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 293Leu Ser Pro Ala Gln
Val Val Ala Ile Ala Ser Gly Ser Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
29432PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 294Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Val Leu
Pro Val Leu Cys Gln Ala His Gly 20 25 30 29532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
295Leu Thr Pro Asp Gln Val Met Ala Ile Ala Asn Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 20 25 30 29632PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 296Leu Thr Pro Glu Gln
Val Val Ala Ile Ala Ser Gly Gly Arg Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30
29732PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 297Leu Thr Pro Ala Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Trp Leu Leu
Pro Val Leu Cys Gln Ala His Gly 20 25 30 29832PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
298Leu Thr Pro Asp Lys Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 29932PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 299Leu Thr Pro Ala Gln
Val Met Ala Ile Ala Asn Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
30032PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 300Leu Thr Gln Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Ala Asn Gly 20 25 30 30132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
301Leu Thr Pro Ala Gln Val Val Ala Ile Ala Ser Gly Gly Lys Pro Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Glu Gln
His Gly 20 25 30 30232PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 302Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Ser Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Met Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
30332PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 303Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Ser Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Arg Gln Asp His Gly 20 25 30 30432PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
304Leu Thr Pro Tyr Gln Val Val Ala Ile Ala Ser Gly Ser Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20
25 30 30532PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 305Leu Thr Pro Tyr Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Ala His Gly 20 25 30 30632PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
306Leu Thr Leu Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Glu
His Gly 20 25 30 30732PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 307Leu Thr Leu Glu Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Leu Val Leu Cys Gln Ala His Gly 20 25 30
30832PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 308Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Arg Arg Leu Leu
Gln Val Leu Cys Gln Asp His Gly 20 25 30 30932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
309Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Arg Gln Asp
His Gly 20 25 30 31032PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 310Leu Thr Pro Asp Gln
Val Val Ser Ile Ala Asn Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30
31132PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 311Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Asn Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Thr His Gly 20 25 30 31232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
312Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Lys Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 20 25 30 31332PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 313Leu Thr Thr Asp Gln
Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
31432PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 314Leu Ile Pro Gln Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30 31532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
315Leu Thr Leu Thr Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 20 25 30 31632PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 316Leu Thr Pro Thr Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
31732PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 317Leu Thr Pro Thr Gln Val Met Ala Ile Ala
Asn Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Ala His Gly 20 25 30 31832PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
318Leu Thr Pro Asp Gln Val Val Ala Val Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 20 25 30 31932PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 319Leu Thr Pro Ala Gln
Val Val Ala Ile Ala Ser Gly Ser Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
32032PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 320Leu Thr Pro Gly Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Arg Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30 32132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
321Leu Thr Pro Asp Gln Val Val Val Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 20 25 30 32232PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 322Leu Pro Pro Asp Gln
Val Val Ala Ile Ala Ser Gly Ser Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
32332PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 323Leu Thr Pro Asp Gln Val Val Thr Ile Ala
Asn Gly Ser Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Ala His Gly 20 25 30 32432PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
324Leu Thr Pro Ala Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Gln Val Leu Cys Gln Asp
His Gly 20 25 30 32532PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 325Leu Thr Pro Asp His
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
32632PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 326Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Gln Val Leu Cys Gln Asp His Gly 20 25 30 32732PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
327Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Gly Gly Arg Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Glu Gln
His Gly 20 25 30 32832PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 328Leu His Pro Gly Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30
32932PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 329Leu Thr Leu Asp Gln Val Val Ser Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30 33032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
330Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Ala Leu Cys Gln Asp
His Gly 20 25 30 33132PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 331Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Pro Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Glu Gln His Gly 20 25 30
33232PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 332Leu Thr Pro Ala Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Lys Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Ala His Gly 20 25 30 33332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
333Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Gly Gly Lys Arg Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 20 25 30 33432PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 334Leu Asn Pro Asp Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 20 25 30
33532PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 335Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Lys Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30 33632PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
336Leu Thr Leu Asp Gln Val Val Ala Ile Ala Asn Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
His Gly 20 25 30 33732PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 337Leu Thr Pro Ala Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Arg Asp His Gly 20 25 30
33832PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 338Leu Thr Pro Ala Gln Val Leu Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Thr Val Leu Cys Gln Asp His Gly 20 25 30 33932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
339Leu Thr Pro Ala Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Met Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 34032PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 340Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Gly Leu Cys Gln Ala His Gly 20 25 30
34132PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 341Leu Thr Arg Glu Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Ala Leu Leu
Pro Val Leu Arg Gln Ala His Gly 20 25 30 34232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
342Leu Thr Pro Ala Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Val
His Gly 20 25 30 34332PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 343Leu Thr Pro Asn Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Leu Val Leu Cys Gln Asp His Gly 20 25 30
34432PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 344Leu Thr Pro Asp Gln Val Met Ala Ile Ala
Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Ala His Gly 20 25 30 34532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
345Leu Thr Arg Glu Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 34633PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 346Leu Ser Thr Ala Gln
Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala 1 5 10 15 Leu Glu Gly
Ile Gly Glu Gln Leu Leu Lys Leu Arg Thr Ala Pro Tyr 20 25 30 Gly
34733PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 347Leu Ser Thr Ala Gln Val Val Ala Val Ala
Ser Gly Gly Lys Pro Ala 1 5 10 15 Leu Glu Ala Val Arg Ala Gln Leu
Leu Ala Leu Arg Ala Ala Pro Tyr 20 25 30 Gly 34832PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
348Leu Thr Gln Val Gln Val Val Ala Ile Ala Ser Gly Gly Lys Gln Ala
1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
His Gly 20 25 30 34932PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 349Leu Thr Pro Asp Gln
Val Val Ala Ile Ala Ser Asn Gly Lys Gln Ala 1 5 10 15 Leu Glu Thr
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 20 25 30
35032PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 350Leu Thr Pro Asp Gln Val Val Ala Ile Ala
Ser Gly Gly Lys Arg Ala 1 5 10 15 Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys Gln Asp His Gly 20 25 30
* * * * *
References