U.S. patent application number 17/473209 was filed with the patent office on 2021-12-30 for arc-based capsids and uses thereof.
This patent application is currently assigned to VNV NEWCO INC.. The applicant listed for this patent is VNV NEWCO INC.. Invention is credited to Jessica CRISP, Adam FRAITES, Zachary GILBERT, Colin MALONE, Ian PEIKON, Andrey PISAREV.
Application Number | 20210403907 17/473209 |
Document ID | / |
Family ID | 1000005899865 |
Filed Date | 2021-12-30 |
United States Patent
Application |
20210403907 |
Kind Code |
A1 |
MALONE; Colin ; et
al. |
December 30, 2021 |
ARC-BASED CAPSIDS AND USES THEREOF
Abstract
Disclosed herein, in certain embodiments, are recombinant Arc
and endogenous Gag polypeptides, and methods of using recombinant
Arc and endogenous Gag polypeptides.
Inventors: |
MALONE; Colin; (Brooklyn,
NY) ; PEIKON; Ian; (Bethpage, NY) ; GILBERT;
Zachary; (Brooklyn, NY) ; PISAREV; Andrey;
(Brooklyn, NY) ; FRAITES; Adam; (Long Beach Twp.,
NJ) ; CRISP; Jessica; (Phoenix, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VNV NEWCO INC. |
New York |
NY |
US |
|
|
Assignee: |
VNV NEWCO INC.
|
Family ID: |
1000005899865 |
Appl. No.: |
17/473209 |
Filed: |
September 13, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17277119 |
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PCT/US2019/051786 |
Sep 18, 2019 |
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17473209 |
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62733015 |
Sep 18, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/20 20170501;
C12N 2310/141 20130101; C12N 2310/531 20130101; C12N 2310/3519
20130101; C12N 15/111 20130101; C12N 2320/32 20130101; C12N 2310/12
20130101 |
International
Class: |
C12N 15/11 20060101
C12N015/11 |
Claims
1. A composition comprising: (a) a capsid that comprises an
endogenous Gag polypeptide and a heterologous cargo, wherein the
endogenous Gag polypeptide is not an Arc polypeptide; and (b) a
delivery component.
2. The composition of claim 1, wherein the delivery component
comprises a microvesicle or a microparticle.
3. The composition of claim 1, wherein the delivery component
further comprises a fusogenic molecule.
4. The composition of claim 1, wherein the delivery component
further comprises a cell-specific binding protein or an engineered
protein that binds to an antigen or cell surface molecule.
5. The composition of claim 1, further comprising a second
polypeptide that is fused to the endogenous Gag polypeptide,
wherein the second polypeptide binds to a target receptor, antigen,
or cell surface molecule.
6. The composition of claim 5, wherein the second polypeptide is an
antibody or antigen-binding fragment thereof, a human protein, a
viral protein, or an engineered protein.
7. The composition of claim 1, wherein the endogenous Gag
polypeptide is a Paraneoplastic Ma antigen family polypeptide.
8. The composition of claim 1, wherein the endogenous Gag
polypeptide is a retrotransposon Gag-like family polypeptide.
9. The composition of claim 8, wherein the retrotransposon Gag-like
family polypeptide is a PEG10 polypeptide.
10. The composition of claim 1, wherein the heterologous cargo
comprises a nucleic acid molecule that comprises or encodes a
component of a CRISPR-Cas system, zinc finger nuclease (ZFN)
system, or transcription activator-like effector nuclease (TALEN)
system.
11. A composition comprising: (a) a capsid that comprises an
endogenous Gag polypeptide, wherein the endogenous Gag polypeptide
is not an Arc polypeptide; and (b) a delivery component that
comprises a liposome or a micelle.
12. The composition of claim 11, wherein the delivery component
comprises the liposome.
13. The composition of claim 12, wherein the liposome comprises a
surface presented lipopeptide.
14. The composition of claim 11, wherein the delivery component
comprises the micelle.
15. The composition of claim 14, wherein the micelle comprises a
hydrophilic polymer, a hydrophilic copolymer, a pH-sensitive
polymer, or a pH-sensitive copolymer.
16. The composition of claim 11, further comprising a fusogenic
molecule.
17. The composition of claim 11, further comprising a cell-specific
binding protein or an engineered protein that binds to an antigen
or cell surface molecule.
18. The composition of claim 11, wherein the endogenous Gag
polypeptide is a Paraneoplastic Ma antigen family polypeptide.
19. The composition of claim 11, wherein the endogenous Gag
polypeptide is a retrotransposon Gag-like family polypeptide.
20. The composition of claim 19, wherein the retrotransposon
Gag-like family polypeptide is a PEG10 polypeptide.
21. The composition of claim 11, further comprising a nucleic acid
molecule that comprises or encodes a component of a CRISPR-Cas
system, zinc finger nuclease (ZFN) system, or transcription
activator-like effector nuclease (TALEN) system.
22. A composition comprising: (a) a capsid that comprises a
recombinant endogenous Gag polypeptide and a cargo, wherein the
recombinant endogenous Gag polypeptide is not an Arc polypeptide;
and (b) a delivery component.
23. The composition of claim 22, wherein the delivery component
comprises a microvesicle or a microparticle.
24. The composition of claim 22, wherein the delivery component
comprises a fusogenic molecule.
25. The composition of claim 22, wherein the delivery component
comprises a cell-specific binding protein or an engineered protein
that binds to an antigen or cell surface molecule.
26. The composition of claim 22, wherein the recombinant endogenous
Gag polypeptide is a Paraneoplastic Ma antigen family
polypeptide.
27. The composition of claim 22, wherein the recombinant endogenous
Gag polypeptide is a retrotransposon Gag-like family
polypeptide.
28. The composition of claim 27, wherein the retrotransposon
Gag-like family polypeptide is a PEG10 polypeptide.
29. The composition of claim 22, wherein the cargo comprises a
nucleic acid molecule that comprises or encodes a component of a
CRISPR-Cas system, zinc finger nuclease (ZFN) system, or
transcription activator-like effector nuclease (TALEN) system.
30. A method of preparing a composition for suitable for delivery
of a cargo, the method comprising: (a) isolating an endogenous Gag
polypeptide, wherein the endogenous Gag polypeptide is not an Arc
polypeptide; (b) incubating the endogenous Gag polypeptide with the
cargo in conditions suitable for capsid formation, thereby
packaging the cargo in a capsid that comprises the endogenous Gag
polypeptide; and (c) formulating the capsid with a delivery
component that comprises a liposome or a micelle.
Description
CROSS REFERENCE
[0001] This application is a continuation of U.S. patent
application Ser. No. 17/277,119, filed Mar. 17, 2021, which is a
national phase entry of International Application No.
PCT/US2019/051786, filed Sep. 18, 2019, which claims the benefit of
U.S. Provisional Patent Application No. 62/733,015, filed Sep. 18,
2018, each of which is incorporated herein by reference in its
entirety.
SEQUENCE LISTING STATEMENT
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 15, 2020, is named 54838_702_303_SL.txt and is 148,382
bytes in size.
SUMMARY OF THE DISCLOSURE
[0003] Disclosed herein, in certain embodiments, are recombinant
and engineered Arc polypeptides and recombinant and engineered
endogenous Gag (endo-Gag) polypeptides. In some embodiments, also
included are Arc-based capsids and endo-Gag based capsids, either
loaded or empty, and methods of preparing the capsids. Additionally
included are methods of delivery of the Arc-based capsids and
endo-Gag-based capsids to a site of interest.
[0004] Disclosed herein, in certain embodiments, is a capsid
comprising a recombinant Arc polypeptide or a recombinant
endogenous Gag polypeptide and a therapeutic agent. In some
embodiments, the therapeutic agent is a nucleic acid. In some
embodiments, the nucleic acid is an RNA. In some embodiments, the
recombinant Arc polypeptide is a human Arc polypeptide comprising
an amino acid sequence that is SEQ ID NO: 1 or an amino acid
sequence that is at least 90% identical to the SEQ ID NO: 1. In
some embodiments, the recombinant Arc polypeptide is an Arc
polypeptide comprising: a) an amino acid sequence that is SEQ ID
NO: 2 or an amino acid sequence that is at least 90% identical to
the SEQ ID NO: 2; b) an amino acid sequence that is SEQ ID NO: 3 or
an amino acid sequence that is at least 90% identical to the SEQ ID
NO: 3; c) an amino acid sequence that is SEQ ID NO: 4 or an amino
acid sequence that is at least 90% identical to the SEQ ID NO: 4;
d) an amino acid sequence that is SEQ ID NO: 5 or an amino acid
sequence that is at least 90% identical to the SEQ ID NO: 5; e) an
amino acid sequence that is SEQ ID NO: 6 or an amino acid sequence
that is at least 90% identical to the SEQ ID NO: 6; f) an amino
acid sequence that is SEQ ID NO: 7 or an amino acid sequence that
is at least 90% identical to the SEQ ID NO: 7; g) an amino acid
sequence that is SEQ ID NO: 8 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 8; h) an amino acid sequence
that is SEQ ID NO: 9 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 9; i) an amino acid sequence that is
SEQ ID NO: 10 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 10; or j) an amino acid sequence that
is SEQ ID NO: 11 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 11; or k) an amino acid sequence that
is SEQ ID NO: 12 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 12; or l) an amino acid sequence that
is SEQ ID NO: 13 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 13; or m) an amino acid sequence that
is SEQ ID NO: 14 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 14; or n) an amino acid sequence that
is SEQ ID NO: 15 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 15. In some embodiments, the
recombinant endogenous Gag polypeptide is a human endogenous Gag
polypeptide. In some embodiments, the recombinant endogenous Gag
polypeptide is an endogenous Gag polypeptide comprising: a) an
amino acid sequence that is SEQ ID NO: 16 or an amino acid sequence
that is at least 90% identical to the SEQ ID NO: 16; b) an amino
acid sequence that is SEQ ID NO: 17 or an amino acid sequence that
is at least 90% identical to the SEQ ID NO: 17; c) an amino acid
sequence that is SEQ ID NO: 18 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 18; d) an amino acid sequence
that is SEQ ID NO: 19 or an amino acid sequence that is at least
90% identical to the SEQ ID NO: 19; e) an amino acid sequence that
is SEQ ID NO: 20 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 20; f) an amino acid sequence that is
SEQ ID NO: 21 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 21; or g) an amino acid sequence that
is SEQ ID NO: 22 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 22; or h) an amino acid sequence that
is SEQ ID NO: 23 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 23; or i) an amino acid sequence that
is SEQ ID NO: 24 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 24; or j) an amino acid sequence that
is SEQ ID NO: 25 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 25; or k) an amino acid sequence that
is SEQ ID NO: 26 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 26; or l) an amino acid sequence that
is SEQ ID NO: 27 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 27 or m) an amino acid sequence that is
SEQ ID NO: 28 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 28.
[0005] Disclosed herein, in certain embodiments, is a capsid
comprising a recombinant Arc polypeptide or a recombinant
endogenous Gag polypeptide, wherein the recombinant Arc polypeptide
is not a rat Arc polypeptide or a human Arc polypeptide. In some
embodiments, the capsid further comprises a cargo. In some
embodiments, the cargo is a nucleic acid. In some embodiments, the
cargo is an RNA. In some embodiments, the cargo is a therapeutic
agent. In some embodiments, the recombinant Arc polypeptide is an
Arc polypeptide comprising: a) an amino acid sequence that is SEQ
ID NO: 2 or an amino acid sequence that is at least 90% identical
to the SEQ ID NO: 2; b) an amino acid sequence that is SEQ ID NO: 3
or an amino acid sequence that is at least 90% identical to the SEQ
ID NO: 3; c) an amino acid sequence that is SEQ ID NO: 4 or an
amino acid sequence that is at least 90% identical to the SEQ ID
NO: 4; d) an amino acid sequence that is SEQ ID NO: 5 or an amino
acid sequence that is at least 90% identical to the SEQ ID NO: 5;
e) an amino acid sequence that is SEQ ID NO: 6 or an amino acid
sequence that is at least 90% identical to the SEQ ID NO: 6; f) an
amino acid sequence that is SEQ ID NO: 7 or an amino acid sequence
that is at least 90% identical to the SEQ ID NO: 7; g) an amino
acid sequence that is SEQ ID NO: 8 or an amino acid sequence that
is at least 90% identical to the SEQ ID NO: 8; h) an amino acid
sequence that is SEQ ID NO: 9 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 9; i) an amino acid sequence
that is SEQ ID NO: 10 or an amino acid sequence that is at least
90% identical to the SEQ ID NO: 10; or j) an amino acid sequence
that is SEQ ID NO: 11 or an amino acid sequence that is at least
90% identical to the SEQ ID NO: 11; or k) an amino acid sequence
that is SEQ ID NO: 12 or an amino acid sequence that is at least
90% identical to the SEQ ID NO: 12; or l) an amino acid sequence
that is SEQ ID NO: 13 or an amino acid sequence that is at least
90% identical to the SEQ ID NO: 13; or m) an amino acid sequence
that is SEQ ID NO: 14 or an amino acid sequence that is at least
90% identical to the SEQ ID NO: 14; or n) an amino acid sequence
that is SEQ ID NO: 15 or an amino acid sequence that is at least
90% identical to the SEQ ID NO: 15. In some embodiments, the
recombinant endogenous Gag polypeptide is an endogenous Gag
polypeptide comprising: a) an amino acid sequence that is SEQ ID
NO: 16 or an amino acid sequence that is at least 90% identical to
the SEQ ID NO: 16; b) an amino acid sequence that is SEQ ID NO: 17
or an amino acid sequence that is at least 90% identical to the SEQ
ID NO: 17; c) an amino acid sequence that is SEQ ID NO: 18 or an
amino acid sequence that is at least 90% identical to the SEQ ID
NO: 18; d) an amino acid sequence that is SEQ ID NO: 19 or an amino
acid sequence that is at least 90% identical to the SEQ ID NO: 19;
e) an amino acid sequence that is SEQ ID NO: 20 or an amino acid
sequence that is at least 90% identical to the SEQ ID NO: 20; f) an
amino acid sequence that is SEQ ID NO: 21 or an amino acid sequence
that is at least 90% identical to the SEQ ID NO: 21; or g) an amino
acid sequence that is SEQ ID NO: 22 or an amino acid sequence that
is at least 90% identical to the SEQ ID NO: 22; or h) an amino acid
sequence that is SEQ ID NO: 23 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 23; or i) an amino acid
sequence that is SEQ ID NO: 24 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 24; or j) an amino acid
sequence that is SEQ ID NO: 25 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 25; or k) an amino acid
sequence that is SEQ ID NO: 26 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 26; or l) an amino acid
sequence that is SEQ ID NO: 27 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 27 or m) an amino acid
sequence that is SEQ ID NO: 28 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 28.
[0006] Disclosed herein, in certain embodiments, is a vector
comprising DNA encoding a recombinant Arc polypeptide or a
recombinant endogenous Gag polypeptide. In some embodiments, the
vector further encodes a therapeutic agent. In some embodiments,
the therapeutic agent is a nucleic acid. In some embodiments, the
nucleic acid is an RNA. In some embodiments, the recombinant Arc
polypeptide is a human Arc polypeptide comprising an amino acid
sequence that is SEQ ID NO: 1 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 1. In some embodiments, the
recombinant Arc polypeptide is an Arc polypeptide comprising: a) an
amino acid sequence that is SEQ ID NO: 2 or an amino acid sequence
that is at least 90% identical to the SEQ ID NO: 2; b) an amino
acid sequence that is SEQ ID NO: 3 or an amino acid sequence that
is at least 90% identical to the SEQ ID NO: 3; c) an amino acid
sequence that is SEQ ID NO: 4 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 4; d) an amino acid sequence
that is SEQ ID NO: 5 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 5; e) an amino acid sequence that is
SEQ ID NO: 6 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 6; f) an amino acid sequence that is
SEQ ID NO: 7 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 7; g) an amino acid sequence that is
SEQ ID NO: 8 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 8; h) an amino acid sequence that is
SEQ ID NO: 9 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 9; i) an amino acid sequence that is
SEQ ID NO: 10 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 10; or j) an amino acid sequence that
is SEQ ID NO: 11 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 11; or k) an amino acid sequence that
is SEQ ID NO: 12 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 12; or l) an amino acid sequence that
is SEQ ID NO: 13 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 13; or m) an amino acid sequence that
is SEQ ID NO: 14 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 14; or n) an amino acid sequence that
is SEQ ID NO: 15 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 15. In some embodiments, the
recombinant endogenous Gag polypeptide is a human endogenous Gag
polypeptide. In some embodiments, the recombinant endogenous Gag
polypeptide is an endogenous Gag polypeptide comprising: a) an
amino acid sequence that is SEQ ID NO: 16 or an amino acid sequence
that is at least 90% identical to the SEQ ID NO: 16; b) an amino
acid sequence that is SEQ ID NO: 17 or an amino acid sequence that
is at least 90% identical to the SEQ ID NO: 17; c) an amino acid
sequence that is SEQ ID NO: 18 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 18; d) an amino acid sequence
that is SEQ ID NO: 19 or an amino acid sequence that is at least
90% identical to the SEQ ID NO: 19; e) an amino acid sequence that
is SEQ ID NO: 20 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 20; f) an amino acid sequence that is
SEQ ID NO: 21 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 21; or g) an amino acid sequence that
is SEQ ID NO: 22 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 22; or h) an amino acid sequence that
is SEQ ID NO: 23 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 23; or i) an amino acid sequence that
is SEQ ID NO: 24 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 24; or j) an amino acid sequence that
is SEQ ID NO: 25 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 25; or k) an amino acid sequence that
is SEQ ID NO: 26 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 26; or l) an amino acid sequence that
is SEQ ID NO: 27 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 27 or m) an amino acid sequence that is
SEQ ID NO: 28 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 28.
[0007] Disclosed herein, in certain embodiments, is a vector
comprising DNA encoding a recombinant Arc polypeptide or a
recombinant endogenous Gag polypeptide, wherein the recombinant Arc
polypeptide is not a rat Arc polypeptide or a human Arc
polypeptide. In some embodiments, the vector further encodes a
cargo. In some embodiments, the cargo is a nucleic acid. In some
embodiments, the cargo is an RNA. In some embodiments, the cargo is
a therapeutic agent. In some embodiments, the recombinant Arc
polypeptide is an Arc polypeptide comprising: a) an amino acid
sequence that is SEQ ID NO: 2 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 2; b) an amino acid sequence
that is SEQ ID NO: 3 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 3; c) an amino acid sequence that is
SEQ ID NO: 4 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 4; d) an amino acid sequence that is
SEQ ID NO: 5 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 5; e) an amino acid sequence that is
SEQ ID NO: 6 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 6; f) an amino acid sequence that is
SEQ ID NO: 7 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 7; g) an amino acid sequence that is
SEQ ID NO: 8 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 8; h) an amino acid sequence that is
SEQ ID NO: 9 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 9; i) an amino acid sequence that is
SEQ ID NO: 10 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 10; or j) an amino acid sequence that
is SEQ ID NO: 11 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 11; or k) an amino acid sequence that
is SEQ ID NO: 12 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 12; or l) an amino acid sequence that
is SEQ ID NO: 13 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 13; or m) an amino acid sequence that
is SEQ ID NO: 14 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 14; or n) an amino acid sequence that
is SEQ ID NO: 15 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 15. In some embodiments, the
recombinant endogenous Gag polypeptide is an endogenous Gag
polypeptide comprising: a) an amino acid sequence that is SEQ ID
NO: 16 or an amino acid sequence that is at least 90% identical to
the SEQ ID NO: 16; b) an amino acid sequence that is SEQ ID NO: 17
or an amino acid sequence that is at least 90% identical to the SEQ
ID NO: 17; c) an amino acid sequence that is SEQ ID NO: 18 or an
amino acid sequence that is at least 90% identical to the SEQ ID
NO: 18; d) an amino acid sequence that is SEQ ID NO: 19 or an amino
acid sequence that is at least 90% identical to the SEQ ID NO: 19;
e) an amino acid sequence that is SEQ ID NO: 20 or an amino acid
sequence that is at least 90% identical to the SEQ ID NO: 20; f) an
amino acid sequence that is SEQ ID NO: 21 or an amino acid sequence
that is at least 90% identical to the SEQ ID NO: 21; or g) an amino
acid sequence that is SEQ ID NO: 22 or an amino acid sequence that
is at least 90% identical to the SEQ ID NO: 22; or h) an amino acid
sequence that is SEQ ID NO: 23 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 23; or i) an amino acid
sequence that is SEQ ID NO: 24 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 24; or j) an amino acid
sequence that is SEQ ID NO: 25 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 25; or k) an amino acid
sequence that is SEQ ID NO: 26 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 26; or l) an amino acid
sequence that is SEQ ID NO: 27 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 27 or m) an amino acid
sequence that is SEQ ID NO: 28 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 28.
[0008] Disclosed herein, in certain embodiments, is a method of
delivering a cargo to a cell comprising administering to the cell a
capsid comprising a recombinant Arc polypeptide or a recombinant
endogenous Gag polypeptide and a therapeutic agent. In some
embodiments, the therapeutic agent is a nucleic acid. In some
embodiments, the nucleic acid is an RNA. In some embodiments, the
recombinant Arc polypeptide is a human Arc polypeptide comprising
an amino acid sequence that is SEQ ID NO: 1 or an amino acid
sequence that is at least 90% identical to the SEQ ID NO: 1. In
some embodiments, the recombinant Arc polypeptide is an Arc
polypeptide comprising: a) an amino acid sequence that is SEQ ID
NO: 2 or an amino acid sequence that is at least 90% identical to
the SEQ ID NO: 2; b) an amino acid sequence that is SEQ ID NO: 3 or
an amino acid sequence that is at least 90% identical to the SEQ ID
NO: 3; c) an amino acid sequence that is SEQ ID NO: 4 or an amino
acid sequence that is at least 90% identical to the SEQ ID NO: 4;
d) an amino acid sequence that is SEQ ID NO: 5 or an amino acid
sequence that is at least 90% identical to the SEQ ID NO: 5; e) an
amino acid sequence that is SEQ ID NO: 6 or an amino acid sequence
that is at least 90% identical to the SEQ ID NO: 6; f) an amino
acid sequence that is SEQ ID NO: 7 or an amino acid sequence that
is at least 90% identical to the SEQ ID NO: 7; g) an amino acid
sequence that is SEQ ID NO: 8 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 8; h) an amino acid sequence
that is SEQ ID NO: 9 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 9; i) an amino acid sequence that is
SEQ ID NO: 10 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 10; or j) an amino acid sequence that
is SEQ ID NO: 11 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 11; or k) an amino acid sequence that
is SEQ ID NO: 12 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 12; or l) an amino acid sequence that
is SEQ ID NO: 13 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 13; or m) an amino acid sequence that
is SEQ ID NO: 14 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 14; or n) an amino acid sequence that
is SEQ ID NO: 15 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 15. In some embodiments, the
recombinant endogenous Gag polypeptide is a human endogenous Gag
polypeptide. In some embodiments, the recombinant endogenous Gag
polypeptide is an endogenous Gag polypeptide comprising: a) an
amino acid sequence that is SEQ ID NO: 16 or an amino acid sequence
that is at least 90% identical to the SEQ ID NO: 16; b) an amino
acid sequence that is SEQ ID NO: 17 or an amino acid sequence that
is at least 90% identical to the SEQ ID NO: 17; c) an amino acid
sequence that is SEQ ID NO: 18 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 18; d) an amino acid sequence
that is SEQ ID NO: 19 or an amino acid sequence that is at least
90% identical to the SEQ ID NO: 19; e) an amino acid sequence that
is SEQ ID NO: 20 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 20; f) an amino acid sequence that is
SEQ ID NO: 21 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 21; or g) an amino acid sequence that
is SEQ ID NO: 22 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 22; or h) an amino acid sequence that
is SEQ ID NO: 23 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 23; or i) an amino acid sequence that
is SEQ ID NO: 24 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 24; or j) an amino acid sequence that
is SEQ ID NO: 25 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 25; or k) an amino acid sequence that
is SEQ ID NO: 26 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 26; or l) an amino acid sequence that
is SEQ ID NO: 27 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 27 or m) an amino acid sequence that is
SEQ ID NO: 28 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 28. In some embodiments, the cell is a
eukaryotic cell. In some embodiments, the cell is a vertebrate
cell. In some embodiments, the cell is a mammalian cell. In some
embodiments, the cell is a human cell. In some embodiments, the
cargo is a nucleic acid. In some embodiments, the cell expresses a
gene encoded by the nucleic acid. In some embodiments, the cargo is
a therapeutic agent.
[0009] Disclosed herein, in certain embodiments, is a method of
delivering a cargo to a cell comprising administering to the cell a
capsid comprising a recombinant Arc polypeptide or a recombinant
endogenous Gag polypeptide, wherein the recombinant Arc polypeptide
is not a rat Arc polypeptide or a human Arc polypeptide. In some
embodiments, the capsid further comprises a cargo. In some
embodiments, the cargo is a nucleic acid. In some embodiments, the
cargo is an RNA. In some embodiments, the cargo is a therapeutic
agent. In some embodiments, the recombinant Arc polypeptide is an
Arc polypeptide comprising: a) an amino acid sequence that is SEQ
ID NO: 2 or an amino acid sequence that is at least 90% identical
to the SEQ ID NO: 2; b) an amino acid sequence that is SEQ ID NO: 3
or an amino acid sequence that is at least 90% identical to the SEQ
ID NO: 3; c) an amino acid sequence that is SEQ ID NO: 4 or an
amino acid sequence that is at least 90% identical to the SEQ ID
NO: 4; d) an amino acid sequence that is SEQ ID NO: 5 or an amino
acid sequence that is at least 90% identical to the SEQ ID NO: 5;
e) an amino acid sequence that is SEQ ID NO: 6 or an amino acid
sequence that is at least 90% identical to the SEQ ID NO: 6; f) an
amino acid sequence that is SEQ ID NO: 7 or an amino acid sequence
that is at least 90% identical to the SEQ ID NO: 7; g) an amino
acid sequence that is SEQ ID NO: 8 or an amino acid sequence that
is at least 90% identical to the SEQ ID NO: 8; h) an amino acid
sequence that is SEQ ID NO: 9 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 9; i) an amino acid sequence
that is SEQ ID NO: 10 or an amino acid sequence that is at least
90% identical to the SEQ ID NO: 10; or j) an amino acid sequence
that is SEQ ID NO: 11 or an amino acid sequence that is at least
90% identical to the SEQ ID NO: 11; or k) an amino acid sequence
that is SEQ ID NO: 12 or an amino acid sequence that is at least
90% identical to the SEQ ID NO: 12; or l) an amino acid sequence
that is SEQ ID NO: 13 or an amino acid sequence that is at least
90% identical to the SEQ ID NO: 13; or m) an amino acid sequence
that is SEQ ID NO: 14 or an amino acid sequence that is at least
90% identical to the SEQ ID NO: 14; or n) an amino acid sequence
that is SEQ ID NO: 15 or an amino acid sequence that is at least
90% identical to the SEQ ID NO: 15. In some embodiments, the
recombinant endogenous Gag polypeptide is an endogenous Gag
polypeptide comprising: a) an amino acid sequence that is SEQ ID
NO: 16 or an amino acid sequence that is at least 90% identical to
the SEQ ID NO: 16; b) an amino acid sequence that is SEQ ID NO: 17
or an amino acid sequence that is at least 90% identical to the SEQ
ID NO: 17; c) an amino acid sequence that is SEQ ID NO: 18 or an
amino acid sequence that is at least 90% identical to the SEQ ID
NO: 18; d) an amino acid sequence that is SEQ ID NO: 19 or an amino
acid sequence that is at least 90% identical to the SEQ ID NO: 19;
e) an amino acid sequence that is SEQ ID NO: 20 or an amino acid
sequence that is at least 90% identical to the SEQ ID NO: 20; f) an
amino acid sequence that is SEQ ID NO: 21 or an amino acid sequence
that is at least 90% identical to the SEQ ID NO: 21; or g) an amino
acid sequence that is SEQ ID NO: 22 or an amino acid sequence that
is at least 90% identical to the SEQ ID NO: 22; or h) an amino acid
sequence that is SEQ ID NO: 23 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 23; or i) an amino acid
sequence that is SEQ ID NO: 24 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 24; or j) an amino acid
sequence that is SEQ ID NO: 25 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 25; or k) an amino acid
sequence that is SEQ ID NO: 26 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 26; or l) an amino acid
sequence that is SEQ ID NO: 27 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 27 or m) an amino acid
sequence that is SEQ ID NO: 28 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 28. In some embodiments, the
cell is a eukaryotic cell. In some embodiments, the cell is a
vertebrate cell. In some embodiments, the cell is a mammalian cell.
In some embodiments, the cell is a human cell. In some embodiments,
the cargo is a nucleic acid. In some embodiments, the cell
expresses a gene encoded by the nucleic acid. In some embodiments,
the cargo is a therapeutic agent.
[0010] Disclosed herein, in certain embodiments, is a method of
transfecting a nucleic acid into a cell comprising administering to
the cell a capsid comprising a recombinant Arc polypeptide or a
recombinant endogenous Gag polypeptide and a therapeutic agent. In
some embodiments, the therapeutic agent is a nucleic acid. In some
embodiments, the nucleic acid is an RNA. In some embodiments, the
recombinant Arc polypeptide is a human Arc polypeptide comprising
an amino acid sequence that is SEQ ID NO: 1 or an amino acid
sequence that is at least 90% identical to the SEQ ID NO: 1. In
some embodiments, the recombinant Arc polypeptide is an Arc
polypeptide comprising: a) an amino acid sequence that is SEQ ID
NO: 2 or an amino acid sequence that is at least 90% identical to
the SEQ ID NO: 2; b) an amino acid sequence that is SEQ ID NO: 3 or
an amino acid sequence that is at least 90% identical to the SEQ ID
NO: 3; c) an amino acid sequence that is SEQ ID NO: 4 or an amino
acid sequence that is at least 90% identical to the SEQ ID NO: 4;
d) an amino acid sequence that is SEQ ID NO: 5 or an amino acid
sequence that is at least 90% identical to the SEQ ID NO: 5; e) an
amino acid sequence that is SEQ ID NO: 6 or an amino acid sequence
that is at least 90% identical to the SEQ ID NO: 6; f) an amino
acid sequence that is SEQ ID NO: 7 or an amino acid sequence that
is at least 90% identical to the SEQ ID NO: 7; g) an amino acid
sequence that is SEQ ID NO: 8 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 8; h) an amino acid sequence
that is SEQ ID NO: 9 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 9; i) an amino acid sequence that is
SEQ ID NO: 10 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 10; or j) an amino acid sequence that
is SEQ ID NO: 11 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 11; or k) an amino acid sequence that
is SEQ ID NO: 12 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 12; or l) an amino acid sequence that
is SEQ ID NO: 13 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 13; or m) an amino acid sequence that
is SEQ ID NO: 14 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 14; or n) an amino acid sequence that
is SEQ ID NO: 15 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 15. In some embodiments, the
recombinant endogenous Gag polypeptide is a human endogenous Gag
polypeptide. In some embodiments, the recombinant endogenous Gag
polypeptide is an endogenous Gag polypeptide comprising: a) an
amino acid sequence that is SEQ ID NO: 16 or an amino acid sequence
that is at least 90% identical to the SEQ ID NO: 16; b) an amino
acid sequence that is SEQ ID NO: 17 or an amino acid sequence that
is at least 90% identical to the SEQ ID NO: 17; c) an amino acid
sequence that is SEQ ID NO: 18 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 18; d) an amino acid sequence
that is SEQ ID NO: 19 or an amino acid sequence that is at least
90% identical to the SEQ ID NO: 19; e) an amino acid sequence that
is SEQ ID NO: 20 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 20; f) an amino acid sequence that is
SEQ ID NO: 21 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 21; or g) an amino acid sequence that
is SEQ ID NO: 22 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 22; or h) an amino acid sequence that
is SEQ ID NO: 23 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 23; or i) an amino acid sequence that
is SEQ ID NO: 24 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 24; or j) an amino acid sequence that
is SEQ ID NO: 25 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 25; or k) an amino acid sequence that
is SEQ ID NO: 26 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 26; or l) an amino acid sequence that
is SEQ ID NO: 27 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 27 or m) an amino acid sequence that is
SEQ ID NO: 28 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 28.
[0011] Disclosed herein, in certain embodiments, is a method of
transfecting a nucleic acid into a cell comprising administering to
the cell a capsid comprising a recombinant Arc polypeptide or a
recombinant endogenous Gag polypeptide, wherein the recombinant Arc
polypeptide is not a rat Arc polypeptide or a human Arc
polypeptide. In some embodiments, the capsid further comprises a
cargo. In some embodiments, the cargo is a nucleic acid. In some
embodiments, the cargo is an RNA. In some embodiments, the cargo is
a therapeutic agent. In some embodiments, the recombinant Arc
polypeptide is an Arc polypeptide comprising: a) an amino acid
sequence that is SEQ ID NO: 2 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 2; b) an amino acid sequence
that is SEQ ID NO: 3 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 3; c) an amino acid sequence that is
SEQ ID NO: 4 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 4; d) an amino acid sequence that is
SEQ ID NO: 5 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 5; e) an amino acid sequence that is
SEQ ID NO: 6 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 6; f) an amino acid sequence that is
SEQ ID NO: 7 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 7; g) an amino acid sequence that is
SEQ ID NO: 8 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 8; h) an amino acid sequence that is
SEQ ID NO: 9 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 9; i) an amino acid sequence that is
SEQ ID NO: 10 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 10; or j) an amino acid sequence that
is SEQ ID NO: 11 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 11; or k) an amino acid sequence that
is SEQ ID NO: 12 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 12; or l) an amino acid sequence that
is SEQ ID NO: 13 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 13; or m) an amino acid sequence that
is SEQ ID NO: 14 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 14; or n) an amino acid sequence that
is SEQ ID NO: 15 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 15. In some embodiments, the
recombinant endogenous Gag polypeptide is an endogenous Gag
polypeptide comprising: a) an amino acid sequence that is SEQ ID
NO: 12 or an amino acid sequence that is at least 90% identical to
the SEQ ID NO: 12; b) an amino acid sequence that is SEQ ID NO: 13
or an amino acid sequence that is at least 90% identical to the SEQ
ID NO: 13; c) an amino acid sequence that is SEQ ID NO: 14 or an
amino acid sequence that is at least 90% identical to the SEQ ID
NO: 14; d) an amino acid sequence that is SEQ ID NO: 15 or an amino
acid sequence that is at least 90% identical to the SEQ ID NO: 15;
e) an amino acid sequence that is SEQ ID NO: 16 or an amino acid
sequence that is at least 90% identical to the SEQ ID NO: 16; f) an
amino acid sequence that is SEQ ID NO: 17 or an amino acid sequence
that is at least 90% identical to the SEQ ID NO: 17; g) an amino
acid sequence that is SEQ ID NO: 18 or an amino acid sequence that
is at least 90% identical to the SEQ ID NO: 18; g) an amino acid
sequence that is SEQ ID NO: 19 or an amino acid sequence that is at
least 90% identical to the SEQ ID NO: 19; g) an amino acid sequence
that is SEQ ID NO: 20 or an amino acid sequence that is at least
90% identical to the SEQ ID NO: 20; g) an amino acid sequence that
is SEQ ID NO: 21 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 21; or h) an amino acid sequence that
is SEQ ID NO: 22 or an amino acid sequence that is at least 90%
identical to the SEQ ID NO: 22.
[0012] Disclosed herein, in certain embodiments, is an engineered
Arc or endo-Gag polypeptide comprising a cargo binding domain and
at least one capsid forming subunit from an Arc or endo-Gag
polypeptide. In some embodiments, the cargo binding domain
comprises a nucleic acid binding domain. In some embodiments, the
cargo binding domain comprises a polypeptide that binds to a small
molecule. In some embodiments, the cargo binding domain comprises a
polypeptide that binds to a protein, a peptide, or an antibody or
binding fragment thereof. In some embodiments, the cargo binding
domain comprises a polypeptide that binds to a peptidomimetic or a
nucleotidomimetic. In some embodiments, the at least one capsid
forming subunit comprises a polypeptide that corresponds to the CA
N-lobe and/or CA C-lobe of SEQ ID NO: 1. In some embodiments, the
engineered Arc or endo-Gag polypeptide further comprises a second
capsid forming subunit from a different species of an Arc or
endo-Gag polypeptide. In some embodiments, the second capsid
forming subunit comprises a polypeptide that corresponds to the
N-lobe and/or C-lobe of SEQ ID NO: 1. In some embodiments, the at
least one capsid forming subunit and the second capsid forming
subunit are each independently selected from a species of Arc or
endo-Gag selected from a mammal, a rodent, a bird, a reptile, a
fish, an insect, a fungus, or a plant. In some embodiments, the at
least one capsid forming subunit and the second capsid forming
subunit are from two different species. In some embodiments, the
cargo binding domain is fused either directly or via a linker to
the C-terminus of the at least one capsid forming subunit. In some
embodiments, the cargo binding domain is fused either directly or
via a linker to the N-terminus of the at least one capsid forming
subunit. In some embodiments, the second capsid forming subunit is
fused either directly or via a linker to the C-terminus of the at
least one capsid forming subunit. In some embodiments, the second
capsid forming subunit is fused either directly or via a linker to
the N-terminus of the at least one capsid forming subunit. In some
embodiments, the cargo binding domain is fused either directly or
via a linker to the N-terminus of the at least one capsid forming
subunit and the second capsid forming subunit is fused either
directly or via a linker to the C-terminus of the at least one
capsid forming subunit. In some embodiments, the cargo binding
domain is fused either directly or via a linker to the C-terminus
of the at least one capsid forming subunit and the second capsid
forming subunit is fused either directly or via a linker to the
N-terminus of the at least one capsid forming subunit. In some
embodiments, the engineered Arc or endo-Gag polypeptide further
comprises a second polypeptide. In some embodiments, the second
polypeptide is fused either directly or via a linker to the at
least one capsid forming subunit. In some embodiments, the second
polypeptide is fused either directly or via a linker to the cargo
binding domain. In some embodiments, the second polypeptide is a
protein or an antibody or its binding fragments thereof. In some
embodiments, the protein is a human protein or a viral protein. In
some embodiments, the protein is a human Gag-like protein. In some
embodiments, the protein is a de novo engineered protein designed
to bind to a target receptor of interest. In some embodiments, the
second polypeptide guides the delivery of a capsid formed by the
engineered Arc or endo-Gag polypeptide to a target site of
interest.
[0013] Disclosed herein, in certain embodiments, is a truncated Arc
or endo-Gag polypeptide wherein a portion that is not involved with
capsid-formation, nucleic acid binding, or delivery is removed. In
some embodiments, the portion comprises a matrix (MA) domain, a
reverse transcriptase (RT) domain, a nucleotide binding domain, or
a combination thereof, provided that the nucleotide binding domain
is not a human Arc RNA binding domain. In some embodiments, the
portion comprises a CA C-lobe domain. In some embodiments, the
portion comprises an N-terminal deletion, a C-terminal deletion, or
a combination thereof. In some embodiments, the N-terminal deletion
comprises a deletion of up to 10 amino acids, 20 amino acids, 30
amino acids, or 50 amino acids. In some embodiments, the C-terminal
deletion comprises a deletion of up to 10 amino acids, 20 amino
acids, 30 amino acids, or 50 amino acids.
[0014] Disclosed herein, in certain embodiments, is an Arc or
endo-Gag-based capsid comprising an engineered Arc or endo-Gag
polypeptide which may be a truncated Arc or endo-Gag polypeptide
and a cargo encapsulated by the capsid formed by the engineered Arc
or endo-Gag polypeptide. In some embodiments, the cargo is a
nucleic acid molecule. In some embodiments, the nucleic acid
molecule is DNA, RNA, or a mixture of DNA and RNA. In some
embodiments, the DNA and the RNA are each independently
single-stranded, double-stranded, or a mixture of single and double
stranded. In some embodiments, the cargo is a small molecule. In
some embodiments, the cargo is a protein. In some embodiments, the
cargo is a peptide. In some embodiments, the cargo is an antibody
or binding fragments thereof. In some embodiments, the cargo is a
peptidomimetic or a nucleotidomimetic. In some embodiments, the Arc
or endo-Gag-based capsid comprises one or more additional capsid
subunits from one or more species of Arc or endo-Gag proteins that
are different than the engineered Arc or endo-Gag polypeptide. In
some embodiments, the Arc-based or endo-Gag-based capsid comprises
one or more additional capsid subunits from non-Arc proteins. In
some embodiments, the one or more additional capsid subunits
comprise Copia protein, ASPRV1 protein, a protein from the SCAN
domain family, a protein encoded by the Paraneoplastic Ma antigen
family (e.g. PNMA5, PNMA6, PNMA6A, and PNMA6B), a protein from the
retrotransposon Gag-like family (e.g. RTL3, RTL6, RTL8A, RTL8B), or
a combination thereof. In some embodiments, the one or more
additional capsid subunits comprise BOP, LDOC1, MOAP1, PEG10,
PNMA3, PNMA5, PNMA6A, PNMA6B, RTL3, RTL6, RTL8A, RTL8B, and ZNF18.
In some embodiments, the capsid has a diameter of at least 1 nm, 2
nm, 3 nm, 4 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 50 nm, 80
nm, 100 nm, 120 nm, 150 nm, 200 nm, 250 nm, 300 nm, 500 nm, 600 nm,
or more. In some embodiments, the capsid has a diameter of from
about 1 nm to about 600 nm, from about 1 nm to about 500 nm, from
about 1 nm to about 200 nm, from about 1 nm to about 100 nm, from
about 1 nm to about 50 nm, or from about 1 nm to about 30 nm. In
some embodiments, the capsid has a reduced off-target effect. In
some embodiments, the capsid does not have an off-target effect. In
some embodiments, the capsid is formed ex-vivo. In some
embodiments, the capsid is formed in-vitro.
[0015] Disclosed herein, in certain embodiments, is a nucleic acid
polymer encoding a recombinant or engineered Arc polypeptide or a
recombinant or engineered endogenous Gag polypeptide described
herein.
[0016] Disclosed herein, in certain embodiments, is a vector
comprising a nucleic acid polymer encoding a recombinant or
engineered Arc polypeptide or a recombinant or engineered
endogenous Gag polypeptide described herein.
[0017] Disclosed herein, in certain embodiments, is a method of
preparing a loaded Arc-based or endo-Gag-based capsid comprising:
incubating a plurality of recombinant or engineered Arc
polypeptides or a plurality of recombinant or engineered endo-Gag
polypeptides with a cargo in a solution for a time sufficient to
generate the loaded capsid. In some embodiments, the method further
comprises mixing the solution comprising the plurality of
engineered Arc or endo-Gag polypeptides with a plurality of non-Arc
or non-endo-Gag capsid forming subunits prior to incubating with
the cargo. In some embodiments, the plurality of non-Arc or
non-endo-Gag capsid forming subunits are mixed with the plurality
of recombinant or engineered Arc or endo-Gag polypeptides at a
ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In
some embodiments, the plurality of non-Arc or non-endo-Gag capsid
forming subunits are mixed with the plurality of engineered Arc or
endo-Gag polypeptides at a ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7,
1:8, 1:9, or 1:10. In some embodiments, the method further
comprises mixing the solution comprising the plurality of truncated
Arc or endo-Gag polypeptides with a plurality of non-Arc or
endo-Gag capsid forming subunits prior to incubating with the
cargo. In some embodiments, the plurality of non-Arc or endo-Gag
capsid forming subunits are mixed with the plurality of truncated
Arc or endo-Gag polypeptides at a ratio of 1:1, 2:1, 3:1, 4:1, 5:1,
6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the plurality of
non-Arc or non-endo-Gag capsid forming subunits are mixed with the
plurality of truncated Arc or endo-Gag polypeptides at a ratio of
1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some
embodiments, the plurality of engineered Arc or endo-Gag
polypeptides is obtained from a bacterial cell system, an insect
cell system, or a mammalian cell system. In some embodiments, the
plurality of engineered Arc or endo-Gag polypeptides is obtained
from a cell-free system. In some embodiments, the plurality of
truncated Arc or endo-Gag polypeptides is obtained from a bacterial
cell system, an insect cell system, or a mammalian cell system. In
some embodiments, the plurality of truncated Arc or endo-Gag
polypeptides is obtained from a cell-free system. In some
embodiments, the loaded Arc-based or endo-Gag capsid is formulated
for systemic administration. In some embodiments, the loaded Arc or
endo-Gag-based capsid is formulated for local administration. In
some embodiments, the loaded Arc or endo-Gag-based capsid is
formulated for parenteral administration. In some embodiments, the
loaded Arc or endo-Gag-based capsid is formulated for oral
administration. In some embodiments, the loaded Arc or
endo-Gag-based capsid is formulated for topical administration. In
some embodiments, the loaded Arc or endo-Gag-based capsid is
formulated for sublingual or aerosol administration.
[0018] Disclosed herein, in certain embodiments, is use of an
engineered or recombinant Arc-based or endo-Gag-based capsid for
delivery of a cargo to a site of interest, comprising contacting a
cell at the site of interest with an Arc-based or endo-Gag-based
capsid for a time sufficient to facilitate cellular uptake of the
capsid. In some embodiments, the cell is a tumor cell. In some
embodiments, the tumor cell is a solid tumor cell. In some
embodiments, the solid tumor cell is a cell from a bladder cancer,
breast cancer, brain cancer, colorectal cancer, kidney cancer,
liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin
cancer, stomach cancer, or thyroid cancer. In some embodiments, the
tumor cell is from a hematologic malignancy. In some embodiments,
the hematologic malignancy is a B-cell malignancy, or a T-cell
malignancy. In some embodiments, the hematologic malignancy is
chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma
(SLL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma,
mantle cell lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma,
or peripheral T cell lymphoma. In some embodiments, the cell is a
somatic cell. In some embodiments, the cell is a stem cell or a
progenitor cell. In some embodiments, the cell is a mesenchymal
stem or progenitor cell. In some embodiments, the cell is a
hematopoietic stem or progenitor cell. In some embodiments, the
cell is a muscle cell, a skin cell, a blood cell, or an immune
cell. In some embodiments, a target protein is overexpressed or is
depleted in the cell. In some embodiments, a target gene in the
cell has one or more mutations. In some embodiments, the cell
comprises an impaired splicing mechanism. In some embodiments, the
use is an in vivo use. In some embodiments, the Arc-based capsid is
administered systemically to a subject. In some embodiments, the
Arc-based or endo-Gag-based capsid is administered via local
administration to a subject. In some embodiments, the Arc-based or
endo-Gag-based capsid is administered parenterally to a subject. In
some embodiments, the Arc-based capsid is administered orally to a
subject. In some embodiments, the Arc-based or endo-Gag-based
capsid is administered topically to a subject. In some embodiments,
the Arc-based or endo-Gag-based capsid is administered via
sublingual or aerosol administration to a subject. In some
embodiments, the use is an in vitro or ex vivo use.
[0019] Disclosed herein, in certain embodiments, is a kit
comprising an engineered Arc or endo-Gag polypeptide, a truncated
Arc or endo-Gag polypeptide, a vector encoding a recombinant or
engineered Arc or endo-Gag polypeptide, or an Arc-based or
endo-Gag-based capsid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Various aspects of the disclosure are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present disclosure will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the disclosure
are utilized, and the accompanying drawings below.
[0021] FIG. 1 is a representation of exemplary Arc
polypeptides.
[0022] FIG. 2 is a representation of exemplary engineered Arc
polypeptides.
[0023] FIGS. 3A and 3B illustrate an exemplary method of
engineering an Arc polypeptide to carry a specific cargo (FIG. 3A)
(e.g., an RNA payload), or remove an off-function effect (FIG.
3B).
[0024] FIG. 4A shows the isolation of 6.times.His-tagged human Arc
by elution from a HisTrap column with an imidazole gradient.
[0025] FIG. 4B shows the separation of 6.times.His-tagged human Arc
from residual nucleic acids on a mono Q column eluted with a NaCl
gradient.
[0026] FIG. 5 shows a transmission electron microscope image of
negatively stained human Arc capsids.
[0027] FIG. 6 shows transmission electron microscope images of
negatively stained capsids formed from recombinantly expressed Arc
orthologs.
[0028] FIG. 7 shows transmission electron microscope images of
negatively stained capsids formed from recombinantly expressed
endo-Gag proteins.
[0029] FIG. 8 shows selective internalization of Alexa594-labeled
Arc capsids by HeLa cells.
[0030] FIG. 9 shows the delivery of Cre RNA to HeLa cells by Arc
capsids.
[0031] FIG. 10 illustrates methods for screening Arc and endo-Gag
gene candidates for the ability to transmit a heterologous RNA
payload.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0032] Administrating diagnostic or therapeutic agents to a site of
interest with precision has presented an ongoing challenge.
Available methods of delivering nucleic acids to cells have myriad
limitations. For example, AAV viral vectors often used for gene
therapy are immunogenic, have a limited payload capacity of <3
kb, suffer from poor bio-distribution, can only be administered by
direct injection, and pose a risk of disrupting host genes by
integration. Non-viral methods have different limitations.
Liposomes are primarily delivered to the liver. Extracellular
vesicles have a limited payload capacity of <1 kb, limited
scalability, and purification difficulties. Thus, there is a
recognized need for new methods of delivering therapeutic
payloads.
[0033] Most molecules do not possess inherent affinity in the body.
In other cases, the administered agents accumulate either in the
liver and the kidney for clearance or in unintended tissue or cell
types. Method for improving delivery includes coating the agent of
choice with hydrophobic compounds or polymers. Such an approach
increases the duration of said agent in circulation and augments
hydrophobicity for cellular uptake. On the other hand, this
approach does not actively direct cargo to the site of interest for
delivery.
[0034] To specifically target sites where therapy is needed,
therapeutic compounds are optionally fused to moieties such as
ligands, antibodies, and aptamers that recognize and bind to
receptors displayed on the surface of targeted cells. Upon reaching
a cell of interest, the therapeutic compound is optionally further
delivered to an intracellular target. For example, a therapeutic
RNA can be translated to a protein if it comes into contact with a
ribosome in the cytoplasm of the cell.
[0035] Arc (activity-regulated cytoskeleton-associated protein)
regulates the endocytic trafficking of
.alpha.-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)
type glutamate receptors. Arc activities have been linked to
synaptic strength and neuronal plasticity. Phenotypes of loss of
Arc in experimental murine model included defective formation of
long-term memory and reduced neuronal activity and plasticity.
[0036] Arc exhibits similar molecular properties to retroviral Gag
proteins. The Arc gene may have originated from the Ty3/gypsy
retrotransposon. An endogenous Gag (endo-Gag) protein is any
protein endogenous to a eukaryotic organism, including Arc, that
has predicted and annotated similarity to viral Gag proteins.
Exemplary endo-Gag proteins are disclosed in Campillos M, Doerks T,
Shah P K, and Bork P, Computational characterization of multiple
Gag-like human proteins, Trends Genet. 2006 November; 22(11):585-9.
An endo-Gag protein is optionally recombinantly expressed by any
host cell, including a prokaryotic or eukaryotic cell, or a
bacterial, yeast, insect, vertebrate, mammalian, or human cell. As
described herein, in some embodiments an endo-Gag protein assembles
into an endo-Gag capsid.
[0037] Disclosed herein, in certain embodiments, are Arc and
endo-Gag polypeptides which assemble into a capsid for delivery of
a cargo of interest. In some embodiments, also described herein are
engineered Arc and endo-Gag polypeptides which assemble into a
capsid for delivery of a cargo of interest. In additional
embodiments, described herein are capsids, e.g., Arc-based or
endo-Gag-based capsids, for delivery of a cargo of interest.
Arc Polypeptides and Endogenous Gag Polypeptides
[0038] In certain embodiments, disclosed herein is an Arc
polypeptide. In certain embodiments, disclosed herein is an
endo-Gag polypeptide. It should be understood that endo-Gag
sequences are optional substitutes for Arc sequences to form any
type of engineered Arc polypeptide described in this section.
[0039] In some instances, Arc is a non-human Arc polypeptide. In
some instances, the Arc polypeptide comprises a full-length Arc
polypeptide (e.g., a full-length non-human Arc polypeptide). In
other instances, the Arc polypeptide comprises a fragment of
non-human Arc, such as a truncated Arc polypeptide, that
participates in the formation of a capsid. In additional instances,
the Arc polypeptide comprises one or more domains of a non-human
Arc polypeptide, in which at least one of the domains participates
in the formation of a capsid. In further instances, the Arc
polypeptide is a recombinant Arc polypeptide.
[0040] In some instances, endo-Gag is a non-human endo-Gag
polypeptide. In some instances, the endo-Gag polypeptide comprises
a full-length endo-Gag polypeptide (e.g., a full-length non-human
endo-Gag polypeptide). In other instances, the endo-Gag polypeptide
comprises a fragment of non-human endo-Gag, such as a truncated
endo-Gag polypeptide, that participates in the formation of a
capsid. In additional instances, the endo-Gag polypeptide comprises
one or more domains of a non-human endo-Gag polypeptide, in which
at least one of the domains participates in the formation of a
capsid. In further instances, the endo-Gag polypeptide is a
recombinant endo-Gag polypeptide.
[0041] In some embodiments, the Arc is a human Arc polypeptide with
at least its RNA binding domain modified to bind to a cargo that is
not native to the human Arc. In some instances, the Arc polypeptide
comprises a full-length human Arc polypeptide with at least its RNA
binding domain modified to bind to a cargo that is not native to
the human Arc protein. In other instances, the Arc polypeptide
comprises a human Arc fragment comprising modification(s) in at
least its RNA binding domain. In additional instances, the Arc
polypeptide comprises one or more domains of a human Arc
polypeptide, in which at least one of the domains participates in
the formation of a capsid and in which the RNA binding domain is
modified to bind to a cargo that native human Arc protein does not
bind to. In further instances, the Arc polypeptide is a recombinant
human Arc polypeptide, with at least the RNA binding domain is
modified to enable loading of a cargo that is not native to the
human Arc protein.
[0042] In some embodiments, the Endo-Gag is a human Endo-Gag
polypeptide with at least its RNA binding domain modified to bind
to a cargo that is not native to the human endo-Gag. In some
instances, the endo-Gag polypeptide comprises a full-length human
endo-Gag polypeptide with at least its RNA binding domain modified
to bind to a cargo that is not native to the human endo-Gag
protein. In other instances, the endo-Gag polypeptide comprises a
human endo-Gag fragment comprising modification(s) in at least its
RNA binding domain to bind to a cargo that a native human endo-Gag
protein does not bind to. In additional instances, the endo-Gag
polypeptide comprises one or more domains of a human endo-Gag
polypeptide, in which at least one of the domains participates in
the formation of a capsid and in which the RNA binding domain is
modified to bind to a cargo that is not native to the human
endo-Gag protein. In further instances, the endo-Gag polypeptide is
a recombinant human endo-Gag polypeptide, with at least the RNA
binding domain is modified to enable loading of a cargo that is not
native to the human endo-Gag protein.
[0043] In some instances, the Arc or endo-Gag polypeptide is an
engineered Arc or endo-Gag polypeptide. As used herein, an
engineered polypeptide is a recombinant polypeptide that is not
identical in sequence to a full length, wild-type polypeptide. In
some instances, the engineered Arc or endo-Gag polypeptide
comprises a fragment of an Arc or endo-Gag polypeptide from a first
species and at least an additional fragment from an Arc or endo-Gag
polypeptide of a second species. In some cases, the first Arc or
endo-Gag polypeptide is selected from a kingdom member of animalia,
plantae, fungi, or protista. In some cases, the first species is
selected from a mammal, a rodent, a bird, a reptile, a fish, a
vertebrate, a eukaryote, an insect, a fungus, or a plant. In some
cases, the second Arc polypeptide is selected from a kingdom member
of animalia, plantae, fungi, or protista that is the same or
different than the first Arc or endo-Gag polypeptide. In some
cases, the second species is selected from a mammal, a rodent, a
bird, a reptile, a fish, a vertebrate, a eukaryote, an insect, a
fungus, or a plant that is different from the first species.
[0044] In some embodiments, an exemplary mammalian Arc or endo-Gag
protein for expression as a recombinant or engineered Arc
polypeptide is from the species Homo sapiens. Additional exemplary
species of primate Arc or endo-Gag protein proteins for expression
as a recombinant or engineered Arc polypeptide include: Gorilla,
Pongo abelii, Pan paniscus, Macaca nemestrina, Chlorocebus sabaeus,
Papio anubis, Rhinopithecus roxellana, Macaca fascicularis,
Nomascus leucogenys, Callithrix jacchus, Aotus nancymaae, Cebus
capucinus imitator, Saimiri boliviensis boliviensis, Otolemur
garnettii, Macaca mulatta, and Macaca fascicularis.
[0045] An exemplary species list of rodent Arc or endo-Gag proteins
for expression as a recombinant or engineered Arc or endo-Gag
polypeptide includes: Fukomys damarensis, Microcebus murinus,
Heterocephalus glaber, Propithecus coquereli, Marmota marmota
marmota, Galeopterus variegatus, Cavia porcellus, Dipodomys ordii,
Octodon degus, Castor canadensis Nannospalax galili, Carlito
syrichta, Chinchilla lanigera, Mus musculus, Ictidomys
tridecemlineatus, Rattus norvegicus, Microtus ochrogaster, Otolemur
garnettii, Meriones unguiculatus, Cricetulus griseus, Rattus
norvegicus, Neotoma lepida, Jaculus jaculus, Mustela putorius furo,
Mesocricetus auratus, Tupaia chinensis, Cricetulus griseus,
Chrysochloris asiatica, Elephantulus edwardii, Erinaceus europaeus,
Ochotona princeps, Sorex araneus, Monodelphis domestica, Echinops
telfairi, and Condylura cristata.
[0046] An exemplary species list of Arc or endo-Gag proteins for
expression as a recombinant or engineered Arc or endo-Gag
polypeptide includes: Vulpes vulpes, Canis lupus dingo, Felis
catus, Panthera pardus, Callorhinus ursinus, Odobenus rosmarus
divergens, Equus asinus, Sus scrofa, Manis javanica, Ceratotherium
simum simum, Leptonychotes weddellii, Enhydra lutris kenyoni,
Lipotes vexillifer, Bos grunniens, Bubalus bubalis, Camelus
dromedarius, Vicugna pacos, Orcinus orca, Neomonachus
schauinslandi, Tursiops truncatus, Bos taurus, Capra hircus,
Delphinapterus leucas, Ovis aries musimon, Balaenoptera
acutorostrata scammoni, Neophocaena asiaeorientalis
asiaeorientalis, Miniopterus natalensis, Pteropus alecto, Physeter
catodon, Loxodonta africana, Orycteropus afer afer, Bos mutus,
Desmodus rotundus, Hipposideros armiger, Ailuropoda melanoleuca,
Trichechus manatus latirostris, Rousettus latirostris, Rousettus
aegyptiacus, Eptesicus fuscus, Rhinolophus sinicus, Cervus elaphus
hippelaphus, Odocoileus virginianus texanus, Pantholops hodgsonii,
Camelus bactrianus, Sarcophilus harrisii, Phascolarctos cinereus,
and Ornithorhynchus anatinus.
[0047] An exemplary species list of bird Arc or endo-Gag proteins
for expression as a recombinant or engineered Arc or endo-Gag
polypeptide includes: Gallus gallus, Corvus cornix, cornix, Panus
major, Corvus brachyrhynchos, Dromaius novaehollandiae, and Apteryx
rowi.
[0048] An exemplary species list of reptile Arc protein for
expression as a recombinant or engineered Arc or endo-Gag
polypeptide includes: Python bivittatus, Pogona vitticeps, Anolis
carolinensis, Protobothrops mucrosquamatus, Alligator sinensis,
Crocodylus porosus, Gavialis gangeticus, Alligator
mississippiensis, Pelodiscus sinensis, Terrapene mexicana
triunguis, Chrysemys picta bellii, Chelonia mydas, Nanorana
parkeri, Xenopus tropicalis, Xenopus laevis, and Latimeria
chalumnae,
[0049] An exemplary species list of fish Arc protein for expression
as a recombinant or engineered Arc or endo-Gag polypeptide
includes: Oncorhynchus mykiss, Acanthochromis polyacanthus,
Oncorhynchus kisutch, Carassius auratus, and Austrofundulus
limnaeus.
[0050] An exemplary species list of insect Arc or endo-Gag proteins
for expression as a recombinant or engineered Arc or endo-Gag
polypeptide includes: Drosophila serrata, Drosophila bipectinata,
Solenopsis invicta, Temnothorax curvispinosus, Drosophila
melanogaster, Agrilus planipennis, Camponotus floridanus,
Pogonomyrmex barbatus, Nilaparvata lugens, Bombyx mori, Tribolium
castaneum, and Leptinotarsa decemlineata.
[0051] An exemplary species list of plant Arc or endo-Gag proteins
for expression as a recombinant or engineered Arc or endo-Gag
polypeptide includes Spinacia oleracea and Erythranthe guttata.
[0052] An exemplary species list of fungi proteins for expression
as a recombinant or engineered Arc or endo-Gag polypeptide
includes: Saccharomyces cerevisiae, Rhizopus delemar, Fusarium
oxysporum, Cryptococcus neoformans, Rhizophagus irregularis,
Fusarium fujikuroi, Candida albicans, Trichophyton rubrum,
Pyrenophora tritici-repentis, Rhizopus microsporus, Rhizoctonia
solani, Aspergillus flavus, Verticillium dahliae, Fusarium
verticillioides, Aspergillus niger, Fusarium graminearum,
Aspergillus fumigatus, Zymoseptoria tritici, and Trichoderma
harzianum.
[0053] An exemplary species list of protists Arc or endo-Gag
proteins for expression as a recombinant or engineered Arc or
endo-Gag polypeptide includes: Entamoeba histolytica, Paulinella
micropora, Guillardia theta, Oxyrrhis marina, Seminavis robusta,
Euglena longa, Naegleria gruberi, and Trichomonas vaginalis.
[0054] In some instances, Arc or endo-Gag comprises a capsid
assembly/forming (CA) domain, a cargo binding domain (e.g., an RNA
binding domain), and optionally a matrix (MA) domain, a reverse
transcriptase (RT) domain, or a combination thereof. In some cases,
the CA domain is further divided into an N-lobe domain and a C-lobe
domain. In some cases, the cargo binding domain comprises an RNA
binding domain, a DNA binding domain, a protein binding domain, a
peptide binding domain, an antibody binding domain, a small
molecule binding domain, or a peptidomimetic/nucleotidomimetic
binding domain. Exemplary cargo binding domains include, but are
not limited to, domains from GPCRs, antibodies or binding fragments
thereof, lipoproteins, integrins, tyrosine kinases, DNA-binding
proteins, RNA-binding proteins, nucleases, ligases, proteases,
integrases, isomerases, phosphatases, GTPases, aromatases,
esterases, adaptor proteins, G-proteins, GEFs, cytokines,
interleukins, interleukin receptors, interferons, interferon
receptors, caspases, transcription factors, neurotrophic factors
and their receptors, growth factors and their receptors, signal
recognition particle and receptor components, extracellular matrix
proteins, integral components of membrane, ribosomal proteins,
translation elongation factors, translation initiation factors,
GPI-anchored proteins, tissue factors, dystrophin, utrophin,
dystrobrevin, any fusions, combinations, subunits, derivatives, or
domains thereof.
[0055] In some embodiments, one or more non-essential regions which
are not involved in capsid formation or nucleic acid binding are
removed from an Arc or endo-Gag protein to generate an Arc or
endo-Gag polypeptide. In such instances, one or more non-essential
regions, e.g., an N-terminal region (e.g., up to 10 amino acids, up
to 20 amino acids, up to 30 amino acids, or up to 50 amino acids),
a C-terminal region (e.g., up to 10 amino acids, up to 20 amino
acids, up to 30 amino acids, or up to 50 amino acids), a RT domain,
a MA domain, or a combination thereof, are deleted from an Arc or
endo-Gag protein to generate an Arc or endo-Gag polypeptide. In
some cases, only the essential regions involved in capsid
assembly/forming and cargo binding remain in an Arc or endo-Gag
polypeptide. In additional cases, only the essential region
involved in capsid assembly/forming (e.g., the N-lobe and/or the
C-lobe) remains in an Arc polypeptide.
[0056] In certain embodiments, the RT domain, the MA domain, and/or
the endogenous RNA binding domain are replaced with other cargo
binding domains: for example, replaced with a DNA binding domain, a
protein binding domain, a peptide binding domain, an antibody
binding domain, a small molecule binding domain, a peptidomimetic
binding domain, or a nucleotidomimetic binding domain. In some
embodiments, an Arc or endo-Gag polypeptide comprises truncations
or modifications of domains involved in capsid forming, nucleic
acid binding, or delivery.
[0057] In some embodiments, the Arc or endo-Gag polypeptide
comprises a MA domain, a CA N-lobe, a CA C-lobe, a cargo binding
domain, and a RT domain. In some instances, the Arc polypeptide
comprises from N-terminus to C-terminus the following domains: the
MA domain, the CA N-lobe, the CA C-lobe, the RT domain, and the
cargo binding domain. In some instances, the Arc or endo-Gag
polypeptide comprises from N-terminus to C-terminus the following
domains: the MA domain, the RT domain, the cargo binding domain,
the CA N-lobe, and the CA C-lobe. In some instances, the Arc or
endo-Gag polypeptide comprises from N-terminus to C-terminus the
following domains: the cargo binding domain, the MA domain, the RT
domain, the CA N-lobe, and the CA C-lobe. In some instances, the
domains are arranged in an order that does not impede capsid
assembly and cargo binding. In some instances, each of the domains
is either directly or indirectly fused to the respective two
flanking domains.
[0058] In some embodiments, the Arc or endo-Gag polypeptide
comprises a MA domain, a CA N-lobe, a CA C-lobe, and a cargo
binding domain. In some instances, the Arc or endo-Gag polypeptide
comprises from N-terminus to C-terminus the following domains: the
MA domain, the CA N-lobe, the CA C-lobe, and the cargo binding
domain. In some instances, the Arc polypeptide comprises from
N-terminus to C-terminus the following domains: the MA domain, the
cargo binding domain, the CA N-lobe, and the CA C-lobe. In some
instances, the Arc or endo-Gag polypeptide comprises from
N-terminus to C-terminus the following domains: the cargo binding
domain, the MA domain, the CA N-lobe, and the CA C-lobe. In some
instances, the domains are arranged in an order that does not
impede capsid assembly and cargo binding. In some instances, each
of the domains is either directly or indirectly fused to the
respective two flanking domains.
[0059] In some embodiments, the Arc or endo-Gag polypeptide
comprises a CA N-lobe, a CA C-lobe, and a cargo binding domain. In
some instances, the Arc or endo-Gag polypeptide comprises from
N-terminus to C-terminus the following domains: the CA N-lobe, the
CA C-lobe, and the cargo binding domain. In some instances, the Arc
or endo-Gag polypeptide comprises from N-terminus to C-terminus the
following domains: the cargo binding domain, the CA N-lobe, and the
CA C-lobe. In some instances, the domains are arranged in an order
that does not impede capsid assembly and cargo binding. In some
instances, each of the domains is either directly or indirectly
fused to the respective two flanking domains.
[0060] In some embodiments, the Arc or endo-Gag polypeptide
comprises a CA N-lobe and a cargo binding domain. In some
instances, the Arc or endo-Gag polypeptide comprises from
N-terminus to C-terminus the following domains: the CA N-lobe and
the cargo binding domain. In some instances, the Arc or endo-Gag
polypeptide comprises from N-terminus to C-terminus the following
domains: the cargo binding domain and the CA N-lobe. In some
instances, the domains are arranged in an order that does not
impede capsid assembly and cargo binding. In some instances, the
two domains are either directly or indirectly fused to each
other.
[0061] In some embodiments, the Arc or endo-Gag polypeptide is
engineered to comprise a cargo binding domain, a CA domain, a MA
domain, or a RT domain from one or more additional species to
generate an engineered Arc polypeptide. For example, the engineered
Arc or endo-Gag polypeptide comprises a cargo binding domain, a CA
domain, a MA domain, or a RT domain from a first species and a
cargo binding domain, a CA domain, a MA domain, or a RT domain from
a second species. In some cases, the first species is selected from
a eukaryote, a vertebrate, a human, a mammal, a rodent, a bird, a
reptile, a fish, an insect, a fungus, or a plant. In some cases,
the second species is selected from a eukaryote, a vertebrate, a
human, a mammal, a rodent, a bird, a reptile, a fish, an insect, a
fungus, or a plant that is different from the first species.
[0062] In some instances, the engineered or endo-Gag Arc
polypeptide comprises a cargo binding domain from a first species
and a CA domain (e.g., a CA N-lobe and optionally a CA C-lobe) from
a second species. The engineered Arc or endo-Gag polypeptide
optionally comprises a MA domain and an RT domain from either the
first species or the second species. In some cases, the first
species is selected from a eukaryote, a vertebrate, a human, a
mammal, a rodent, a bird, a reptile, a fish, an insect, a fungus,
or a plant. In some cases, the second species is selected from a
eukaryote, a vertebrate, a human, a mammal, a rodent, a bird, a
reptile, a fish, an insect, a fungus, or a plant that is different
from the first species.
[0063] In some instances, the engineered Arc or endo-Gag
polypeptide comprises a cargo binding domain, a first CA domain, a
second CA domain, and optionally a MA domain and/or a RT domain. In
some cases, the cargo binding domain, the first CA domain, and
optionally a MA domain and/or a RT domain are from a first species
and the second CA domain is from a second species. In some cases,
the first CA domain is from a first species and the cargo binding
domain, the second CA domain, and optionally a MA domain and/or a
RT domain are from a second species. In some instances, the domains
are arranged in an order that does not impede capsid assembly and
cargo binding. In some instances, each of the domains is either
directly or indirectly fused to the respective two adjacent
domains.
[0064] In some instances, the engineered Arc or endo-Gag
polypeptide comprises a cargo binding domain, a first CA domain,
and a second CA domain. In some cases, the cargo binding domain and
the first CA domain are from a first species and the second CA
domain is from a second species. In some cases, the first CA domain
is from a first species and the cargo binding domain and the second
CA domain are from a second species. In such cases, the engineered
Arc or endo-Gag polypeptide comprises from the N-terminus to the
C-terminus the following domains: a cargo binding domain, a first
CA domain, and a second CA domain. In such cases, the engineered
Arc or endo-Gag polypeptide comprises from the N-terminus to the
C-terminus the following domains: a first CA domain, a cargo
binding domain, and a second CA domain. In such cases, the
engineered Arc or endo-Gag polypeptide comprises from the
N-terminus to the C-terminus the following domains: a first CA
domain, a second CA domain, and a cargo binding domain. In some
instances, the domains are arranged in an order that does not
impede capsid assembly and cargo binding. In some instances, each
of the domains is either directly or indirectly fused to the
respective two flanking domains.
[0065] In some instances, the engineered Arc or endo-Gag
polypeptide further comprises a second polypeptide. In some
instances, the second polypeptide is fused directly or indirectly
via a linker to one or more of: a cargo binding domain, a first CA
domain, a second CA domain, a MA domain if present, or a RT domain
if present. In some cases, the second polypeptide is a protein
(e.g., a human protein), an antibody or binding fragment thereof, a
viral protein, a Gag-like protein (e.g., a human Gag-like protein),
or a de novo engineered protein designed to bind to a target
receptor of interest. In some instances, the antibody or binding
fragment thereof comprises a humanized antibody or binding
fragments thereof, a murine antibody or binding fragment thereof, a
chimeric antibody or binding fragment thereof, a monoclonal
antibody or binding fragment thereof, a multi-specific antibody or
binding fragment thereof, a bispecific antibody or biding fragment
thereof, a monovalent Fab', a divalent Fab.sub.2, F(ab)'.sub.3
fragments, a single-chain variable fragment (scFv), a bis-scFv, an
(scFv).sub.2, a diabody, a minibody, a nanobody, a triabody, a
tetrabody, a disulfide stabilized Fv protein (dsFv), a
single-domain antibody (sdAb), an Ig NAR, a camelid antibody or
binding fragment thereof, or a chemically modified derivative
thereof. In some instances, the second polypeptide guides the
delivery of a capsid formed by the engineered Arc polypeptide to a
target site of interest.
[0066] In some embodiments, a nucleic acid sequence or amino acid
sequence of the disclosure (for example, encoding an Arc
polypeptide or endo-Gag polypeptide) has at least 70% homology, at
least 71% homology, at least 72% homology, at least 73% homology,
at least 74% homology, at least 75% homology, at least 76%
homology, at least 77% homology, at least 78% homology, at least
79% homology, at least 80% homology, at least 81% homology, at
least 82% homology, at least 83% homology, at least 84% homology,
at least 85% homology, at least 86% homology, at least 87%
homology, at least 88% homology, at least 89% homology, at least
90% homology, at least 91% homology, at least 92% homology, at
least 93% homology, at least 94% homology, at least 95% homology,
at least 96% homology, at least 97% homology, at least 98%
homology, at least 99% homology, at least 99.1% homology, at least
99.2% homology, at least 99.3% homology, at least 99.4% homology,
at least 99.5% homology, at least 99.6% homology, at least 99.7%
homology, at least 99.8% homology, at least 99.9% or at least
99.99% homology to an amino acid sequence provided herein. Various
methods and software programs are used to determine the homology
between two or sequences, such as NCBI BLAST, Clustal W, MAFFT,
Clustal Omega, AlignMe, Praline, or another suitable method or
algorithm.
[0067] In certain embodiments, the Arc polypeptide is a human
polypeptide having the amino acid sequence of SEQ ID NO: 1 or a
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity to SEQ ID NO: 1.
[0068] In certain embodiments, the Arc polypeptide is a killer
whale polypeptide having the amino acid sequence of SEQ ID NO: 2 or
a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity to SEQ ID NO: 2.
[0069] In certain embodiments, the Arc polypeptide is a white
tailed deer polypeptide having the amino acid sequence of SEQ ID
NO: 3 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identity to SEQ ID NO: 3.
[0070] In certain embodiments, the Arc polypeptide is a platypus
polypeptide having the amino acid sequence of SEQ ID NO: 4 or a
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity to SEQ ID NO: 4.
[0071] In certain embodiments, the Arc polypeptide is a goose
polypeptide having the amino acid sequence of SEQ ID NO: 5 or a
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity to SEQ ID NO: 5.
[0072] In certain embodiments, the Arc polypeptide is a Dalmatian
pelican polypeptide having the amino acid sequence of SEQ ID NO: 6
or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity to SEQ ID NO: 6.
[0073] In certain embodiments, the Arc polypeptide is a white
tailed eagle polypeptide having the amino acid sequence of SEQ ID
NO: 7 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identity to SEQ ID NO: 7.
[0074] In certain embodiments, the Arc polypeptide is a king cobra
polypeptide having the amino acid sequence of SEQ ID NO: 8 or a
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity to SEQ ID NO: 8.
[0075] In certain embodiments, the Arc polypeptide is a ray finned
fish polypeptide having the amino acid sequence of SEQ ID NO: 9 or
a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity to SEQ ID NO: 9.
[0076] In certain embodiments, the Arc polypeptide is a sperm whale
polypeptide having the amino acid sequence of SEQ ID NO: 10 or a
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity to SEQ ID NO: 10.
[0077] In certain embodiments, the Arc polypeptide is a turkey
polypeptide having the amino acid sequence of SEQ ID NO: 11 or a
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity to SEQ ID NO: 11.
[0078] In certain embodiments, the Arc polypeptide is a central
bearded dragon polypeptide having the amino acid sequence of SEQ ID
NO: 12 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identity to SEQ ID NO: 12.
[0079] In certain embodiments, the Arc polypeptide is a Chinese
alligator polypeptide having the amino acid sequence of SEQ ID NO:
13 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity to SEQ ID NO: 13.
[0080] In certain embodiments, the Arc polypeptide is an American
alligator polypeptide having the amino acid sequence of SEQ ID NO:
14 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity to SEQ ID NO: 14.
[0081] In certain embodiments, the Arc polypeptide is a Japanese
gekko polypeptide having the amino acid sequence of SEQ ID NO: 15
or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity to SEQ ID NO: 15.
[0082] In certain embodiments, the endo-Gag polypeptide is a human
PNMA3 polypeptide having the amino acid sequence of SEQ ID NO: 16
or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity to SEQ ID NO: 16.
[0083] In certain embodiments, the endo-Gag polypeptide is a human
PNMA5 polypeptide having the amino acid sequence of SEQ ID NO: 17
or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity to SEQ ID NO: 17.
[0084] In certain embodiments, the endo-Gag polypeptide is a human
PNMA6A polypeptide having the amino acid sequence of SEQ ID NO: 18
or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity to SEQ ID NO: 18.
[0085] In certain embodiments, the endo-Gag polypeptide is a human
PNMA6B polypeptide having the amino acid sequence of SEQ ID NO: 19
or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity to SEQ ID NO: 19.
[0086] In certain embodiments, the endo-Gag polypeptide is a human
RTL3 polypeptide having the amino acid sequence of SEQ ID NO: 20 or
a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity to SEQ ID NO: 20.
[0087] In certain embodiments, the endo-Gag polypeptide is a human
RTL6 polypeptide having the amino acid sequence of SEQ ID NO: 21 or
a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity to SEQ ID NO: 21.
[0088] In certain embodiments, the endo-Gag polypeptide is a human
RTL8A polypeptide having the amino acid sequence of SEQ ID NO: 22
or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity to SEQ ID NO: 22.
[0089] In certain embodiments, the endo-Gag polypeptide is a human
RTL8B polypeptide having the amino acid sequence of SEQ ID NO: 23
or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity to SEQ ID NO: 23.
[0090] In certain embodiments, the endo-Gag polypeptide is a human
BOP polypeptide having the amino acid sequence of SEQ ID NO: 24 or
a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity to SEQ ID NO: 24.
[0091] In certain embodiments, the endo-Gag polypeptide is a human
LDOC1 polypeptide having the amino acid sequence of SEQ ID NO: 25
or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity to SEQ ID NO: 25.
[0092] In certain embodiments, the endo-Gag polypeptide is a human
ZNF18 polypeptide having the amino acid sequence of SEQ ID NO: 26
or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity to SEQ ID NO: 26.
[0093] In certain embodiments, the endo-Gag polypeptide is a human
MOAP1 polypeptide having the amino acid sequence of SEQ ID NO: 27
or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity to SEQ ID NO: 27.
[0094] In certain embodiments, the endo-Gag polypeptide is a human
PEG10 polypeptide having the amino acid sequence of SEQ ID NO: 28
or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity to SEQ ID NO: 28.
[0095] In some cases, the recombinant Arc or endo-Gag polypeptide
is an Arc polypeptide illustrated in FIG. 1.
[0096] In some cases, the engineered Arc or endo-Gag polypeptide is
an engineered Arc polypeptide illustrated in FIG. 2.
Linkers
[0097] In certain embodiments, a polypeptide of the disclosure
comprises a linker. In some embodiments, the linker is a peptide
linker. In some instances, the linker is a rigid linker. In other
instances, the linker is a flexible linker. In some cases, the
linker is a non-cleavable linker. In other cases, the linker is a
cleavable linker. In additional cases, the linker comprises a
linear structure, or a non-linear structure (e.g., a cyclic
structure).
[0098] In certain embodiments, non-cleavable linkers comprise short
peptides of varying lengths. Exemplary non-cleavable linkers
include (EAAAK)n (SEQ ID NO: 70), or (EAAAR)n (SEQ ID NO: 71),
where n is from 1 to 5, and up to 30 residues of glutamic
acid-proline or lysine-proline repeats. In some embodiments, the
non-cleavable linker comprises (GGGGS)n (SEQ ID NO: 72) or (GGGS)n
(SEQ ID NO: 73), wherein n is 1 to 10; KESGSVSSEQLAQFRSLD (SEQ ID
NO: 74); or EGKSSGSGSESKST (SEQ ID NO: 75). In some embodiments,
the non-cleavable linker comprises a poly-Gly/Ala polymer.
[0099] In certain embodiments, the linker is a cleavable linker,
e.g., an extracellular cleavable linker or an intracellular
cleavable linker. In some instances, the linker is designed for
cleavage in the presence of particular conditions or in a
particular environment (e.g., under physiological condition). For
example, the design of a linker for cleavage by specific
conditions, such as by a specific enzyme, allows the targeting of
cellular uptake to a specific location.
[0100] In some embodiments, the linker is a pH-sensitive linker. In
one instance, the linker is cleaved under basic pH conditions. In
other instance, the linker is cleaved under acidic pH
conditions.
[0101] In some embodiments, the linker is cleaved in vivo by
endogenous enzymes (e.g., proteases) such as serine proteases
including but not limited to thrombin, metalloproteases, furin,
cathepsin B, necrotic enzymes (e.g., calpains), and the like.
Exemplary cleavable linkers include, but are not limited to,
GGAANLVRGG (SEQ ID NO: 76); SGRIGFLRTA (SEQ ID NO: 77); SGRSA (SEQ
ID NO: 78); GFLG (SEQ ID NO: 79); ALAL (SEQ ID NO: 80); FK;
PIC(Et)F-F (SEQ ID NO: 81), where C(Et) indicates S-ethylcysteine;
PR(S/T)(L/I)(S/T) (SEQ ID NO: 82); DEVD (SEQ ID NO: 83); GWEHDG
(SEQ ID NO: 84); RPLALWRS (SEQ ID NO: 85); or a combination
thereof.
Capsids
[0102] In some embodiments, disclosed herein is a capsid. In some
instances, the capsid comprises an Arc polypeptide and/or an
endo-Gag polypeptide such as a Copia protein, ASPRV1 protein, a
protein from the SCAN domain family, a protein encoded by the
Paraneoplastic Ma antigen family, a protein or a combination of
proteins chosen from the retrotransposon Gag-like family, or a
combination thereof. Exemplary endo-Gag polypeptides are BOP,
LDOC1, MOAP1, PEG10, PNMA3, PNMA5, PNMA6A, PNMA6B, RTL3, RTL6,
RTL8A, RTL8B, and ZNF18. In some instances, the Arc polypeptide,
the Copia protein, the ASPRV1 protein, the protein from the SCAN
domain family, the protein encoded by the Paraneoplastic Ma antigen
family, and the protein or a combination of proteins chosen from
the retrotransposon Gag-like family are each independently a
full-length polypeptide. In other instances, the Arc polypeptide,
the Copia protein, the ASPRV1 protein, the protein from the SCAN
domain family, the protein encoded by the Paraneoplastic Ma antigen
family, and the protein or a combination of proteins chosen from
the retrotransposon Gag-like family are each independently a
functional fragment thereof, e.g., that is capable of forming a
subunit of a capsid.
Arc-Based Capsids and Endo-Gag-Based Capsids
[0103] In some embodiments, the capsid comprises an Arc-based
capsid. In some embodiments, the capsid comprises an endo-Gag-based
capsid. In some instances, the Arc-based and/or endo-Gag capsid
comprises a plurality of recombinant Arc polypeptides and/or
endo-Gag polypeptides described above, a plurality of engineered
Arc polypeptides and/or endo-Gag polypeptides described above, or a
combination thereof. In some cases, the Arc-based capsid comprises
a plurality of recombinant Arc polypeptides. In other cases, the
Arc-based capsid comprises a plurality of engineered Arc
polypeptides. In some cases, the endo-Gag-based capsid comprises a
plurality of recombinant endo-Gag polypeptides. In other cases, the
endo-Gag-based capsid comprises a plurality of engineered endo-Gag
polypeptides.
[0104] In some embodiments, the Arc-based or endo-Gag-based capsid
comprises a first plurality of Arc and/or endo-Gag polypeptides
from a first species and a second plurality of Arc and/or endo-Gag
polypeptides from at least a second species. In some cases, the
first species is selected from a eukaryote, a vertebrate, a human,
a mammal, a rodent, a bird, a reptile, a fish, an insect, a fungus,
or a plant. In some cases, the second species is selected from a
eukaryote, a vertebrate, a human, a mammal, a rodent, a bird, a
reptile, a fish, an insect, a fungus, or a plant that is different
from the first species.
[0105] In some instances, the ratio of the first plurality of Arc
or endo-Gag polypeptides to the second plurality of Arc or endo-Gag
polypeptides is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,
20:1, 50:1, or 100:1. In some cases, the ratio of the first
plurality of Arc or endo-Gag polypeptides to the second plurality
of Arc or endo-Gag polypeptides is 1:1. In some cases, the ratio of
the first plurality of Arc or endo-Gag polypeptides to the second
plurality of Arc or endo-Gag polypeptides is 2:1. In some cases,
the ratio of the first plurality of Arc or endo-Gag polypeptides to
the second plurality of Arc or endo-Gag polypeptides is 4:1. In
some cases, the ratio of the first plurality of Arc or endo-Gag
polypeptides to the second plurality of Arc or endo-Gag
polypeptides is 5:1. In some cases, the ratio of the first
plurality of Arc or endo-Gag polypeptides to the second plurality
of Arc or endo-Gag polypeptides is 8:1. In some cases, the ratio of
the first plurality of Arc or endo-Gag polypeptides to the second
plurality of Arc polypeptides is 10:1. In some cases, the ratio of
the first plurality of Arc or endo-Gag polypeptides to the second
plurality of Arc or endo-Gag polypeptides is 20:1. In some cases,
the ratio of the first plurality of Arc or endo-Gag polypeptides to
the second plurality of Arc or endo-Gag polypeptides is 50:1. In
some cases, the ratio of the first plurality of Arc or endo-Gag
polypeptides to the second plurality of Arc or endo-Gag
polypeptides is 100:1. In some instances, the ratio is the
comparison in molar concentration. In some instances, the ratio is
the comparison in the number of capsid forming subunits (e.g., each
of the or engineered Arc polypeptide forms a capsid subunit).
[0106] In some instances, the ratio of the first plurality of Arc
or endo-Gag polypeptides to the second plurality of Arc or endo-Gag
polypeptides is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20,
or 1:50. In some cases, the ratio of the first plurality of Arc or
endo-Gag polypeptides to the second plurality of Arc or endo-Gag
polypeptides is 1:2. In some cases, the ratio of the first
plurality of Arc or endo-Gag polypeptides to the second plurality
of Arc or endo-Gag polypeptides is 1:5. In some cases, the ratio of
the first plurality of Arc or endo-Gag polypeptides to the second
plurality of Arc or endo-Gag polypeptides is 1:8. In some cases,
the ratio of the first plurality of Arc or endo-Gag polypeptides to
the second plurality of Arc or endo-Gag polypeptides is 1:10. In
some cases, the ratio of the first plurality of Arc or endo-Gag
polypeptides to the second plurality of Arc or endo-Gag
polypeptides is 1:20. In some cases, the ratio of the first
plurality of Arc or endo-Gag polypeptides to the second plurality
of Arc or endo-Gag polypeptides is 1:50. In some instances, the
ratio is the comparison in molar concentration. In some instances,
the ratio is the comparison in the number of capsid forming
subunits (e.g., each of the recombinant or engineered Arc or
endo-Gag polypeptide forms a capsid subunit).
[0107] In some embodiments, the Arc-based capsid or endo-Gag-based
capsid comprises a plurality of recombinant or engineered Arc
polypeptides and a plurality of non-Arc proteins. Exemplary species
of non-Arc proteins include but are not limited to, Copia, ASPRV1,
a protein or a combination of proteins chosen from the SCAN domain
family, a protein or a combination of proteins chosen from the
Paraneoplastic Ma antigen family, and a protein or a combination of
proteins chosen from the retrotransposon Gag-like family. Exemplary
species of non-Arc proteins include BOP, LDOC1, MOAP1, PEG10,
PNMA3, PNMA5, PNMA6A, PNMA6B, RTL3, RTL6, RTL8A, RTL8B, and
ZNF18.
[0108] In some instances, the ratio of the plurality of recombinant
or engineered Arc polypeptides to the plurality of non-Arc proteins
is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1,
or 100:1. In some cases, the ratio of the plurality of recombinant
or engineered Arc polypeptides to the plurality of non-Arc proteins
is 1:1. In some cases, the ratio of the plurality of recombinant or
engineered Arc polypeptides to the plurality of non-Arc proteins is
2:1. In some cases, the ratio of the plurality of recombinant or
engineered Arc polypeptides to the plurality of non-Arc proteins is
4:1. In some cases, the ratio of the plurality of recombinant or
engineered Arc polypeptides to the plurality of non-Arc proteins is
5:1. In some cases, the ratio of the plurality of recombinant or
engineered Arc polypeptides to the plurality of non-Arc proteins is
8:1. In some cases, the ratio of the plurality of recombinant or
engineered Arc polypeptides to the plurality of non-Arc proteins is
10:1. In some cases, the ratio of the plurality of recombinant or
engineered Arc polypeptides to the plurality of non-Arc proteins is
20:1. In some cases, the ratio of the plurality of recombinant or
engineered Arc polypeptides to the plurality of non-Arc proteins is
50:1. In some cases, the ratio of the plurality of recombinant or
engineered Arc polypeptides to the plurality of non-Arc proteins is
100:1. In some instances, the ratio is the comparison in molar
concentration. In some instances, the ratio is the comparison in
the number of capsid forming subunits (e.g., each of the
recombinant or engineered Arc polypeptide forms a capsid
subunit).
[0109] In some instances, the ratio of the plurality of recombinant
or engineered Arc polypeptides to the plurality of non-Arc proteins
is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, or 1:50. In
some cases, the ratio of the plurality of recombinant or engineered
Arc polypeptides to the plurality of non-Arc proteins is 1:2. In
some cases, the ratio of the plurality of recombinant or engineered
Arc polypeptides to the plurality of non-Arc proteins is 1:5. In
some cases, the ratio of the plurality of recombinant or engineered
Arc polypeptides to the plurality of non-Arc proteins is 1:8. In
some cases, the ratio of the plurality of recombinant or engineered
Arc polypeptides to the plurality of non-Arc proteins is 1:10. In
some cases, the ratio of the plurality of recombinant or engineered
Arc polypeptides to the plurality of non-Arc proteins is 1:20. In
some cases, the ratio of the plurality of recombinant or engineered
Arc polypeptides to the plurality of non-Arc proteins is 1:50. In
some instances, the ratio is the comparison in molar concentration.
In some instances, the ratio is the comparison in the number of
capsid forming subunits (e.g., each of the recombinant or
engineered Arc polypeptide forms a capsid subunit).
[0110] In some embodiments, the capsid has a diameter of at least 1
nm, or more. In some instances, the capsid has a diameter of at
least 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40
nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 300
nm, 400 nm, 500 nm, 600 nm, or more. In some instances, the capsid
has a diameter of at least 5 nm, or more. In some cases, the capsid
has a diameter of at least 10 nm, or more. In some instances, the
capsid has a diameter of at least 20 nm, or more. In some cases,
the capsid has a diameter of at least 30 nm, or more. In some
cases, the capsid has a diameter of at least 40 nm, or more. In
some cases, the capsid has a diameter of at least 50 nm, or more.
In some cases, the capsid has a diameter of at least 80 nm, or
more. In some cases, the capsid has a diameter of at least 100 nm,
or more. In some cases, the capsid has a diameter of at least 200
nm, or more. In some cases, the capsid has a diameter of at least
300 nm, or more. In some cases, the capsid has a diameter of at
least 400 nm, or more. In some cases, the capsid has a diameter of
at least 500 nm, or more. In some cases, the capsid has a diameter
of at least 600 nm, or more.
[0111] In some embodiments, the capsid has a diameter of at most 1
nm, or less. In some instances, the capsid has a diameter of at
most 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40
nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 300
nm, 400 nm, 500 nm, 600 nm, or less. In some instances, the capsid
has a diameter of at most 5 nm, or less. In some cases, the capsid
has a diameter of at most 10 nm, or less. In some instances, the
capsid has a diameter of at most 20 nm, or less. In some cases, the
capsid has a diameter of at most 30 nm, or less. In some cases, the
capsid has a diameter of at least 40 nm, or less. In some cases,
the capsid has a diameter of at least 50 nm, or less. In some
cases, the capsid has a diameter of at least 80 nm, or less. In
some cases, the capsid has a diameter of at least 100 nm, or less.
In some cases, the capsid has a diameter of at least 200 nm, or
less. In some cases, the capsid has a diameter of at least 300 nm,
or less. In some cases, the capsid has a diameter of at least 400
nm, or less. In some cases, the capsid has a diameter of at least
500 nm, or less. In some cases, the capsid has a diameter of at
least 600 nm, or less.
[0112] In some embodiments, the capsid has a diameter of about 1
nm, 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40
nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 300
nm, 400 nm, 500 nm, or 600 nm. In some instances, the capsid has a
diameter of about 5 nm. In some cases, the capsid has a diameter of
about 10 nm. In some instances, the capsid has a diameter of about
20 nm. In some cases, the capsid has a diameter of about 30 nm. In
some cases, the capsid has a diameter of about 40 nm. In some
cases, the capsid has a diameter of about 50 nm. In some cases, the
capsid has a diameter of about 80 nm. In some cases, the capsid has
a diameter of about 100 nm. In some cases, the capsid has a
diameter of about 200 nm. In some cases, the capsid has a diameter
of about 300 nm. In some cases, the capsid has a diameter of about
400 nm. In some cases, the capsid has a diameter of about 500 nm.
In some cases, the capsid has a diameter of about 600 nm.
[0113] In some embodiments, the capsid has a diameter of from about
1 nm to about 600 nm. In some instances, the capsid has a diameter
of from about 2 nm to about 500 nm, from about 2 nm to about 400
nm, from about 2 nm to about 300 nm, from about 2 nm to about 200
nm, from about 2 nm to about 100 nm, from about 2 nm to about 50
nm, from about 2 nm to about 30 nm, from about 20 nm to about 400
nm, from about 20 nm to about 300 nm, from about 20 nm to about 200
nm, from about 20 nm to about 100 nm, from about 20 nm to about 50
nm, from about 20 nm to about 30 nm, from about 30 nm to about 500
nm, from about 30 nm to about 400 nm, from about 30 nm to about 300
nm, from about 30 nm to about 200 nm, from about 30 nm to about 100
nm, from about 30 nm to about 50 nm, from about 50 nm to about 300
nm, from about 50 nm to about 200 nm, from about 50 nm to about 100
nm, from about 2 nm to about 25 nm, from about 2 nm to about 20 nm,
from about 2 nm to about 10 nm, from about 5 nm to about 25 nm,
from about 5 nm to about 20 nm, from about 5 nm to about 10 nm,
from about 10 nm to about 25 nm, or from about 10 nm to about 20
nm.
[0114] In some embodiments, the capsid has a reduced off-target
effect. In some cases, the off-target effect is less than 10%, 5%,
4%, 3%, 2%, 1%, or 0.5%. In some cases, the off-target effect is no
more than 10%, 5%, 4%, 3%, 2%, 1%, or 0.5%.
[0115] In some cases, the capsid does not have an off-target
effect.
[0116] In certain embodiments, the formation of Arc and/or
endo-Gag-based capsids occurs either ex vivo or in vitro.
[0117] In some instances, the Arc and/or endo-Gag-based capsids is
assembled in vivo.
[0118] In some instances, the Arc and/or endo-Gag-based capsids is
stable at room temperature. In some cases, the Arc and/or
endo-Gag-based capsids is empty. In other cases, the Arc and/or
endo-Gag-based capsids is loaded (for example, loaded with a cargo
and/or a therapeutic agent, e.g., a DNA or an RNA).
[0119] In some instances, the Arc and/or endo-Gag-based capsids is
stable at a temperature from about 2.degree. C. to about 37.degree.
C. In some instances, the Arc and/or endo-Gag-based capsids is
stable at a temperature from about 2.degree. C. to about 8.degree.
C., about 2.degree. C. to about 4.degree. C., about 20.degree. C.
to about 37.degree. C., about 25.degree. C. to about 37.degree. C.,
about 20.degree. C. to about 30.degree. C., about 25.degree. C. to
about 30.degree. C., or about 30.degree. C. to about 37.degree. C.
In some cases, the Arc and/or endo-Gag-based capsid is empty. In
other cases, the Arc and/or endo-Gag-based capsids is loaded (for
example, loaded with a cargo and/or a therapeutic agent, e.g., a
DNA or an RNA).
[0120] In some instances, the Arc and/or endo-Gag-based capsids is
stable for at least about 1 day, 2 days, 4 days, 5 days, 7 days, 14
days, 28 days, 30 days, 60 days, 2 months, 3 months, 4 months, 5
months, 6 months, 12 months, 18 months, 24 months, 3 years, 5
years, or longer. In some case, the Arc and/or endo-Gag-based
capsids has minimum degradation, e.g., less than 10%, 5%, 4%, 3%,
2%, 1%, 0.5% based on the total population of the Arc and/or
endo-Gag-based capsids that is degraded. In some cases, the Arc
and/or endo-Gag-based capsid is empty. In other cases, the Arc
and/or endo-Gag-based capsids is loaded (for example, loaded with a
therapeutic agent, e.g., a DNA or an RNA).
Additional Capsids
[0121] In some embodiments, the capsid comprises the Copia protein.
In some instances, the Copia protein is from Drosophila
melanogaster (UniProtKB-P04146), Ceratitis capitate
(UniProtKB-W8BHY5), or Drosophila simulans (UniProtKB-Q08461).
[0122] In some embodiments, the capsid comprises the protein
ASPRV1. The ASPRV1 protein is a structural protein that
participates in the development and maintenance of the skin
barrier. In some instances, the protein ASPRV1 is from Homo sapiens
(UniProtKB-Q53RT3).
[0123] In some embodiments, the capsid comprises a protein from the
SCAN domain family. SCAN domain is a superfamily of zinc finger
transcription factors. SCAN domain is also known as leucine rich
region (LeR) and functions as protein interaction domain that
mediates self-association or selective association with other
proteins.
[0124] In some embodiments, the capsid comprises a protein from the
Paraneoplastic Ma antigen family. The Paraneoplastic Ma antigen
family comprises about 14 members of neuro- and testis-specific
proteins.
[0125] In some embodiments, the capsid comprises a protein encoded
by a Retrotransposon Gag-like gene.
[0126] In some embodiments, the capsid comprises BOP, LDOC1, MOAP1,
PEG10, PNMA3, PNMA5, PNMA6A, PNMA6B, RTL3, RTL6, RTL8A, RTL8B,
and/or ZNF18.
Cargos
[0127] In some embodiments, a composition of the disclosure (for
example, a capsid) comprises a cargo. In some embodiments, the
cargo is a therapeutic agent. In some embodiments, the cargo is a
nucleic acid molecule, a small molecule, a protein, a peptide, an
antibody or binding fragment thereof, a peptidomimetic, or a
nucleotidomimetic. In some instances, the cargo is a therapeutic
cargo, comprising e.g., one or more drugs. In some instances, the
cargo comprises a diagnostic tool, for profiling, e.g., one or more
markers (such as markers associates with one or more disease
phenotypes). In additional instances, the cargo comprises an
imaging tool.
[0128] In some instances, the cargo is a nucleic acid molecule.
Exemplary nucleic acid molecules include DNA, RNA, or a mixture of
DNA and RNA. In some instances, the nucleic acid molecule is a DNA
polymer. In some cases, the DNA is a single stranded DNA polymer.
In other cases, the DNA is a double stranded DNA polymer. In
additional cases, the DNA is a hybrid of single and double stranded
DNA polymer.
[0129] In some embodiments, the nucleic acid molecule is a RNA
polymer, e.g., a single stranded RNA polymer, a double stranded RNA
polymer, or a hybrid of single and double stranded RNA polymers. In
some instances, the RNA comprises and/or encodes an antisense
oligoribonucleotide, a siRNA, an mRNA, a tRNA, an rRNA, a snRNA, a
shRNA, microRNA, or a non-coding RNA.
[0130] In some embodiments, the nucleic acid molecule comprises a
hybrid of DNA and RNA.
[0131] In some embodiments, the nucleic acid molecule is an
antisense oligonucleotide, optionally comprising DNA, RNA, or a
hybrid of DNA and RNA.
[0132] In some instances, the nucleic acid molecule comprises
and/or encodes an mRNA molecule.
[0133] In some embodiments, the nucleic acid molecule comprises
and/or encodes an RNAi molecule. In some cases, the RNAi molecule
is a microRNA (miRNA) molecule. In other cases, the RNAi molecule
is a siRNA molecule. The miRNA and/or siRNA are optionally
double-stranded or as a hairpin, and further optionally
encapsulated as precursor molecules.
[0134] In some embodiments, the nucleic acid molecule is for use in
a nucleic acid-based therapy. In some instances, the nucleic acid
molecule is for regulating gene expression (e.g., modulating mRNA
translation or degradation), modulating RNA splicing, or RNA
interference. In some cases, the nucleic acid molecule comprises
and/or encodes an antisense oligonucleotide, microRNA molecule,
siRNA molecule, mRNA molecule, for use in regulation of gene
expression, modulating RNA splicing, or RNA interference.
[0135] In some instances, the nucleic acid molecule is for use in
gene editing. Exemplary gene editing systems include, but are not
limited to, CRISPR-Cas systems, zinc finger nuclease (ZFN) systems,
and transcription activator-like effector nuclease (TALEN) systems.
In some cases, the nucleic acid molecule comprises and/or encodes a
component involved in the CRISPR-Cas systems, ZFN systems, or the
TALEN systems.
[0136] In some cases, the nucleic acid molecule is for use in
antigen production for therapeutic and/or prophylactic vaccine
production. For example, the nucleic acid molecule encodes an
antigen that is expressed and elicits a desirable immune response
(e.g., a pro-inflammatory immune response, an anti-inflammatory
immune response, an B cell response, an antibody response, a T cell
response, a CD4+ T cell response, a CD8+ T cell response, a Th1
immune response, a Th2 immune response, a Th17 immune response, a
Treg immune response, or a combination thereof).
[0137] In some cases, the nucleic acid molecule comprises a nucleic
acid enzyme. Nucleic acid enzymes are RNA molecules (e.g.,
ribozymes) or DNA molecules (e.g., deoxyribozymes) that have
catalytic activities. In some instances, the nucleic acid molecule
is a ribozyme. In other instances, the nucleic acid molecule is a
deoxyribozyme. In some cases, the nucleic acid molecule is a
MNAzyme, which functions as a biosensor and/or a molecular switch
(see, e.g., Mokany, et al., "MNAzymes, a versatile new class of
nucleic acid enzymes that can function as biosensors and molecular
switches," JACS 132(2): 1051-1059 (2010)).
[0138] In some instances, exemplary targets of the nucleic acid
molecule include, but are not limited to, UL123 (human
cytomegalovirus), APOB, AR (androgen receptor) gene, KRAS, PCSK9,
CFTR, and SMN (e.g., SMN2).
[0139] In some embodiments, the nucleic acid molecule is at least 5
nucleotides or more in length. In some instances, the nucleic acid
molecule is at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 60,
70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 1000, 1500, 2000,
3000, 4000, 5000, 6000, 7000, 8000, 9000 nucleotides or more in
length. In some instances, the nucleic acid molecule is at least 10
nucleotides or more in length. In some instances, the nucleic acid
molecule is at least 12 nucleotides or more in length. In some
instances, the nucleic acid molecule is at least 15 nucleotides or
more in length. In some instances, the nucleic acid molecule is at
least 18 nucleotides or more in length. In some instances, the
nucleic acid molecule is at least 19 nucleotides or more in length.
In some instances, the nucleic acid molecule is at least 20
nucleotides or more in length. In some instances, the nucleic acid
molecule is at least 21 nucleotides or more in length. In some
instances, the nucleic acid molecule is at least 22 nucleotides or
more in length. In some instances, the nucleic acid molecule is at
least 23 nucleotides or more in length. In some instances, the
nucleic acid molecule is at least 24 nucleotides or more in length.
In some instances, the nucleic acid molecule is at least 25
nucleotides or more in length. In some instances, the nucleic acid
molecule is at least 26 nucleotides or more in length. In some
instances, the nucleic acid molecule is at least 27 nucleotides or
more in length. In some instances, the nucleic acid molecule is at
least 28 nucleotides or more in length. In some instances, the
nucleic acid molecule is at least 29 nucleotides or more in length.
In some instances, the nucleic acid molecule is at least 30
nucleotides or more in length. In some instances, the nucleic acid
molecule is at least 40 nucleotides or more in length. In some
instances, the nucleic acid molecule is at least 50 nucleotides or
more in length. In some instances, the nucleic acid molecule is at
least 100 nucleotides or more in length. In some instances, the
nucleic acid molecule is at least 200 nucleotides or more in
length. In some instances, the nucleic acid molecule is at least
300 nucleotides or more in length. In some instances, the nucleic
acid molecule is at least 500 nucleotides or more in length. In
some instances, the nucleic acid molecule is at least 1000
nucleotides or more in length. In some instances, the nucleic acid
molecule is at least 2000 nucleotides or more in length. In some
instances, the nucleic acid molecule is at least 3000 nucleotides
or more in length. In some instances, the nucleic acid molecule is
at least 4000 nucleotides or more in length. In some instances, the
nucleic acid molecule is at least 5000 nucleotides or more in
length. In some instances, the nucleic acid molecule is at least
8000 nucleotides or more in length.
[0140] In some embodiments, the nucleic acid molecule is at most 12
nucleotides or less in length. In some instances, the nucleic acid
molecule is at most 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200,
250, 300, 400, 500, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000,
8000, 9000 nucleotides or less in length. In some instances, the
nucleic acid molecule is at most 15 nucleotides or less in length.
In some instances, the nucleic acid molecule is at most 18
nucleotides or less in length. In some instances, the nucleic acid
molecule is at most 19 nucleotides or less in length. In some
instances, the nucleic acid molecule is at most 20 nucleotides or
less in length. In some instances, the nucleic acid molecule is at
most 21 nucleotides or less in length. In some instances, the
nucleic acid molecule is at most 22 nucleotides or less in length.
In some instances, the nucleic acid molecule is at most 23
nucleotides or less in length. In some instances, the nucleic acid
molecule is at most 24 nucleotides or less in length. In some
instances, the nucleic acid molecule is at most 25 nucleotides or
less in length. In some instances, the nucleic acid molecule is at
most 26 nucleotides or less in length. In some instances, the
nucleic acid molecule is at most 27 nucleotides or less in length.
In some instances, the nucleic acid molecule is at most 28
nucleotides or less in length. In some instances, the nucleic acid
molecule is at most 29 nucleotides or less in length. In some
instances, the nucleic acid molecule is at most 30 nucleotides or
less in length. In some instances, the nucleic acid molecule is at
most 40 nucleotides or less in length. In some instances, the
nucleic acid molecule is at most 50 nucleotides or less in length.
In some instances, the nucleic acid molecule is at most 100
nucleotides or less in length. In some instances, the nucleic acid
molecule is at most 200 nucleotides or less in length. In some
instances, the nucleic acid molecule is at most 300 nucleotides or
less in length. In some instances, the nucleic acid molecule is at
most 500 nucleotides or less in length. In some instances, the
nucleic acid molecule is at most 1000 nucleotides or less in
length. In some instances, the nucleic acid molecule is at most
2000 nucleotides or less in length. In some instances, the nucleic
acid molecule is at most 3000 nucleotides or less in length. In
some instances, the nucleic acid molecule is at most 4000
nucleotides or less in length. In some instances, the nucleic acid
molecule is at most 5000 nucleotides or less in length. In some
instances, the nucleic acid molecule is at most 8000 nucleotides or
less in length.
[0141] In some embodiments, the nucleic acid molecule is about 5
nucleotides in length. In some instances, the nucleic acid molecule
is about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 60, 70, 80, 90,
100, 150, 200, 250, 300, 400, 500, 1000, 1500, 2000, 3000, 4000,
5000, 6000, 7000, 8000, 9000 nucleotides in length. In some
instances, the nucleic acid molecule is about 10 nucleotides in
length. In some instances, the nucleic acid molecule is about 12
nucleotides in length. In some instances, the nucleic acid molecule
is about 15 nucleotides in length. In some instances, the nucleic
acid molecule is about 18 nucleotides in length. In some instances,
the nucleic acid molecule is about 19 nucleotides in length. In
some instances, the nucleic acid molecule is about 20 nucleotides
in length. In some instances, the nucleic acid molecule is about 21
nucleotides in length. In some instances, the nucleic acid molecule
is about 22 nucleotides in length. In some instances, the nucleic
acid molecule is about 23 nucleotides in length. In some instances,
the nucleic acid molecule is about 24 nucleotides in length. In
some instances, the nucleic acid molecule is about 25 nucleotides
in length. In some instances, the nucleic acid molecule is about 26
nucleotides in length. In some instances, the nucleic acid molecule
is about 27 nucleotides in length. In some instances, the nucleic
acid molecule is about 28 nucleotides in length. In some instances,
the nucleic acid molecule is about 29 nucleotides in length. In
some instances, the nucleic acid molecule is about 30 nucleotides
in length. In some instances, the nucleic acid molecule is about 40
nucleotides in length. In some instances, the nucleic acid molecule
is about 50 nucleotides in length. In some instances, the nucleic
acid molecule is about 100 nucleotides in length. In some
instances, the nucleic acid molecule is about 200 nucleotides in
length. In some instances, the nucleic acid molecule is about 300
nucleotides in length. In some instances, the nucleic acid molecule
is about 500 nucleotides in length. In some instances, the nucleic
acid molecule is about 1000 nucleotides in length. In some
instances, the nucleic acid molecule is about 2000 nucleotides in
length. In some instances, the nucleic acid molecule is about 3000
nucleotides in length. In some instances, the nucleic acid molecule
is about 4000 nucleotides in length. In some instances, the nucleic
acid molecule is about 5000 nucleotides in length. In some
instances, the nucleic acid molecule is about 8000 nucleotides in
length.
[0142] In some embodiments, the nucleic acid molecule is from about
5 to about 10,000 nucleotides in length. In some instances, the
nucleic acid molecule is from about 5 to about 9000 nucleotides in
length, from about 5 to about 8000 nucleotides in length, from
about 5 to about 7000 nucleotides in length, from about 5 to about
6000 nucleotides in length, from about 5 to about 5000 nucleotides
in length, from about 5 to about 4000 nucleotides in length, from
about 5 to about 3000 nucleotides in length, from about 5 to about
2000 nucleotides in length, from about 5 to about 1000 nucleotides
in length, from about 5 to about 500 nucleotides in length, from
about 5 to about 100 nucleotides in length, from about 5 to about
50 nucleotides in length, from about 5 to about 40 nucleotides in
length, from about 5 to about 30 nucleotides in length, from about
5 to about 25 nucleotides in length, from about 5 to about 20
nucleotides in length, from about 10 to about 8000 nucleotides in
length, from about 10 to about 7000 nucleotides in length, from
about 10 to about 6000 nucleotides in length, from about 10 to
about 5000 nucleotides in length, from about 10 to about 4000
nucleotides in length, from about 10 to about 3000 nucleotides in
length, from about 10 to about 2000 nucleotides in length, from
about 10 to about 1000 nucleotides in length, from about 10 to
about 500 nucleotides in length, from about 10 to about 100
nucleotides in length, from about 10 to about 50 nucleotides in
length, from about 10 to about 40 nucleotides in length, from about
10 to about 30 nucleotides in length, from about 10 to about 25
nucleotides in length, from about 10 to about 20 nucleotides in
length, from about 18 to about 8000 nucleotides in length, from
about 18 to about 7000 nucleotides in length, from about 18 to
about 6000 nucleotides in length, from about 18 to about 5000
nucleotides in length, from about 18 to about 4000 nucleotides in
length, from about 18 to about 3000 nucleotides in length, from
about 18 to about 2000 nucleotides in length, from about 18 to
about 1000 nucleotides in length, from about 18 to about 500
nucleotides in length, from about 18 to about 100 nucleotides in
length, from about 18 to about 50 nucleotides in length, from about
18 to about 40 nucleotides in length, from about 18 to about 30
nucleotides in length, from about 18 to about 25 nucleotides in
length, from about 12 to about 50 nucleotides in length, from about
20 to about 40 nucleotides in length, from about 20 to about 30
nucleotides in length, or from about 25 to about 30 nucleotides in
length.
[0143] In some embodiments, the nucleic acid molecule comprises
natural, synthetic, or artificial nucleotide analogues or bases. In
some cases, the nucleic acid molecule comprises combinations of
DNA, RNA and/or nucleotide analogues. In some instances, the
synthetic or artificial nucleotide analogues or bases comprise
modifications at one or more of ribose moiety, phosphate moiety,
nucleoside moiety, or a combination thereof.
[0144] In some embodiments, a nucleotide analogue or artificial
nucleotide base described above comprises a nucleic acid with a
modification at a 2' hydroxyl group of the ribose moiety. In some
instances, the modification includes an H, OR, R, halo, SH, SR,
NH2, NHR, NR2, or CN, wherein R is an alkyl moiety. Exemplary alkyl
moiety includes, but is not limited to, halogens, sulfurs, thiols,
thioethers, thioesters, amines (primary, secondary, or tertiary),
amides, ethers, esters, alcohols and oxygen. In some instances, the
alkyl moiety further comprises a modification. In some instances,
the modification comprises an azo group, a keto group, an aldehyde
group, a carboxyl group, a nitro group, a nitroso, group, a nitrile
group, a heterocycle (e.g., imidazole, hydrazino or hydroxylamino)
group, an isocyanate or cyanate group, or a sulfur containing group
(e.g., sulfoxide, sulfone, sulfide, or disulfide). In some
instances, the alkyl moiety further comprises a hetero
substitution. In some instances, the carbon of the heterocyclic
group is substituted by a nitrogen, oxygen or sulfur. In some
instances, the heterocyclic substitution includes but is not
limited to, morpholino, imidazole, and pyrrolidino.
[0145] In some instances, the modification at the 2' hydroxyl group
is a 2'-O-methyl modification or a 2'-O-methoxyethyl (2'-O-MOE)
modification. In some cases, the 2'-O-methyl modification adds a
methyl group to the 2' hydroxyl group of the ribose moiety whereas
the 2'O-methoxyethyl modification adds a methoxyethyl group to the
2' hydroxyl group of the ribose moiety.
[0146] In some instances, the modification at the 2' hydroxyl group
is a 2'-O-aminopropyl modification in which an extended amine group
comprising a propyl linker binds the amine group to the 2' oxygen.
In some instances, this modification neutralizes the
phosphate-derived overall negative charge of the oligonucleotide
molecule by introducing one positive charge from the amine group
per sugar and thereby improves cellular uptake properties due to
its zwitterionic properties.
[0147] In some instances, the modification at the 2' hydroxyl group
is a locked or bridged ribose modification (e.g., locked nucleic
acid or LNA) in which the oxygen molecule bound at the 2' carbon is
linked to the 4' carbon by a methylene group, thus forming a
2'-C,4'-C-oxy-methylene-linked bicyclic ribonucleotide monomer.
[0148] In some embodiments, additional modifications at the 2'
hydroxyl group include 2'-deoxy, T-deoxy-2'-fluoro,
2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),
2'-O-dimethylaminopropyl (2'-O-DMAP),
T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O-N-methylacetamido (2'-O-NMA).
[0149] In some embodiments, a nucleotide analogue comprises a
modified base such as, but not limited to, 5-propynyluridine,
5-propynylcytidine, 6-methyladenine, 6-methylguanine, N, N,
-dimethyladenine, 2-propyladenine, 2propylguanine, 2-aminoadenine,
1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine
and other nucleotides having a modification at the 5 position,
5-(2-amino) propyl uridine, 5-halocytidine, 5-halouridine,
4-acetylcytidine, 1-methyladenosine, 2-methyladenosine,
3-methylcytidine, 6-methyluridine, 2-methylguanosine,
7-methylguanosine, 2, 2-dimethylguanosine,
5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides
(such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, or
6-azothymidine), 5-methyl-2-thiouridine, other thio bases (such as
2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine,
pseudouridine, queuosine, archaeosine, naphthyl and substituted
naphthyl groups, any O- and N-alkylated purines and pyrimidines
(such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine
5-oxyacetic acid, pyridine-4-one, or pyridine-2-one), phenyl and
modified phenyl groups such as aminophenol or 2,4, 6-trimethoxy
benzene, modified cytosines that act as G-clamp nucleotides,
8-substituted adenines and guanines, 5-substituted uracils and
thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides,
carboxyalkylaminoalkyi nucleotides, and alkylcarbonylalkylated
nucleotides. Modified nucleotides also include those nucleotides
that are modified with respect to the sugar moiety, as well as
nucleotides having sugars or analogs thereof that are not ribosyl.
For example, the sugar moieties, in some cases are or are based on,
mannoses, arabinoses, glucopyranoses, galactopyranoses,
4'-thioribose, and other sugars, heterocycles, or carbocycles. The
term nucleotide also includes universal bases. By way of example,
universal bases include but are not limited to 3-nitropyrrole,
5-nitroindole, or nebularine.
[0150] In some embodiments, a nucleotide analogue further comprises
a morpholino, a peptide nucleic acid (PNA), a methylphosphonate
nucleotide, a thiolphosphonate nucleotide, a 2'-fluoro
N3-P5'-phosphoramidite, or a 1', 5'-anhydrohexitol nucleic acid
(HNA). Morpholino or phosphorodiamidate morpholino oligo (PMO)
comprises synthetic molecules whose structure mimics natural
nucleic acid structure but deviates from the normal sugar and
phosphate structures. In some instances, the five member ribose
ring is substituted with a six member morpholino ring containing
four carbons, one nitrogen, and one oxygen. In some cases, the
ribose monomers are linked by a phosphordiamidate group instead of
a phosphate group. In such cases, the backbone alterations remove
all positive and negative charges making morpholinos neutral
molecules capable of crossing cellular membranes without the aid of
cellular delivery agents such as those used by charged
oligonucleotides.
[0151] In some embodiments, peptide nucleic acid (PNA) does not
contain sugar ring or phosphate linkage and the bases are attached
and appropriately spaced by oligoglycine-like molecules, therefore,
eliminating a backbone charge.
[0152] In some embodiments, one or more modifications optionally
occur at the internucleotide linkage. In some instances, modified
internucleotide linkage includes, but is not limited to,
phosphorothioates; phosphorodithioates; methylphosphonates;
5'-alkylenephosphonates; 5'-methylphosphonate; 3'-alkylene
phosphonates; borontrifluoridates; borano phosphate esters and
selenophosphates of 3'-5'linkage or 2'-5'linkage; phosphotriesters;
thionoalkylphosphotriesters; hydrogen phosphonate linkages; alkyl
phosphonates; alkylphosphonothioates; arylphosphonothioates;
phosphoroselenoates; phosphorodiselenoates; phosphinates;
phosphoramidates; 3'-alkylphosphoramidates;
aminoalkylphosphoramidates; thionophosphoramidates;
phosphoropiperazidates; phosphoroanilothioates;
phosphoroanilidates; ketones; sulfones; sulfonamides; carbonates;
carbamates; methylenehydrazos; methylenedimethylhydrazos;
formacetals; thioformacetals; oximes; methyleneiminos;
methylenemethyliminos; thioamidates; linkages with riboacetyl
groups; aminoethyl glycine; silyl or siloxane linkages; alkyl or
cycloalkyl linkages with or without heteroatoms of, for example, 1
to 10 carbons that are saturated or unsaturated and/or substituted
and/or contain heteroatoms; linkages with morpholino structures,
amides, or polyamides wherein the bases are attached to the aza
nitrogens of the backbone directly or indirectly; and combinations
thereof
[0153] In some embodiments, one or more modifications comprise a
modified phosphate backbone in which the modification generates a
neutral or uncharged backbone. In some instances, the phosphate
backbone is modified by alkylation to generate an uncharged or
neutral phosphate backbone. As used herein, alkylation includes
methylation, ethylation, and propylation. In some cases, an alkyl
group, as used herein in the context of alkylation, refers to a
linear or branched saturated hydrocarbon group containing from 1 to
6 carbon atoms. In some instances, exemplary alkyl groups include,
but are not limited to, methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl,
neopentyl, hexyl, isohexyl, 1, 1-dimethylbutyl, 2,2-dimethylbutyl,
3.3-dimethylbutyl, and 2-ethylbutyl groups. In some cases, a
modified phosphate is a phosphate group as described in U.S. Pat.
No. 9,481,905.
[0154] In some embodiments, additional modified phosphate backbones
comprise methylphosphonate, ethylphosphonate,
methylthiophosphonate, or methoxyphosphonate. In some cases, the
modified phosphate is methylphosphonate. In some cases, the
modified phosphate is ethylphosphonate. In some cases, the modified
phosphate is methylthiophosphonate. In some cases, the modified
phosphate is methoxyphosphonate.
[0155] In some embodiments, one or more modifications further
optionally include modifications of the ribose moiety, phosphate
backbone and the nucleoside, or modifications of the nucleotide
analogues at the 3' or the 5' terminus. For example, the 3'
terminus optionally include a 3' cationic group, or by inverting
the nucleoside at the 3'-terminus with a 3'-3' linkage. In another
alternative, the 3'-terminus is optionally conjugated with an
aminoalkyl group, e.g., a 3' C5-aminoalkyl dT. In an additional
alternative, the 3'-terminus is optionally conjugated with an
abasic site, e.g., with an apurinic or apyrimidinic site. In some
instances, the 5'-terminus is conjugated with an aminoalkyl group,
e.g., a 5'-O-alkylamino substituent. In some cases, the 5'-terminus
is conjugated with an abasic site, e.g., with an apurinic or
apyrimidinic site.
[0156] In some embodiments, exemplary nucleic acid cargos include,
but are not limited to, Fomivirsen, Mipomersen, AZD5312
(AstraZeneca), Nusinersen, and SB010 (Sterna Biologicals).
Small Molecules
[0157] In some embodiments, the cargo is a small molecule. In some
instances, the small molecule is an inhibitor (e.g., a pan
inhibitor or a selective inhibitor). In other instances, the small
molecule is an activator. In additional cases, the small molecule
is an agonist, antagonist, a partial agonist, a mixed
agonist/antagonist, or a competitive antagonist.
[0158] In some embodiments, the small molecule is a drug that falls
under the class of analgesics, antianxiety drugs, antiarrhythmics,
antibacterials, antibiotics, anticoagulants and thrombolytics,
anticonvulsants, antidepressants, antidiarrheals, antiemetics,
antifungals, antihistamines, antihypertensives,
anti-inflammatories, antineoplastics, antipsychotics, antipyretics,
antivirals, barbiturates, beta-blockers, bronchodilators, cold
cures, corticosteroids, cough suppressants, cytotoxics,
decongestants, diuretics, expectorant, hormones, hypoglycemics,
immunosuppressives, laxatives, muscle relaxants, sex hormones,
sleeping drugs, or tranquilizers.
[0159] In some embodiments, the small molecule is an inhibitor,
e.g., an inhibitor of a kinase pathway such as the Tyrosine kinase
pathway or a Serine/Threonine kinase pathway. In some cases, the
small molecule is a dual protein kinase inhibitor. In some cases,
the small molecule is a lipid kinase inhibitor.
[0160] In some cases, the small molecule is a neuraminidase
inhibitor.
[0161] In some cases, the small molecule is a carbonic anhydrase
inhibitor.
[0162] In some embodiments, exemplary targets of the small molecule
include, but are not limited to, vascular endothelial growth factor
receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2
(VEGFR2), vascular endothelial growth factor receptor 3 (VEGFR3),
fibroblast growth factor receptor 1 (FGFR1), fibroblast growth
factor receptor 2 (FGFR2), fibroblast growth factor receptor 3
(FGFR3), fibroblast growth factor receptor 4 (FGFR4),
cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6),
a receptor tyrosine kinase, a phosphoinositide 3-kinase (PI3K)
isoform (e.g., PI3K.delta., also known as p110.delta.), Janus
kinase 1 (JAK1), Janus kinase 3 (JAK3), a receptor from the family
of platelet-derived growth factor receptors (PDFG-R), and carbonic
anhydrase (e.g., carbonic anhydrase I).
[0163] In some embodiments, the small molecule targets a viral
protein, e.g., a viral envelope protein. In some embodiments, the
small molecule decreases viral adsorption to a host cell. In some
embodiments, the small molecule decreases viral entry into a host
cell. In some embodiments, the small molecule decreases viral
replication in a host or a host cell. In some embodiments, the
small molecule decreases viral assembly.
[0164] In some embodiments, exemplary small molecule cargos
include, but are not limited to, lenvatinib, palbociclib,
regorafenib, idelalisib, tofacitinib, nintedanib, zanamivir,
ethoxzolamide, and artemisinin.
Proteins
[0165] In some embodiments, the cargo is a protein. In some
instances, the protein is a full-length protein. In other
instances, the protein is a fragment, e.g., a functional fragment.
In some cases, the protein is a naturally occurring protein. In
additional cases, the protein is a de novo engineered protein. In
further cases, the protein is a fusion protein. In further cases,
the protein is a recombinant protein. Exemplary proteins include,
but are not limited to, Fc fusion proteins, anticoagulants, blood
factors, bone morphogenetic proteins, enzymes, growth factors,
hormones, interferons, interleukins, and thrombolytics.
[0166] In some instances, the protein is for use in an enzyme
replacement therapy.
[0167] In some cases, the protein is for use in antigen production
for therapeutic and/or prophylactic vaccine production. For
example, the protein comprises an antigen that elicits a desirable
immune response (e.g., a pro-inflammatory immune response, an
anti-inflammatory immune response, an B cell response, an antibody
response, a T cell response, a CD4+ T cell response, a CD8+ T cell
response, a Th1 immune response, a Th2 immune response, a Th17
immune response, a Treg immune response, or a combination
thereof).
[0168] In some instances, exemplary protein cargos include, but are
not limited to, romiplostim, liraglutide, a human growth hormone
(rHGH), human insulin (BHI), follicle-stimulating hormone (FSH),
Factor VIII, erythropoietin (EPO), granulocyte colony-stimulating
factor (G-CSF), alpha-galactosidase A, alpha-L-iduronidase,
N-acetylgalactosamine-4-sulfatase, dornase alfa, tissue plasminogen
activator (TPA), glucocerebrosidase, interferon-beta-1a,
insulin-like growth factor 1 (IGF-1), or rasburicase.
Peptides
[0169] In some embodiments, the cargo is a peptide. In some
instances, the peptide is a naturally occurring peptide. In other
instances, the peptide is an artificial engineered peptide or a
recombinant peptide. In some cases, the peptide targets a G-protein
coupled receptor, an ion channel, a microbe, an anti-microbial
target, a catalytic or other Ig-family of receptors, an
intracellular target, a membrane-anchored target, or an
extracellular target.
[0170] In some cases, the peptide comprises at least 2 amino acids.
In some cases, the peptide comprises at least 3, 4, 5, 6, 7, 8, 9,
10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 amino
acids. In some cases, the peptide comprises at least 10 amino
acids. In some cases, the peptide comprises at least 15 amino
acids. In some cases, the peptide comprises at least 20 amino
acids. In some cases, the peptide comprises at least 30 amino
acids. In some cases, the peptide comprises at least 40 amino
acids. In some cases, the peptide comprises at least 50 amino
acids. In some cases, the peptide comprises at least 60 amino
acids. In some cases, the peptide comprises at least 70 amino
acids. In some cases, the peptide comprises at least 80 amino
acids. In some cases, the peptide comprises at least 90 amino
acids. In some cases, the peptide comprises at least 100 amino
acids.
[0171] In some cases, the peptide comprises at most 3 amino acids.
In some cases, the peptide comprises at most 4, 5, 6, 7, 8, 9, 10,
15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 amino acids. In
some cases, the peptide comprises at most 10 amino acids. In some
cases, the peptide comprises at most 15 amino acids. In some cases,
the peptide comprises at most 20 amino acids. In some cases, the
peptide comprises at most 30 amino acids. In some cases, the
peptide comprises at most 40 amino acids. In some cases, the
peptide comprises at most 50 amino acids. In some cases, the
peptide comprises at most 60 amino acids. In some cases, the
peptide comprises at most 70 amino acids. In some cases, the
peptide comprises at most 80 amino acids. In some cases, the
peptide comprises at most 90 amino acids. In some cases, the
peptide comprises at most 100 amino acids.
[0172] In some cases, the peptide comprises from about 1 to about
10 kDa. In some cases, the peptide comprises from about 1 to about
9 kDa, about 1 to about 6 kDa, about 1 to about 5 kDa, about 1 to
about 4 kDa, about 1 to about 3 kDa, about 2 to about 8 kDa, about
2 to about 6 kDa, about 2 to about 4 kDa, about 1.2 to about 2.8
kDa, about 1.5 to about 2.5 kDa, or about 1.5 to about 2 kDa.
[0173] In some embodiments, the peptide is a cyclic peptide. In
some instances, the cyclic peptide is a macrocyclic peptide. In
other instances, the cyclic peptide is a constrained peptide. The
cyclic peptides are assembled with varied linkages, such as for
example, head-to-tail, head-to-side-chain, side-chain to tail, and
side-chain to side-chain linkages. In some instances, a cyclic
peptide (e.g., a macrocyclic or a constrained peptide) has a
molecular weight from about 500 Dalton to about 2000 Dalton. In
other instances, a cyclic peptide (e.g., a macrocyclic or a
constrained peptide) ranges from about 10 amino acids to about 100
amino acids, from about 10 amino acids to about 70 amino acids, or
from about 10 amino acids to about 50 amino acids.
[0174] In some cases, the peptide is for use in antigen production
for therapeutic and/or prophylactic vaccine production. For
example, the peptide comprises an antigen that elicits a desirable
immune response (e.g., a pro-inflammatory immune response, an
anti-inflammatory immune response, an B cell response, an antibody
response, a T cell response, a CD4+ T cell response, a CD8+ T cell
response, a Th1 immune response, a Th2 immune response, a Th17
immune response, a Treg immune response, or a combination
thereof).
[0175] In some embodiments, the peptide comprises natural amino
acids, unnatural amino acids, or a combination thereof. In some
instances, an amino acid residue refers to a molecule containing
both an amino group and a carboxyl group. Suitable amino acids
include, without limitation, both the D- and L-isomers of the
naturally-occurring amino acids, as well as non-naturally occurring
amino acids prepared by organic synthesis or other metabolic
routes. The term amino acid, as used herein, includes, without
limitation, .alpha.-amino acids, natural amino acids, non-natural
amino acids, and amino acid analogs.
[0176] In some instances, .alpha.-amino acid refers to a molecule
containing both an amino group and a carboxyl group bound to a
carbon which is designated the .alpha.-carbon.
[0177] In some instances, .beta.-amino acid refers to a molecule
containing both an amino group and a carboxyl group in a .beta.
configuration.
[0178] In some embodiments, an amino acid analog is a racemic
mixture. In some instances, the D isomer of the amino acid analog
is used. In some cases, the L isomer of the amino acid analog is
used. In some instances, the amino acid analog comprises chiral
centers that are in the R or S configuration.
[0179] In some embodiments, exemplary peptide cargos include, but
are not limited to, peginesatide, insulin, adrenocorticotropic
hormone (ACTH), calcitonin, oxytocin, vasopressin, octreolide, and
leuprorelin.
[0180] In some embodiments, exemplary peptide cargos include, but
are not limited to, Telavancin, Dalbavancin, Oritavancin,
Anidulafungin, Lanreotide, Pasireotide, Romidepsin, Linaclotide,
and Peginesatide.
Antibodies
[0181] In some embodiments, the cargo is an antibody or a binding
fragment thereof. In some instances, the antibody or binding
fragment thereof comprises a humanized antibody or binding fragment
thereof, murine antibody or binding fragment thereof, chimeric
antibody or binding fragment thereof, monoclonal antibody or
binding fragment thereof, bispecific antibody or biding fragment
thereof, monovalent Fab', divalent Fab.sub.2, F(ab)'.sub.3
fragments, single-chain variable fragment (scFv), bis-scFv,
(scFv).sub.2, diabody, minibody, nanobody, triabody, tetrabody,
disulfide stabilized Fv protein (dsFv), single-domain antibody
(sdAb), Ig NAR, camelid antibody or binding fragment thereof, or a
chemically modified derivative thereof.
[0182] In some instances, the antibody or binding fragment thereof
recognizes a cell surface protein. In some instances, the cell
surface protein is an antigen expressed by a cancerous cell. In
some instances, the cell surface protein is a neoepitope. In some
instances, the cell surface protein comprises one or more mutations
compared to a wild-type protein. Exemplary cancer antigens include,
but are not limited to, alpha fetoprotein, ASLG659, B7-H3, BAFF-R,
Brevican, CA125 (MUC16), CA15-3, CA19-9, carcinoembryonic antigen
(CEA), CA242, CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,
teratocarcinoma-derived growth factor), CTLA-4, CXCR5, E16 (LAT1,
SLC7A5), FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing
phosphatase anchor protein 1a), SPAP1B, SPAP1C), epidermal growth
factor, ETBR, Fc receptor-like protein 1 (FCRH1), GEDA, HLA-DOB
(Beta subunit of MHC class II molecule (Ia antigen), human
chorionic gonadotropin, ICOS, IL-2 receptor, IL20R.alpha.,
Immunoglobulin superfamily receptor translocation associated 2
(IRTA2), L6, Lewis Y, Lewis X, MAGE-1, MAGE-2, MAGE-3, MAGE 4,
MART1, mesothelin, MDP, MPF (SMR, MSLN), MCP1 (CCL2), macrophage
inhibitory factor (MIF), MPG, MSG783, mucin, MUC1-KLH, Napi3b
(SLC34A2), nectin-4, Neu oncogene product, NCA, placental alkaline
phosphatase, prostate specific membrane antigen (PMSA), prostatic
acid phosphatase, PSCA hlg, anti-transferrin receptor, p97,
Purinergic receptor P2X ligand-gated ion channel 5 (P2X5), LY64
(Lymphocyte antigen 64 (RP105), gp100, P21, six transmembrane
epithelial antigen of prostate (STEAP1), STEAP2, Sema 5b,
tumor-associated glycoprotein 72 (TAG-72), TrpM4 (BR22450,
FLJ20041, TRPM4, TRPM4B, transient receptor potential cation
channel, subfamily M, member 4) and the like.
[0183] In some instances, the cell surface protein comprises
clusters of differentiation (CD) cell surface markers. Exemplary CD
cell surface markers include, but are not limited to, CD1, CD2,
CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c,
CD11d, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19,
CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30,
CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41,
CD42, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48,
CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53,
CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L
(L-selectin), CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d,
CD66e, CD71, CD79 (e.g., CD79a, CD79b), CD90, CD95 (Fas), CD103,
CD104, CD125 (IL5RA), CD134 (0X40), CD137 (4-1BB), CD152 (CTLA-4),
CD221, CD274, CD279 (PD-1), CD319 (SLAMF7), CD326 (EpCAM), and the
like.
[0184] In some embodiments, exemplary antibodies or binding
fragments thereof include, but are not limited to, zalutumumab
(HuMax-EFGr, Genmab), abagovomab (Menarini), abituzumab (Merck),
adecatumumab (MT201), alacizumab pegol, alemtuzumab (Campath.RTM.,
MabCampath, or Campath-1H; Leukosite), AlloMune (BioTransplant),
amatuximab (Morphotek, Inc.), anti-VEGF (Genetech), anatumomab
mafenatox, apolizumab (hulD10), ascrinvacumab (Pfizer Inc.),
atezolizumab (MPDL3280A; Genentech/Roche), B43.13 (OvaRex, AltaRex
Corporation), basiliximab (Simulect.RTM., Novartis), belimumab
(Benlysta.RTM., GlaxoSmithKline), bevacizumab (Avastin.RTM.,
Genentech), blinatumomab (Blincyto, AMG103; Amgen), BEC2 (ImGlone
Systems Inc.), carlumab (Janssen Biotech), catumaxomab (Removab,
Trion Pharma), CEAcide (Immunomedics), Cetuximab (Erbitux.RTM.,
ImClone), citatuzumab bogatox (VB6-845), cixutumumab (IMC-A12,
ImClone Systems Inc.), conatumumab (AMG 655, Amgen), dacetuzumab
(SGN-40, huS2C6; Seattle Genetics, Inc.), daratumumab
(Darzalex.RTM., Janssen Biotech), detumomab, drozitumab
(Genentech), durvalumab (MedImmune), dusigitumab (MedImmune),
edrecolomab (MAb17-1A, Panorex, Glaxo Wellcome), elotuzumab
(Empliciti.TM., Bristol-Myers Squibb), emibetuzumab (Eli Lilly),
enavatuzumab (Facet Biotech Corp.), enfortumab vedotin (Seattle
Genetics, Inc.), enoblituzumab (MGA271, MacroGenics, Inc.),
ensituxumab (Neogenix Oncology, Inc.), epratuzumab (LymphoCide,
Immunomedics, Inc.), ertumaxomab (Rexomun.RTM., Trion Pharma),
etaracizumab (Abegrin, MedImmune), farletuzumab (MORAb-003,
Morphotek, Inc), FBTA05 (Lymphomun, Trion Pharma), ficlatuzumab
(AVEO Pharmaceuticals), figitumumab (CP-751871, Pfizer),
flanvotumab (ImClone Systems), fresolimumab (GC1008,
Aanofi-Aventis), futuximab, glaximab, ganitumab (Amgen),
girentuximab (Rencarex.RTM., Wilex AG), IMAB362 (Claudiximab,
Ganymed Pharmaceuticals AG), imalumab (Baxalta), IMC-1C11 (ImClone
Systems), IMC-C225 (Imclone Systems Inc.), imgatuzumab
(Genentech/Roche), intetumumab (Centocor, Inc.), ipilimumab
(Yervoy.RTM., Bristol-Myers Squibb), iratumumab (Medarex, Inc.),
isatuximab (SAR650984, Sanofi-Aventis), labetuzumab (CEA-CIDE,
Immunomedics), lexatumumab (ETR2-ST01, Cambridge Antibody
Technology), lintuzumab (SGN-33, Seattle Genetics), lucatumumab
(Novartis), lumiliximab, mapatumumab (HGS-ETR1, Human Genome
Sciences), matuzumab (EMD 72000, Merck), milatuzumab (hLL1,
Immunomedics, Inc.), mitumomab (BEC-2, ImClone Systems), narnatumab
(ImClone Systems), necitumumab (Portrazza.TM., Eli Lilly),
nesvacumab (Regeneron Pharmaceuticals), nimotuzumab (h-R3, BIOMAb
EGFR, TheraClM, Theraloc, or CIMAher; Biotech Pharmaceutical Co.),
nivolumab (Opdivo.RTM., Bristol-Myers Squibb), obinutuzumab (Gazyva
or Gazyvaro; Hoffmann-La Roche), ocaratuzumab (AME-133v, LY2469298;
Mentrik Biotech, LLC), ofatumumab (Arzerra.RTM., Genmab),
onartuzumab (Genentech), Ontuxizumab (Morphotek, Inc.), oregovomab
(OvaRex.RTM., AltaRex Corp.), otlertuzumab (Emergent BioSolutions),
panitumumab (ABX-EGF, Amgen), pankomab (Glycotope GMBH),
parsatuzumab (Genentech), patritumab, pembrolizumab (Keytruda.RTM.,
Merck), pemtumomab (Theragyn, Antisoma), pertuzumab (Perj eta,
Genentech), pidilizumab (CT-011, Medivation), polatuzumab vedotin
(Genentech/Roche), pritumumab, racotumomab (Vaxira.RTM., Recombio),
ramucirumab (Cyramza.RTM., ImClone Systems Inc.), rituximab
(Rituxan.RTM., Genentech), robatumumab (Schering-Plough),
Seribantumab (Sanofi/Merrimack Pharmaceuticals, Inc.),
sibrotuzumab, siltuximab (Sylvant.TM., Janssen Biotech), Smart MI95
(Protein Design Labs, Inc.), Smart ID10 (Protein Design Labs,
Inc.), tabalumab (LY2127399, Eli Lilly), taplitumomab paptox,
tenatumomab, teprotumumab (Roche), tetulomab, TGN1412
(CD28-SuperMAB or TAB08), tigatuzumab (CD-1008, Daiichi Sankyo),
tositumomab, trastuzumab (Herceptin.RTM.), tremelimumab
(CP-672,206; Pfizer), tucotuzumab celmoleukin (EMD
Pharmaceuticals), ublituximab, urelumab (BMS-663513, Bristol-Myers
Squibb), volociximab (M200, Biogen Idec), and zatuximab.
[0185] In some instances, the antibody or binding fragments thereof
is an antibody-drug conjugate (ADC). In some cases, the payload of
the ADC comprises, for example, but is not limited to, an
auristatin derivative, maytansine, a maytansinoid, a taxane, a
calicheamicin, cemadotin, a duocarmycin, a pyrrolobenzodiazepine
(PDB), or a tubulysin. In some instances, the payload comprises
monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).
In some instances, the payload comprises DM2 (mertansine) or DM4.
In some instances, the payload comprises a pyrrolobenzodiazepine
dimer.
Additional Cargos
[0186] In some embodiments, the cargo is a peptidomimetic. A
peptidomimetic is a small protein-like polymer designed to mimic a
peptide. In some instances, the peptidomimetic comprises
D-peptides. In other instances, the peptidomimetic comprises
L-peptides. Exemplary peptidomimetics include peptoids and
.beta.-peptides.
[0187] In some embodiments, the cargo is a nucleotidomimetic.
Vectors and Expression Systems
[0188] In certain embodiments, the Arc polypeptides, endo-Gag
polypeptides, engineered Arc and engineered endo-Gag polypeptides
described supra are encoded by plasmid vectors. In some
embodiments, vectors include any suitable vectors derived from
either a eukaryotic or prokaryotic sources. In some cases, vectors
are obtained from bacteria (e.g. E. coli), insects, yeast (e.g.
Pichia pastoris), algae, or mammalian sources.
[0189] Exemplary bacterial vectors include pACYC177, pASK75, pBAD
vector series, pBADM vector series, pET vector series, pETM vector
series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE
vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series,
pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG
Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.
[0190] Exemplary insect vectors include pFastBac1, pFastBac DUAL,
pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac
M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393
M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or
pPolh-MAT 2, or MAT vectors such as pPolh-MAT1, or pPolh-MAT2.
[0191] In some cases, yeast vectors include Gateway.RTM. pDEST.TM.
14 vector, Gateway.RTM. pDEST.TM. 15 vector, Gateway.RTM. pDEST.TM.
17 vector, Gateway.RTM. pDEST.TM. 24 vector, Gateway.RTM.
pYES-DEST52 vector, pBAD-DEST49 Gateway.RTM. destination vector,
pAO815 Pichia vector, pFLD1 Pichi pastoris vector, pGAPZA, B, &
C Pichia pastoris vector, pPIC3.5K Pichia vector, pPIC6 A, B, &
C Pichia vector, pPIC9K Pichia vector, pTEF1/Zeo, pYES2 yeast
vector, pYES2/CT yeast vector, pYES2/NT A, B, & C yeast vector,
or pYES3/CT yeast vector.
[0192] Exemplary algae vectors include pChlamy-4 vector or MCS
vector.
[0193] Examples of mammalian vectors include transient expression
vectors or stable expression vectors. Mammalian transient
expression vectors include p3xFLAG-CMV 8, pFLAG-Myc-CMV 19,
pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1,
pFLAG-CMV 5a,b,c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV
24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4.
Mammalian stable expression vector include pFLAG-CMV 3, p3xFLAG-CMV
9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV
4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22,
p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.
[0194] In some instances, a cell-free system is a mixture of
cytoplasmic and/or nuclear components from a cell and is used for
in vitro nucleic acid synthesis. In some cases, a cell-free system
utilizes either prokaryotic cell components or eukaryotic cell
components. Sometimes, a nucleic acid synthesis is obtained in a
cell-free system based on for example Drosophila cell, Xenopus egg,
or HeLa cells (ATCC.RTM. CCL-2.TM.). Exemplary cell-free systems
include, but are not limited to, E. coli S30 Extract system, E.
coli T7 S30 system, or PURExpress.RTM..
Host Cells
[0195] In some embodiments, a host cell includes any suitable cell
such as a naturally derived cell or a genetically modified cell. In
some instances, a host cell is a production host cell. In some
instances, a host cell is a eukaryotic cell. In other instances, a
host cell is a prokaryotic cell. In some cases, a eukaryotic cell
includes fungi (e.g., a yeast cell), an animal cell, or a plant
cell. In some cases, a prokaryotic cell is a bacterial cell.
Examples of bacterial cell include gram-positive bacteria or
gram-negative bacteria. In some embodiments the gram-negative
bacteria is anaerobic, rod-shaped, or both.
[0196] In some instances, gram-positive bacteria include
Actinobacteria, Firmicutes or Tenericutes. In some cases,
gram-negative bacteria include Aquificae, Deinococcus-Thermus,
Fibrobacteres-Chlorobi/Bacteroidetes (FCB group), Fusobacteria,
Gemmatimonadetes, Nitrospirae,
Planctomycetes-Verrucomicrobia/Chlamydiae (PVC group),
Proteobacteria, Spirochaetes or Synergistetes. In some embodiments,
bacteria is Acidobacteria, Chloroflexi, Chrysiogenetes,
Cyanobacteria, Deferribacteres, Dictyoglomi, Thermodesulfobacteria
or Thermotogae. In some embodiments, a bacterial cell is
Escherichia coli, Clostridium botulinum, or Coli bacilli.
[0197] Exemplary prokaryotic host cells include, but are not
limited to, BL21, Mach1.TM., DH10B.TM., TOP10, DH5.alpha.,
DH10Bac.TM., OmniMax.TM., MegaX.TM., DH12S.TM., INV110, TOP10F',
INV.alpha.F, TOP10/P3, ccdB Survival, PIR1, PIR2, Stbl2.TM.,
Stbl3.TM., or Stbl4.TM..
[0198] In some instances, animal cells include a cell from a
vertebrate or from an invertebrate. In some cases, an animal cell
includes a cell from a marine invertebrate, fish, insects,
amphibian, reptile, mammal, or human. In some cases, a fungus cell
includes a yeast cell, such as brewer's yeast, baker's yeast, or
wine yeast.
[0199] Fungi include ascomycetes such as yeast, mold, filamentous
fungi, basidiomycetes, or zygomycetes. In some instances, yeast
includes Ascomycota or Basidiomycota. In some cases, Ascomycota
includes Saccharomycotina (true yeasts, e.g. Saccharomyces
cerevisiae (baker's yeast)) or Taphrinomycotina (e.g.
Schizosaccharomycetes (fission yeasts)). In some cases,
Basidiomycota includes Agaricomycotina (e.g. Tremellomycetes) or
Pucciniomycotina (e.g. Microbotryomycetes).
[0200] Exemplary yeast or filamentous fungi include, for example,
the genus: Saccharomyces, Schizosaccharomyces, Candida, Pichia,
Hansenula, Kluyveromyces, Zygosaccharomyces, Yarrowia,
Trichosporon, Rhodosporidi, Aspergillus, Fusarium, or Trichoderma.
Exemplary yeast or filamentous fungi include, for example, the
species: Saccharomyces cerevisiae, Schizosaccharomyces pombe,
Candida utilis, Candida boidini, Candida albicans, Candida
tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei,
Candida parapsilosis, Candida guilliermondii, Candida viswanathii,
Candida lusitaniae, Rhodotorula mucilaginosa, Pichia metanolica,
Pichia angusta, Pichia pastoris, Pichia anomala, Hansenula
polymorpha, Kluyveromyces lactis, Zygosaccharomyces rouxii,
Yarrowia hpolytica, Trichosporon pullulans, Rhodosporidium
toru-Aspergillus niger, Aspergillus nidulans, Aspergillus awamori,
Aspergillus oryzae, Trichoderma reesei, Yarrowia hpolytica,
Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces
pombe, Torulaspora delbrueckii, Zygosaccharomyces bailii,
Cryptococcus neoformans, Cryptococcus gattii, or Saccharomyces
boulardii.
[0201] Exemplary yeast host cells include, but are not limited to,
Pichia pastoris yeast strains such as GS115, KM71H, SMD1168,
SMD1168H, and X-33; and Saccharomyces cerevisiae yeast strain such
as INVScl.
[0202] In some instances, additional animal cells include cells
obtained from a mollusk, arthropod, annelid or sponge. In some
cases, an additional animal cell is a mammalian cell, e.g., from a
human, primate, ape, equine, bovine, porcine, canine, feline or
rodent. In some cases, a rodent includes mouse, rat, hamster,
gerbil, hamster, chinchilla, fancy rat, or guinea pig.
[0203] Exemplary mammalian host cells include, but are not limited
to, 293A cell line, 293FT cell line, 293F cells, 293 H cells, CHO
DG44 cells, CHO-S cells, CHO-Kl cells, Expi293F.TM. cells,
Flp-In.TM. T-REx.TM. 293 cell line, Flp-In.TM.-293 cell line,
Flp-In.TM.-3T3 cell line, Flp-In.TM.-BHK cell line, Flp-In.TM.-CHO
cell line, Flp-In.TM.-CV-1 cell line, Flp-In.TM.-Jurkat cell line,
FreeStyle.TM. 293-F cells, FreeStyle.TM. CHO-S cells, GripTite.TM.
293 MSR cell line, GS-CHO cell line, HepaRG.TM. cells, T-REx.TM.
Jurkat cell line, Per.C6 cells, T-REx.TM.-293 cell line,
T-REx.TM.-CHO cell line, and T-REx.TM.-HeLa cell line.
[0204] In some instances, a mammalian host cell is a primary cell.
In some instances, a mammalian host cell is a stable cell line, or
a cell line that has incorporated a genetic material of interest
into its own genome and has the capability to express the product
of the genetic material after many generations of cell division. In
some cases, a mammalian host cell is a transient cell line, or a
cell line that has not incorporated a genetic material of interest
into its own genome and does not have the capability to express the
product of the genetic material after many generations of cell
division.
[0205] Exemplary insect host cell include, but are not limited to,
Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five.TM. cells,
and expresSF+.RTM. cells.
[0206] In some instances, plant cells include a cell from algae.
Exemplary insect cell lines include, but are not limited to,
strains from Chlamydomonas reinhardtii 137c, or Synechococcus
elongatus PPC 7942.
Methods of Use
[0207] Disclosed herein, in certain embodiments, are methods of
preparing a capsid which encapsulates a cargo. In some embodiments,
the method comprises incubating a plurality of Arc or endo-Gag
polypeptides, engineered Arc or endo-Gag polypeptides, and/or
recombinant Arc or endo-Gag polypeptides with a cargo in a solution
for a time sufficient to generate a loaded Arc-based capsid or
endo-Gag-based capsid.
[0208] In some instances, the method comprises mixing a solution
comprising a plurality of engineered and/or recombinant Arc
polypeptides with a plurality of non-Arc capsid forming subunits
prior to incubating with the cargo. In some cases, the plurality of
non-Arc capsid forming subunits are mixed with the plurality of
engineered and/or recombinant Arc polypeptides at a ratio of 1:1,
2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In other cases,
the plurality of non-Arc capsid forming subunits are mixed with the
plurality of engineered and/or recombinant Arc polypeptides at a
ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.
[0209] In some cases, the time sufficient to generate a loaded
Arc-based capsid or endo-Gag-based capsid is at least about 5
minutes, at least about 10 minutes, at least about 20 minutes, at
least about 30 minutes, at least about 1 hour, at least about 2
hours, at least about 4 hours, at least about 6 hours, at least
about 10 hours, at least about 12 hours, at least about 24 hours,
or more.
[0210] In some cases, the Arc-based capsid or endo-Gag-based capsid
is prepared at a temperature from about 2.degree. C. to about
37.degree. C. In some instances, the Arc-based capsid or
endo-Gag-based capsid is prepared at a temperature from about
2.degree. C. to about 8.degree. C., about 2.degree. C. to about
4.degree. C., about 20.degree. C. to about 37.degree. C., about
25.degree. C. to about 37.degree. C., about 20.degree. C. to about
30.degree. C., about 25.degree. C. to about 30.degree. C., or about
30.degree. C. to about 37.degree. C.
[0211] In some cases, the Arc-based capsid or endo-Gag-based capsid
is prepared at room temperature.
[0212] In some instances, the Arc-based capsid or endo-Gag-based
capsid is further formulated for systemic administration.
[0213] In some instances, the Arc-based capsid or endo-Gag-based
capsid is further formulated for local administration.
[0214] In some instances, the Arc-based capsid or endo-Gag-based
capsid is further formulated for parenteral (e.g., intra-arterial,
intra-articular, intradermal, intralesional, intramuscular,
intraocular, intraosseous infusion, intraperitoneal, intrathecal,
intravenous, intravitreal, or subcutaneous) administration.
[0215] In some instances, the Arc-based capsid or endo-Gag-based
capsid is further formulated for topical administration.
[0216] In some instances, the Arc-based capsid or endo-Gag-based
capsid is further formulated for oral administration.
[0217] In some instances, the Arc-based capsid or endo-Gag-based
capsid is further formulated for sublingual administration.
[0218] In some instances, the Arc-based capsid or endo-Gag-based
capsid is further formulated for aerosol administration.
[0219] In certain embodiments, also described herein is a use of an
Arc-based capsid or endo-Gag-based capsid for delivery of a cargo
to a site of interest. In some instances, the method comprises
contacting a cell at the site of interest with an Arc-based capsid
or endo-Gag-based capsid for a time sufficient to facilitate
cellular uptake of the capsid.
[0220] In some cases, the cell is a muscle cell, a skin cell, a
blood cell, or an immune cell (e.g., a T cell or a B cell).
[0221] In some instances, the cell is a tumor cell, e.g., a solid
tumor cell or a cell from a hematologic malignancy. In some cases,
the solid tumor cell is a cell from a bladder cancer, breast
cancer, brain cancer, colorectal cancer, kidney cancer, liver
cancer, lung cancer, pancreatic cancer, prostate cancer, skin
cancer, stomach cancer, or thyroid cancer. In some cases, the cell
from a hematologic malignancy is from a B-cell malignancy or a
T-cell malignancy. In some cases, the cell is from a leukeuma, a
lymphoma, a myeloma, chronic lymphocytic leukemia (CLL), small
lymphocytic lymphoma (SLL), diffuse large B cell lymphoma (DLBCL),
follicular lymphoma, mantle cell lymphoma, Burkitt lymphoma,
cutaneous T-cell lymphoma, peripheral T cell lymphoma, multiple
myeloma, plasmacytoma, acute lymphoblastic leukemia (ALL), acute
myeloid leukemia (AML), or chronic myeloid leukemia (CML).
[0222] In some embodiments, the cell is a somatic cell. In some
instances, the cell is a blood cell, a skin cell, a connective
tissue cell, a bone cell, a muscle cell, or a cell from an
organ.
[0223] In some embodiments, the cell is an epithelial cell, a
connective tissue cell, a muscular cell, or a neuron.
[0224] In some instances, the cell is an endodermal cell, a
mesodermal cell, or an ectodermal. In some instances, the endoderm
comprises cells of the respiratory system, the intestine, the
liver, the gallbladder, the pancreas, the islets of Langerhans, the
thyroid, or the hindgut. In some cases, the mesoderm comprises
osteochondroprogenitor cells, muscle cells, cells from the
digestive system, renal stem cells, cells from the reproductive
system, cells from the circulatory system (such as endothelial
cells). Exemplary cells from the ectoderm comprise epithelial
cells, cells of the anterior pituitary, cells of the peripheral
nervous system, cells of the neuroendocrine system, cells of the
eyes, cells of the central nervous system, cells of the ependymal,
or cells of the pineal gland. In some cases, cells derived from the
central and peripheral nervous system comprise neurons, Schwann
cells, satellite glial cells, oligodendrocytes, or astrocytes. In
some cases, neurons further comprise interneurons, pyramidal
neurons, gabaergic neurons, dopaminergic neurons, serotoninergic
neurons, glutamatergic neurons, motor neurons from the spinal cord,
or inhibitory spinal neurons.
[0225] In some embodiments, the cell is a stem cell or a progenitor
cell. In some cases, the cell is a mesenchymal stem or progenitor
cell. In other cases, the cell is a hematopoietic stem or
progenitor cell.
[0226] In some cases, a target protein is overexpressed or is
depleted in the cell. In some cases, the target protein is
overexpressed in the cell. In additional cases, the target protein
is depleted in the cell.
[0227] In some cases, a target gene in the cell has one or more
mutations.
[0228] In some cases, the cell comprises an impaired splicing
mechanism.
[0229] In some instances, the Arc-based capsid is administered
systemically to a subject in need thereof.
[0230] In other instances, the Arc-based capsid or endo-Gag-based
capsid is administered locally to a subject in need thereof.
[0231] In some embodiments, the Arc-based capsid or endo-Gag-based
capsid is administered parenterally, orally, topically, via
sublingual, or by aerosol to a subject in need thereof. In some
cases, the Arc-based capsid or endo-Gag-based capsid is
administered parenterally to a subject in need thereof. In other
cases, the Arc-based capsid or endo-Gag-based capsid is
administered orally to a subject in need thereof. In additional
cases, the Arc-based capsid or endo-Gag-based capsid is
administered topically, via sublingual, or by aerosol to a subject
in need thereof.
[0232] In some embodiments, a delivery component is combined with
an Arc-based capsid or endo-Gag-based capsid for a targeted
delivery to a site of interest. In some instances, the delivery
component comprises a carrier, e.g., an extracellular vesicle such
as a micelle, a liposome, or a microvesicle; or a viral
envelope.
[0233] In some instances, the delivery component serves as a
primary delivery vehicle for an Arc-based capsid or endo-Gag-based
capsid which does not comprise its own delivery component (e.g., in
which the second polypeptide is not present). In such cases, the
delivery component directs the Arc-based capsid or endo-Gag-based
capsid to a target site of interest and optionally facilitates
intracellular uptake.
[0234] In other instances, the delivery component enhances target
specificity and/or sensitivity of an Arc-based capsid's second
polypeptide. In such cases, the delivery component enhances the
specificity and/or affinity of the Arc-based capsid or
endo-Gag-based capsid to the target site. In additional cases, the
delivery components enhances the specificity and/or affinity by
about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,
9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 200-fold,
500-fold, or more. In further cases, the delivery components
enhances the specificity and/or affinity by about 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or more. Further
still, the delivery component optionally minimizes off-target
effect by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,
8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold,
200-fold, 500-fold, or more. Further still, the delivery component
optionally minimizes off-target effect by about 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or more.
[0235] In additional instances, the delivery component serves as a
first vehicle that transports an Arc-based capsid to a general
target region (e.g., a tumor microenvironment) and the Arc-based or
endo-Gag-based capsid's second polypeptide serves as a second
delivery molecule that drives the Arc-based capsid or
endo-Gag-based capsid to the specific target site and optionally
facilitates intracellular uptake. In such cases, the delivery
component minimizes off-target effect by about 2-fold, 3-fold,
4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,
30-fold, 50-fold, 100-fold, 200-fold, 500-fold, or more. In such
cases, the delivery component minimizes off-target effect by about
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or
more.
[0236] In further instances, the delivery component serves as a
first vehicle that transports an Arc-based capsid to a target site
of interest and the Arc-based or endo-Gag-based capsid's second
polypeptide serves as a second delivery molecule that facilitates
intracellular uptake.
[0237] In some embodiments, the delivery component comprises an
extracellular vesicle. In some instances, the extracellular vesicle
comprises a microvesicle, a liposome, or a micelle. In some
instances, the extracellular vesicle has a diameter of from about
10 nm to about 2000 nm, from about 10 nm to about 1000 nm, from
about 10 nm to about 800 nm, from about 20 nm to about 600 nm, from
about 30 nm to about 500 nm, from about 50 nm to about 200 nm, or
from about 80 nm to about 100 nm.
[0238] In some embodiments, the delivery component comprises a
microvesicle. Also known as circulating microvesicles or
microparticles, microvesicles are membrane-bound vesicles that
comprise phospholipids. In some instances, the microvesicle has a
diameter of from about 50 nm to about 1000 nm, from about 100 nm to
about 800 nm, from about 200 nm to about 500 nm, or from about 50
nm to about 400 nm.
[0239] In some instances, the microvesicle is originated from cell
membrane inversion, exocytosis, shedding, blebbing, or budding. In
some instances, the microvesicles are generated from differentiated
cells. In other instances, the microvesicles are generated from
undifferentiated cells, e.g., by blast cells, progenitor cells, or
stem cells.
[0240] In some embodiments, the delivery component comprises a
liposome. In some instances, the liposome comprises a plurality of
lipopeptides, which are presented on the surface of the liposome,
for targeted delivery to a site or region of interest. In some
cases, the liposomes fuse with the target cell, whereby the
contents of the liposome are then emptied into the target cell. In
some cases, a liposome is endocytosed by cells that are phagocytic.
Endocytosis is then followed by intralysosomal degradation of
liposomal lipids and release of the encapsulated agents.
[0241] Exemplary liposomes suitable for incorporation include, and
are not limited to, multilamellar vesicles (MLV), oligolamellar
vesicles (OLV), unilamellar vesicles (UV), small unilamellar
vesicles (SUV), medium-sized unilamellar vesicles (MUV), large
unilamellar vesicles (LUV), giant unilamellar vesicles (GUV),
multivesicular vesicles (MVV), single or oligolamellar vesicles
made by reverse-phase evaporation method (REV), multilamellar
vesicles made by the reverse-phase evaporation method (MLV-REV),
stable plurilamellar vesicles (SPLV), frozen and thawed MLV
(FATMLV), vesicles prepared by extrusion methods (VET), vesicles
prepared by French press (FPV), vesicles prepared by fusion (FUV),
dehydration-rehydration vesicles (DRV), and bubblesomes (BSV). In
some instances, a liposome comprises Amphipol (A8-35). Techniques
for preparing liposomes are described in, for example, COLLOIDAL
DRUG DELIVERY SYSTEMS, vol. 66 (J. Kreuter ed., Marcel Dekker, Inc.
(1994)).
[0242] Depending on the method of preparation, liposomes are
unilamellar or multilamellar, and vary in size with diameters
ranging from about 20 nm to greater than about 1000 nm.
[0243] In some instances, liposomes provided herein also comprise
carrier lipids. In some embodiments the carrier lipids are
phospholipids. Carrier lipids capable of forming liposomes include,
but are not limited to, dipalmitoylphosphatidylcholine (DPPC),
phosphatidylcholine (PC; lecithin), phosphatidic acid (PA),
phosphatidylglycerol (PG), phosphatidylethanolamine (PE), or
phosphatidylserine (PS). Other suitable phospholipids further
include distearoylphosphatidylcholine (DSPC),
dimyristoylphosphatidylcholine (DMPC),
dipalmitoylphosphatidyglycerol (DPPG),
distearoylphosphatidyglycerol (DSPG),
dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidic
acid (DPPA); dimyristoylphosphatidic acid (DMPA),
distearoylphosphatidic acid (DSPA), dipalmitoylphosphatidylserine
(DPPS), dimyristoylphosphatidylserine (DMPS),
distearoylphosphatidylserine (DSPS),
dipalmitoylphosphatidyethanolamine (DPPE),
dimyristoylphosphatidylethanolamine (DMPE),
distearoylphosphatidylethanolamine (DSPE) and the like, or
combinations thereof. In some embodiments, the liposomes further
comprise a sterol (e.g., cholesterol) which modulates liposome
formation. The carrier lipids are optionally any non-phosphate
polar lipids.
[0244] In some embodiments, the delivery component comprises a
micelle. In some instances, the micelle has a diameter from about 2
nm to about 250 nm, from about 20 nm to about 200 nm, from about 20
nm to about 100 nm, or from about 50 to about 100 nm.
[0245] In some instances, the micelle is a polymeric micelle,
characterized by a core shell structure, in which the hydrophobic
core is surrounded by a hydrophilic shell. In some cases, the
hydrophilic shell further comprises a hydrophilic polymer or
copolymer and a pH sensitive component.
[0246] Exemplary hydrophilic polymers or copolymers include, but
are not limited to, poly(N-substituted acrylamides),
poly(N-acryloyl pyrrolidine), poly(N-acryloyl piperidine),
poly(N-acryl-L-amino acid amides), poly(ethyl oxazoline),
methylcellulose, hydroxypropyl acrylate, hydroxyalkyl cellulose
derivatives and poly(vinyl alcohol), poly(N-isopropylacrylamide),
poly(N-vinyl-2-pyrrolidone), polyethyleneglycol derivatives, and
combinations thereof.
[0247] The pH-sensitive moiety includes, but is not limited to, an
alkylacrylic acid such as methacrylic acid, ethylacrylic acid,
propyl acrylic acid and butyl acrylic acid, or an amino acid such
as glutamic acid.
[0248] In some instances, the hydrophobic moiety constitutes the
core of the micelle and includes, for example, a single alkyl
chain, such as octadecyl acrylate or a double chain alkyl compound
such as phosphatidylethanolamine or dioctadecylamine. In some
cases, the hydrophobic moiety is optionally a water insoluble
polymer such as a poly(lactic acid) or a poly(e-caprolactone).
[0249] Polymeric micelles exhibiting pH-sensitive properties are
also contemplated and are formed, e.g., by using pH-sensitive
polymers including, but not limited to, copolymers from methacrylic
acid, methacrylic acid esters and acrylic acid esters, polyvinyl
acetate phthalate, hydroxypropyl methyl cellulose phthalate,
cellulose acetate phthalate, or cellulose acetate trimellitate.
[0250] In some embodiments, the delivery component comprises a
viral envelope. Viral envelopes comprise glycoproteins,
phospholipids, and additional proteins obtained from a host. In
some instances, the viral envelope is permissive to a wide range of
target cells. In other instances, the viral envelope is
non-permissive and is specific to a target cell of interest. In
some cases, the viral envelope comprises a cell-specific binding
protein and optionally a fusogenic molecule that aids in the fusion
of the cargo into a target cell. In some cases, the viral envelope
comprises an endogenous viral envelope. In other cases, the viral
envelope is a modified envelop, comprising one or more foreign
proteins.
[0251] In some instances, the viral envelope is derived from a DNA
virus. Exemplary enveloped DNA viruses include viruses from the
family of Herpesviridae, Poxviridae, and Hepadnavirdae.
[0252] In other instances, the viral envelope is derived from an
RNA virus. Exemplary enveloped RNA viruses include viruses from the
family of Bunyaviridae, Coronaviridae, Filoviridae, Flaviviridae,
Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, and
Togaviridae.
[0253] In additional instances, the viral envelope is derived from
a virus from the family of Retroviridae.
[0254] In some embodiments, the viral envelope is from an oncolytic
virus, such as an oncolytic DNA virus from the family of
Herpesviridae (for example, HSV1) or Poxviridae (for example,
Vaccinia virus and myxoma virus); or an oncolytic RNA virus from
the family of Rhabdoviridae (for example, VSV) or Paramyxoviridae
(for example MV and NDV).
[0255] In some instances, the viral envelope further comprises a
foreign or engineered protein that binds to an antigen or a cell
surface molecule. Exemplary antigens and cell surface molecules for
targeting include, but are not limited to, P-glycoprotein,
Her2/Neu, erythropoietin (EPO), epidermal growth factor receptor
(EGFR), vascular endothelial growth factor receptor (VEGF-R),
cadherin, carcinoembryonic antigen (CEA), CD4. CD8, CD19. CD20,
CD33, CD34, CD45, CD117 (c-kit), CD133, HLA-A, HLA-B, HLA-C,
chemokine receptor 5 (CCRS), stem cell marker ABCG2 transporter,
ovarian cancer antigen CA125, immunoglobulins, integrins, prostate
specific antigen (PSA), prostate stem cell antigen (PSCA),
dendritic cell-specific intercellular adhesion molecule 3-grabbing
nonintegrin (DC-SIGN), thyroglobulin, granulocyte-macrophage colony
stimulating factor (GM-CSF), myogenic differentiation promoting
factor-1 (MyoD-1), Leu-7 (CD57), LeuM-1, cell
proliferation-associated human nuclear antigen defined by the
monoclonal antibody Ki-67 (Ki-67), viral envelope proteins, HIV
gp120, or transferrin receptor.
[0256] In some embodiments, the Arc-based capsid or endo-Gag-based
capsid is for in vitro use.
[0257] In some instances, the Arc-based capsid or endo-Gag-based
capsid is for ex vivo use.
[0258] In some cases, the Arc-based capsid or endo-Gag-based capsid
is for in vivo use.
Kits/Article of Manufacture
[0259] Disclosed herein, in certain embodiments, are kits and
articles of manufacture for use with one or more methods described
herein. Such kits include a carrier, package, or container that is
compartmentalized to receive one or more containers such as vials,
tubes, and the like, each of the container(s) comprising one of the
separate elements to be used in a method described herein. Suitable
containers include, for example, bottles, vials, syringes, and test
tubes. In one embodiment, the containers are formed from a variety
of materials such as glass or plastic.
[0260] For example, the container(s) include a recombinant or
engineered Arc or endo-Gag polypeptide described above. Such kits
optionally include an identifying description or label or
instructions relating to its use in the methods described herein.
For example, a kit typically includes labels listing contents
and/or instructions for use, and package inserts with instructions
for use. A set of instructions will also typically be included.
CERTAIN TERMINOLOGIES
[0261] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood. It is
to be understood that the detailed description are exemplary and
explanatory only and are not restrictive of any subject matter
claimed. In this application, the use of the singular includes the
plural unless specifically stated otherwise. It must be noted that,
as used in the specification, the singular forms "a," "an" and
"the" include plural referents unless the context clearly dictates
otherwise. In this application, the use of "or" means "and/or"
unless stated otherwise. Furthermore, use of the term "including"
as well as other forms, such as "include", "includes," and
"included," is not limiting.
[0262] Although various features of the invention may be described
in the context of a single embodiment, the features may also be
provided separately or in any suitable combination. Conversely,
although the invention may be described herein in the context of
separate embodiments for clarity, the invention may also be
implemented in a single embodiment.
[0263] Reference in the specification to "some embodiments", "an
embodiment", "one embodiment" or "other embodiments" means that a
particular feature, structure, or characteristic described in
connection with the embodiments is included in at least some
embodiments, but not necessarily all embodiments, of the
inventions.
[0264] As used herein, ranges and amounts can be expressed as
"about" a particular value or range. About also includes the exact
amount. Hence "about 5 .mu.L" means "about 5 .mu.L" and also "5
.mu.L." Generally, the term "about" includes an amount that would
be expected to be within experimental error.
[0265] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described.
[0266] As used herein, the sequence of a CA N-lobe described herein
corresponds to the human CA N-lobe. In some instances, the human CA
N-lobe comprises residues 207-278 of SEQ ID NO: 1. In some
instances, a CA N-lobe described herein comprises about 30%, 40%,
50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or
99% sequence identity to residue 207-278 of SEQ ID NO: 1. In some
cases, a CA N-lobe described herein shares a structural similarity
with the human CA N-lobe. For example, a CA N-lobe described herein
shares about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%
or 99% structural similarity with the human CA N-lobe. In some
cases, the CA N-lobe shares a high structural similarity (e.g.,
80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% structural
similarity) but does not share a high sequence identity (e.g., the
sequence identity is lower than 80%, lower than 70%, lower than
60%, lower than 50%, lower than 40%, or lower than 30%). In some
cases, the CA N-lobe comprises residues 207-278 of SEQ ID NO:
1.
[0267] As used herein, the sequence of a CA C-lobe described herein
corresponds to the human CA C-lobe. In some instances, the human CA
C-lobe comprises residues 278-370 of SEQ ID NO: 1. In some
instances, a CA C-lobe described herein comprises about 30%, 40%,
50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or
99% sequence identity to residue 278-370 of SEQ ID NO: 1. In some
cases, a CA C-lobe described herein shares a structural similarity
with the human CA C-lobe. For example, a CA C-lobe described herein
shares about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%
or 99% structural similarity with the human CA C-lobe. In some
cases, the CA C-lobe shares a high structural similarity (e.g.,
80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% structural
similarity) but does not share a high sequence identity (e.g., the
sequence identity is lower than 80%, lower than 70%, lower than
60%, lower than 50%, lower than 40%, or lower than 30%). In some
cases, the CA C-lobe comprises residues 278-370 of SEQ ID NO:
1.
[0268] As used herein, the terms "individual(s)", "subject(s)" and
"patient(s)" mean any mammal. In some embodiments, the mammal is a
human. In some embodiments, the mammal is a non-human. None of the
terms require or are limited to situations characterized by the
supervision (e.g. constant or intermittent) of a health care worker
(e.g. a doctor, a registered nurse, a nurse practitioner, a
physician's assistant, an orderly or a hospice worker).
EXAMPLES
[0269] These examples are provided for illustrative purposes only
and not to limit the scope of the claims provided herein.
Example 1--Construction of DNA Vectors Encoding Recombinant Arc
Proteins and Engineered Arc Proteins
[0270] To construct recombinant DNA vectors for Arc expression,
full length cDNA open reading frames, excluding the initial
methionine, are inserted into a cloning vector and subsequently
transferred into an expression vector according to standard
methods. The same approach is used to construct recombinant DNA
vectors for expressing endo-Gag proteins. Human Arc cDNA includes
an annotated matrix domain (MA) and a capsid domain. The capsid
domain has an N-terminal lobe (NTD) and a C-terminal lobe (CTD).
FIG. 1 illustrates the structure of the Human Arc protein and the
predicted structure of Arc from Python, Platypus, and Orca.
[0271] cDNAs encoding engineered Arc proteins are optionally
generated by recombining Arc sequences from different species (FIG.
2), by inserting functional domains from other proteins into an Arc
protein (FIG. 3A), by modifying the sequence of an Arc protein
(FIG. 3B), and/or by any combination of the approaches exemplified
in FIGS. 2-3. cDNAs encoding engineered endo-Gag proteins are
likewise generated by recombining endo-Gag sequences from different
species, by inserting functional domains from other proteins into
an endo-Gag protein, by modifying the sequence of an endo-Gag
protein, and/or by any combination of these approaches.
Furthermore, an engineered endo-Gag protein optionally contains Arc
sequences and an engineered Arc protein optionally contains
endo-Gag sequences. Engineered Arc and endo-Gag protein monomers
assemble into capsids.
[0272] cDNAs encoding the Arc and endo-Gag proteins of Table 1 were
inserted into an expression vector derived from pET-41 a(+) (EMD
Millipore (Novagen) Cat #70566). The entire cloning site of pET-41
a(+) was removed and replaced with the DNA having the nucleotide
sequence of SEQ ID NO: 57, which encodes an alternative N-terminal
tag having the amino acid sequence of SEQ ID NO: 58 and comprising
a 6.times.His tag (SEQ ID NO: 59), a 6 amino acid spacer (SEQ ID
NO: 60), and an AcTEV.TM. cleavage site (SEQ ID NO: 61). Arc and
endo-Gag open reading frames without their starting methionine
codon were inserted after the AcTEV.TM. cleavage site by Gibson
assembly. Gibson D G, Young L, Chuang R Y, Venter J C, Hutchison C
A 3rd, Smith HO (2009). "Enzymatic assembly of DNA molecules up to
several hundred kilobases". Nature Methods. 6 (5): 343-345. After
expression and AcTEV.TM. cleavage, the N-terminus of the resulting
Arc or endo-Gag protein has a single residual Glycine from the
AcTEV.TM. cleavage site.
TABLE-US-00001 SEQ ID NO: 57
ATGCATCACCATCACCATCACGGCTCAGGGTCTGGTAGCGAAAATCTGTA CTTCCAGGGG SEQ
ID NO: 58 MHHHHHHGSGSGSENLYFQG SEQ ID NO: 59 HHHHHH SEQ ID NO: 60
GSGSGS SEQ ID NO: 61 ENLYFQG
TABLE-US-00002 TABLE 1 Sequences of Arc and endo-Gag polypeptides
and nucleotides. SEQ ID NO: Gene Species Amino Name Common name
Proper name Sequence ID acid DNA Arc Human Homo sapiens NP_056008.1
1 29 Arc Killer Whale Orcinus orca XP_004265337.1 2 30 Arc White
Tailed Deer Odocoileus XP_020755692.1 3 31 virginianus texanus Arc
Platypus Ornithorhynchus XP_001512750.1 4 32 anatinus Arc Goose
Anser cygnoides XP_013046406.1 5 33 domesticus Arc Dalmation
Pelican Pelecanus crispus KFQ60200.1 6 34 Arc White Tailed Eagle
Haliaeetus albicilla KFQ04633.1 7 35 Arc King Cobra Ophiophagus
ETE60609.1 8 36 hannah Arc Ray Finned Fish Austrofundulus
XP_013881732.1 9 37 limnaeus Arc Sperm Whale Physeter catodon
XP_007119193.2 10 38 Arc Turkey Meleagris XP_010707654.1 11 39
gallopavo Arc Central Bearded Pogona vitticeps XP_020633722.1 12 40
Dragon Arc Chinese Alligator Alligator sinensis XP_006027442.1 13
41 Arc American Alligator Alligator XP_019337372.1 14 42
mississippiensis Arc Japanese Gekko Gekko japonicus XP_015273745.1
15 43 PNMA3 Human Homo sapiens NP_001269464.1 16 44 PNMA5 Human
Homo sapiens NP_001096620.1 17 45 PNMA6A Human Homo sapiens
NP_116271.3 18 46 PNMA6B Human Homo sapiens SP_ P0C5W0.1 19 47 RTL3
Human Homo sapiens NP_689907.1 20 48 RTL6 Human Homo sapiens
NP_115663.2 21 49 RTL8A Human Homo sapiens NP_001071640.1 22 50
RTL8B Human Homo sapiens NP_001071641.1 23 51 BOP Human Homo
sapiens NP_078903.3 24 52 LDOC1 Human Homo sapiens NP_036449.1 25
53 ZNF18 Human Homo sapiens NP_001290210.1 26 54 MOAP1 Human Homo
sapiens AAG31786.1 27 55 PEG10 Human Homo sapiens NP_055883.2 28
56
Example 2--Expression and Purification of Arc and Endo-Gag
Proteins
[0273] Expression vectors constructs comprising Arc and endo-Gag
open reading frames were transformed into the Rosetta 2 (DE3)pLysS
E. coli strain (Millipore Sigma, Cat #71403). Arc or endo-Gag
expression was induced with 0.1 mM IPTG followed by a 16-hour
incubation at 16.degree. C. Cell pellets were lysed by sonication
in 20 mM sodium phosphate pH 7.4, 0.1M NaCl, 40 mM imidazole, 1 mM
DTT, and 10% glycerol. The lysate was treated with excess TURBO
DNase (Thermo Fisher Scientific, Cat #AM2238), RNase Cocktail
(Thermo Fisher Scientific, Cat #AM2286), and Benzonase Nuclease
(Millipore Sigma, Cat #71205) to eliminate nucleic acids. NaCl was
added to lysate in order to adjust the NaCl concentration to 0.5 M
followed by centrifugation and filtration to remove cellular
debris. 6.times.His-tagged recombinant protein was loaded onto a
HisTrap HP column (GE Healthcare, Cat #17-5247-01), washed with
buffer A (20 mM sodium phosphate pH 7.4, 0.5M NaCl, 40 mM
imidazole, and 10% glycerol), and eluted with a linear gradient of
buffer B (20 mM sodium phosphate pH 7.4, 0.5M NaCl, 500 mM
imidazole, and 10% glycerol). Collection tubes were supplemented in
advance with 10 .mu.l of 0.5 M EDTA pH 8.0 per 1 ml eluate. The
resulting Arc or endo-Gag protein is generally more than 95% pure
as revealed by SDS-PAGE analysis, with a yield of up to 50 mg per 1
L of bacterial culture. FIG. 4A.
[0274] Residual nucleic acid was removed by anion exchange
chromatography on a mono Q 5/50 GL column (GE Healthcare, Cat
#17516601). Before loading to the column, recombinant protein was
buffer exchanged to buffer C (20 mM Tris-HCl pH 8.0, 100 mM NaCl,
and 10% glycerol) using "Pierce Protein Concentrator PES, 10K MWCO,
5-20 ml" (Thermo Scientific, Cat #88528) according to the
manufacturer's protocol. After loading, the mono Q resin was washed
with 2 ml of buffer C. Arc and endo-Gag proteins were eluted using
a linear gradient of buffer D (20 mM Tris-HCl pH 8.0, 500 mM NaCl,
and 10% glycerol). RNA efficiently separated from Arc and eluted at
600 mM NaCl (FIG. 4B).
[0275] The N-terminal 6.times.His tag and spacer were removed from
concentrating peak fractions of the mono Q purified Arc using a 10
kDa MWCO PES concentrator and then treating with 10% v/v of
AcTEV.TM. Protease (Invitrogen.TM. #12575023). The cleavage
efficiency is above 99% as revealed by SDS-PAGE assay. The protein
is then diluted into HisTrap Buffer A and cleaned with HisTrap HP
resin. The resulting purified Arc has an N-terminal Glycine residue
and does not contain the initial methionine.
Example 3--Capsid Assembly
[0276] Cleaved Arc protein (1 mg/mL) was loaded into a 20 kDa MWCO
dialysis cassette and dialyzed overnight in 1M sodium phosophate
(pH 7.5) at room temperature. The following day, the solution was
removed from the cassette, transferred to microcentrifuge tubes,
and spun at max speed for 5 minutes in a tabletop centrifuge. The
supernatant was transferred to a 100 kDa MWCO Regenerated Cellulose
Amicon Ultrafiltration Centrifugal concentrator. The buffer was
exchanged to PBS pH 7.5 and the volume was reduced 20-fold.
[0277] Capsid assembly was assayed by transmission electron
microscopy. EM grids (Carbon Support Film, Square Grid, 400 mesh,
5-6 nm, Copper, CF400-Cu-UL) were prepared by glow discharge. A 5
.mu.L sample of purified Arc was applied to the grid for 20 seconds
and then wicked away using filter paper. The grid was then washed
with MilliQ H.sub.2O, stained with 5 .mu.L of 1% Uranyl Acetate in
H.sub.2O for 30 seconds, and air dried for 1 minute. Images of Arc
capsids were acquired using a FEI Talos L120C TEM equipped with a
Gatan 4k.times.4k OneView camera. FIG. 5 shows concentrated human
Arc capsids. FIG. 6 shows capsids formed from recombinantly
expressed Arc orthologs from other vertebrate species. FIG. 7 shows
capsids formed from recombinantly expressed endo-Gag genes from
other vertebrate species.
Example 4--Selective Cellular Internalization of Arc Capsids
[0278] Capsids assembled from isolated recombinant human Arc
protein (0.5 mg/ml) were fluorescently labeled by reacting with a
50-molar excess of NHS ester Alexa Fluor.TM. 594-NHS dye
(Invitrogen.TM. #A20004) (dissolved in DMSO) in PBS (pH 8.5).
Reactions were allowed to proceed for 2-hours in the dark.
Alexa594-labeled capsids were then dialyzed with PBS (pH 7.5)
overnight at room temperature in the dark with at least two buffer
exchanges to remove any unlabeled dye.
[0279] HeLa cells (ATCC.RTM. CCL-2.TM.) were seeded 24-hours prior
to the experiment in 96-well plates at counts such that they reach
.about.80% confluency for treatment. Labeled-capsids were then
spiked into complete tissue culture media to a final capsid
concentration of 0.05 mg/ml. Treatments proceed for 4-hours at
37.degree. C., and then cells are washed 3-times with imaging media
(DMEM, no phenol red, with 10% FBS and 20 mM HEPES) containing 10
ug/ml Hoechst nuclear stain prior to imaging. Fluorescence
microscopy revealed a punctate staining pattern, suggesting that
the Arc capsids were internalized by the HeLa cells (FIG. 8).
Little or no intracellular staining was observed after
administration of Alexa Fluor.TM. 594-labeled bovine serum albumin
(BSA) (final concentration of 0.05 mg/ml) or 45.6 .mu.M Alexa
Fluor.TM. 594 under identical conditions.
Example 5--Heterologous RNA Delivery by Arc Capsids
[0280] Human Arc capsids were loaded with Cre RNA by spiking in
excess RNA during capsid formation (by dialysis into 1M sodium
phosphate). Cre RNA-loaded capsids were administered to HeLa cells
in biological triplicate at a final capsid concentration of 0.05
mg/ml for 4-hours at 37.degree. C. The cells were then washed
3-times with ice-cold 1.times.PBS prior to RNA extraction
(Invitrogen.TM. TRIzol.TM. Reagent #15596026). Purified
cell-associated RNA was quantified by qPCR in technical triplicate,
normalizing values to cellular GAPDH-levels, and comparing to
Escherichia coli rrsA mRNA and Arc RNA that could have carried over
from protein purification. Table 2 shows primers used for the PCR
reaction. The amount of cell-associated Cre RNA detected was
>27-fold higher when Arc capsid were loaded with Cre RNA
compared to control capsids not loaded with Cre RNA (FIG. 9).
TABLE-US-00003 TABLE 2 Primers for qPCR quantification of RNA
delivered by Arc capsids to HeLa cells Gene - SEQ ID Primer
Sequence NO: GAPDH-F AAGCTCATTTCCTGGTATGACAACGA 62 GAPDH-R
AGGGTCTCTCTCTTCCTCTTGTGCT 63 rrsA-F GCTCAACCTGGGAACTGCATCTGAT 64
rrsA-R TAATCCTGTTTGCTCCCCACGCTTT 65 Arc CDS-F
GGCCCCTCAGCTCCAGTGATTC 66 Arc CDS-R CCTGTTGTCACTCTCCTGGCTCTGA 67
Cre CDS-F GCCAAGACATAAGAAACCTCGCCT 68 Cre CDS-R
GTGAATCAACATCCTCCCTCCGTC 69
[0281] FIG. 10 illustrates an alternative method of demonstrating
the delivery of a heterologous RNA by an Arc or endo-Gag capsid.
6.times.His-tagged Arc or endo-Gag genes are expressed in a host
cell. The resulting Arc monomers are mixed with translatable Cre
mRNA under capsid forming conditions to form Cre mRNA loaded
capsids. Cre-loaded capsids are then administered to
LoxP-luciferase reporter mice. Upon successful delivery of Cre mRNA
into mouse cells and subsequent translation of Cre recombinase
protein, LoxP sites of the reporter are recombined, leading to
luciferase expression, which is optionally detected by
bioluminescence imaging upon administration of luciferin. This
method is used to test the transmission potential of candidate Arc
and endo-Gag genes. A positive luciferase signal indicates that the
candidate Arc or endo-Gag gene encodes an Arc or endo-Gag protein
capable of assembling into capsids that incorporate a heterologous
cargo and deliver that cargo to a target cell.
[0282] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
TABLE-US-00004 TABLE 3 Arc and endo-Gag amino acid and nucleotide
sequences SEQ ID NO: 1
GELDHRTSGGLHAYPGPRGGQVAKPNVILQIGKCRAEMLEHVRRTHRHLLAEVSKQVERELKGLHRSVGKLES
NLDGYVPTSDSQRWKKSIKACLCRCQETIANLERWVKREMHVWREVFYRLERWADRLESTGGKYPVGSESARH
TVSVGVGGPESYCHEADGYDYTVSPYAITPPPAAGELPGQEPAEAQQYQPWVPGEDGQPSPGVDTQIFEDPRE
FLSHLEEYLRQVGGSEEYWLSQIQNHMNGPAKKWWEFKQGSVKNWVEFKKEFLQYSEGTLSREAIQRELDLPQ
KQGEPLDQFLWRKRDLYQTLYVDADEEEIIQYVVGTLQPKLKRFLRHPLPKTLEQLIQRGMEVQDDLEQAAEP
AGPHLPVEDEAETLTPAPNSESVASDRTQPE SEQ ID NO: 2
GELDQRTTGGLHAYPAPRGGPVAKPNVILQIGKCRAEMLEHVRRTHRHLLTEVSKQVERELKGLHRSVGKLES
NLDGYVPTGDSQRWRKSIKACLCRCQETIANLERWVKREMHVWREVFYRLERWADRLESMGGKYPVGSNPSRH
TTSVGVGGPESYGHEADTYDYTVSPYAITPPPAAGELPGQEAVEAQQYPPWGLGEDGQPSPGVDTQIFEDPRE
FLSHLEEYLRQVGGSEEYWLSQIQNHMNGPAKKWWEYKQGSVKNWVEFKKEFLQYSEGALSREAVQRELDLPQ
KQGEPLDQFLWRKRDLYQTLYVDADEEEIIQYVVGTLQPKLKRFLRPPLPKTLEQLIQKGMEVEDGLEQVAEP
ASPHLPTEEESEALTPALTSESVASDRTQPE SEQ ID NO: 3
GELDHRTTGGLHAYPAPRGGPAAKPNVILQIGKCRAEMLEHVRRTHRHLLAEVSKQVERELKGLHRSVGKLES
NLDGYVPTGDSQRWKKSIKACLSRCQETIANLERWVKREMHVWREVFYRLERWADRLESGGGKYPVGSDPARH
TVSVGVGGPESYCQDADNYDYTVSPYAITPPPAAGQLPGQEEVEAQQYPPWAPGEDGQLSPGVDTQVFEDPRE
FLRHLEDYLRQVGGSEEYWLSQIQNHMNGPAKKWWEYKQGSVKNWVEFKKEFLQYSEGTLSREAIQRELDLPQ
KQGEPLDQFLWRKRDLYQTLYVDAEEEEIIQYVVGTLQPKLKRFLRPPLPKTLEQLIQKGMEVQDGLEQAAEP
AAEEAEALTPALTNESVASDRTQPE SEQ ID NO: 4
GELDRLNPSSGLHPSSGLHPYPGLRGGATAKPNVILQIGKCRAEMLEHVRKTHRHLLTEVSRQVERELKGLHK
SVGKLESNLDGYVPSSDSQRWKKSIKACLSRCQETIAHLERWVKREMNVWREVFYRLERWADRLEAMGGKYPA
GEQARRTVSVGVGGPETCCPGDESYDCPISPYAVPPSTGESPESLDQGDQHYQQWFALPEESPVSPGVDTQIF
EDPREFLRHLEKYLKQVGGTEEDWLSQIQNHMNGPAKKWWEYKQGSVKNWLEFKKEFLQYSEGTLTRDALKRE
LDLPQKQGEPLDQFLWRKRDLYQTLYVDADEEEIIQYVVGTLQPKLKRFLHHPLPKTLEQLIQRGQEVQNGLE
PTDDPAGQRTQSEDNDESLTPAVTNESTASEGTLPE SEQ ID NO: 5
GQLDNVTNAGIHSFQGHRGVANKPNVILQIGKCRAEMLEHVRRTHRHLLSEVSKQVERELKGLQKSVGKLENN
LEDHVPTDNQRWKKSIKACLARCQETIAHLERWVKREMNVWKEVFFRLEKWADRLESMGGKYCPGEHGKQTVS
VGVGGPEIRPSEGEIYDYALDMSQMYALTPPPGEMPSIPQAHDSYQWVSVSEDAPASPVETQVFEDPREFLSH
LEEYLKQVGGTEEYWLSQIQNHMNGPAKKWWEYKQDSVKNWVEFKKEFLQYSEGTLTRDAIKRELDLPQKEGE
PLDQFLWRKRDLYQTLYVDADEEEIIQYVVGTLQPKLKRFLSYPLPKTLEQLIQRGKEVQGNMDHSDEPSPQR
TPEIQSGDSVESMPPSTTASPVPSNGTQPEPPSPPATVI SEQ ID NO: 6
GQLDNVTNAGIHSFQGHRGVANKPNVILQIGKCRAEMLEHVRRTHRHLLSEVSKQVERELKGLQKSVGKLENN
LEDHVPTDNQRWKKSIKACLARCQETIAHLERWVKREMNVWKEVFFRLEKWADRLESMGGKYCPGEHGKQTVS
VGVGGPEIRPSEGEIYDYALDMSQMYALTPPPGEVPSIPQAHDSYQWVSVSEDAPASPVETQVFEDPREFLSH
LEEYLKQVGGTEEYWLSQIQNHMNGPAKKWWEYKQDSVKNWVEFKKEFLQYSEGTLTRDAIKRELDLPQKEGE
PLDQFLWRKRDLYQTLYVDADEEEIIQYVVGTLQPKLKRFLSYPLPKTLEQLIQRGKEVQGNMDHSEEPSPQR
TPEIQSGDSVDSVPPSTTASPVPSNGTQPE SEQ ID NO: 7
GQLDNVTNAGIHSFQGHRGVANKPNVILQIGKCRAEMLEHVRRTHRHLLSEVSKQVERELKGLQKSVGKLENN
LEDHVPTDNQRWKKSIKACLARCQETIAHLERWVKREMNVWKEVFFRLEKWADRLESMGGKYCPGDHGKQTVS
VGVGGPEIRPSEGEIYDYALDMSQMYALTPPPGEVPSIPQAHDSYQWVSTSEDAPASPVETQVFEDPREFLSH
LEEYLKQVGGTEEYWLSQIQNHMNGPAKKWWEYKQDSVKNWVEFKKEFLQYSEGTLTRDAIKRELDLPQKEGE
PLDQFLWRKRDLYQTLYVDADEEEIIQYVVGTLQPKLKRFLSYPLPKTLEQLIQRGKEVQGNMDHSEEPSPQR
TPEIQSGDSVDSVPPSTTASPVPSNGTQPE SEQ ID NO: 8
GSWGLQRHVADERRGLATPTYGAVCSIREKKASQLSGQSCLEKELLGWKCTEAIVEMMQVDNFNHGNLHSCQG
HRGMANHKPNVILQIGKCRAEMLDHVRRTHRHLLTEVSKQVERELKSLQKSVGKLENNLEDHVPSAAENQRWK
KSIKACLARCQETIAHLERWVKREINVWKEVFFRLEKWADRLESGGGKYGPGDQSRQTVSVGVGAPEIQPRKE
EIYDYALDMSQMYALTPPPMGEDPNVPQSHDSYQWITISDDSPPSPVETQIFEDPREFLTHLEDYLKQVGGTE
EYWLSQIQNHMNGPAKKWWEYKQDSVKNWLEFKKEFLQYSEGTLTRDAIKQELDLPQKDGEPLDQFLWRKRDL
YQTLYIDAEEEEVIQYVVGTLQPKLKRFLSHPYPKTLEQLIQRGKEVEGNLDNSEEPSPQRSPKHQLGGSVES
LPPSSTASPVASDETHPDVSAPPVTVI SEQ ID NO: 9
GDGETQAENPSTSLNNTDEDILEQLKKIVMDQQHLYQKELKASFEQLSRKMFSQMEQMNSKQTDLLLEHQKQT
VKHVDKRVEYLRAQFDASLGWRLKEQHADITTKIIPEIIQTVKEDISLCLSTLCSIAEDIQTSRATTVTGHAA
VQTHPVDLLGEHHLGTTGHPRLQSTRVGKPDDVPESPVSLFMQGEARSRIVGKSPIKLQFPTFGKANDSSDPL
QYLERCEDFLALNPLTDEELMATLRNVLHGTSRDWWDVARHKIQTWREFNKHFRAAFLSEDYEDELAERVRNR
IQKEDESIRDFAYMYQSLCKRWNPAICEGDVVKLILKNINPQLPSQLRSRVTTVDELVRLGQQLEKDRQNQLQ
YELRKSSGKIIQKSSSCETSALPNTKSTPNQQNPATSNRPPQVYCWRCKGHHAPASCPQWKADKHRAQPSRSS
GPQTLTNLQAQDI SEQ ID NO: 10
GELDQRAAGGLRAYPAPRGGPVAKPSVILQIGKCRAEMLEHVRRTHRHLLTEVSKQVERELKGLHRSVGKLEG
NLDGYVPTGDSQRWKKSIKACLCRCQETIANLERWVKREMHVWREVFYRLERWADRLESMGGKYPVGTNPSRH
TVSVGVGGPEGYSHEADTYDYTVSPYAITPPPAAGELPGQEAVEAQQYPPWGLGEDGQPGPGVDTQIFEDPRE
FLSHLEEYLRQVGGSEEYWLSQIQNHMNGPAKKWWEFKQGSVKNWVEFKKEFLQYSEGTLSREAIQRELDLPQ
KQGEPLDQFLWRKRDLYQTLYVDAEEEEIIQYVVGTLQPKLKRFLRPPLPKTLEQLIQKGMEVQDGLEQAAEP
ASPRLPPEEESEALTPALTSESVASDRTQPE SEQ ID NO: 11
GQLDNVTNAGIHSFQGHRGVANKPNVILQIGKCRAEMLEHVRRTHRHLLSEVSKQVERELKGLQKSVGKLENN
LEDHVPTDNQRWKKSIKACLARCQETIAHLERWVKREMNVWKEVFFRLEKWADRLESMGGKYCPGEHGKQTVS
VGVGGPEIRPSEGEIYDYALDMSQMYALTPGPGEVPSIPQAHDSYQWVSVSEDAPASPVETQIFEDPHEFLSH
LEEYLKQVGGTEEYWLSQIQNHMNGPAKKWWEYKQDSVKNWVEFKKEFLQYSEGTLTRDAIKRELDLPQKEGE
PLDQFLWRKRDLYQTLYVDADEEEIIQYVVGTLQPKLKRFLSYPLPKTLEQLIQRGKEVQGNMDHSEEPSPQR
TPEIQSGDSVESMPPSTTASPVPSNGTQPEPPSPPATVI SEQ ID NO: 12
GQLENINQGSLHAFQGHRGVVHNNKPNVILQIGKCRAEMLEHVRRTHRHLLTEVSKQVERELKGLQKSVGKLE
NNLEDHVPSAAENQRWKKSIKACLARCQETIANLERWVKREMNVWKEVFFRLERWADRLESGGGKYCHADQGR
QTVSVGVGGPEVRPSEGEIYDYALDMSQMYALTPPPMGDVPVIPQPHDSYQWVTDPEEAPPSPVETQIFEDPR
EFLTHLEDYLKQVGGTEEYWLSQIQNHMNGPAKKWWEYKQDSVKNWLEFKKEFLQYSEGTLTRDAIKQELDLP
QKEGEPLDQFLWRKRDLYQTLYVEAEEEEVIQYVVGTLQPKLKRFLSHPYPKTLEQLIQRGKEVEGNLDNSEE
PSPQRTPEHQLGDSVESLPPSTTASPAGSDKTQPEISLPPTTVI SEQ ID NO: 13
GQLDSVTNAGVHTYQGHRSVANKPNVILQIGKCRTEMLEHVRRTHRHLLTEVSKQVERELKGLQKSVGKLENN
LEDHVPTDNQRWKKSIKACLARCQETIAHLERWVKREMNVWKEVFFRLERWADRLESMGGKYCPTDSARQTVS
VGVGGPEIRPSEGEIYDYALDMSQMYALTPSPGELPSVPQPHDSYQWVTSPEDAPASPVETQVFEDPREFLCH
LEEYLKQVGGTEEYWLSQIQNHMNGPAKKWWEYKQDTVKNWVEFKKEFLQYSEGTLTRDAIKRELDLPQKDGE
PLDQFLWRKRDLYQTLYIDADEEQIIQYVVGTLQPKLKRFLSYPLPKTLEQLIQKGKEVQGSLDHSEEPSPQR
ASEARTGDSVETLPPSTTTSPNTSSGTQPEAPSPPATVI SEQ ID NO: 14
GQLDSVTNAGVHTYQGHRGVANKPNVILQIGKCRTEMLEHVRRTHRHLLTEVSKQVERELKGLQKSVGKLENN
LEDHVPTDNQRWKKSIKACLARCQETIAHLERWVKREMNVWKEVFFRLERWADRLESMGGKYCPTDSARQTVS
VGVGGPEIRPSEGEIYDYALDMSQMYALTPSPGELPSIPQPHDSYQWVTSPEDAPASPVETQVFEDPREFLCH
LEEYLKQVGGTEEYWLSQIQNHMNGPAKKWWEYKQDTVKNWVEFKKEFLQYSEGTLTRDAIKRELDLPQKDGE
PLDQFLWRKRDLYQTLYIDADEEQIIQYVVGTLQPKLKRFLSYPLPKTLEQLIQKGKEVQGSLDHSEEPSPQR
ASEARTGDSVESLPPSTTTSPNASSGTQPEAPSPPATVI SEQ ID NO: 15
GQLENVNHGNLHSFQGHRGGVANKPNVILQIGKCRAEMLDHVRRTHRHLLTEVSKQVERELKGLQKSVGKLEN
NLEDHVPSAVENQRWKKSIKACLSRCQETIAHLERWVKREMNVWKEVFFRLERWADRLESGGGKYCHGDNHRQ
TVSVGVGGPEVRPSEGEIYDYALDMSQMYALTPPSPGDVPVVSQPHDSYQWVTVPEDTPPSPVETQIFEDPRE
FLTHLEDYLKQVGGTEEYWLSQIQNHMNGPAKKWWEYKQDSVKNWLEFKKEFLQYSEGTLTRDAIKEELDLPQ
KDGEPLDQFLWRKRDLYQTLYVEADEEEVIQYVVGTLQPKLKRFLSHPYPKTLEQLIQRGKEVEGNLDNSEEP
TPQRTPEHQLCGSVESLPPSSTVSPVASDGTQPETSPLPATVI SEQ ID NO: 16
GPLTLLQDWCRGEHLNTRRCMLILGIPEDCGEDEFEETLQEACRHLGRYRVIGRMFRREENAQAILLELAQDI
DYALLPREIPGKGGPWEVIVKPRNSDGEFLNRLNRFLEEERRTVSDMNRVLGSDTNCSAPRVTISPEFWTWAQ
TLGAAVQPLLEQMLYRELRVFSGNTISIPGALAFDAWLEHTTEMLQMWQVPEGEKRRRLMECLRGPALQVVSG
LRASNASITVEECLAALQQVFGPVESHKIAQVKLCKAYQEAGEKVSSFVLRLEPLLQRAVENNVVSRRNVNQT
RLKRVLSGATLPDKLRDKLKLMKQRRKPPGFLALVKLLREEEEWEATLGPDRESLEGLEVAPRPPARITGVGA
VPLPASGNSFDARPSQGYRRRRGRGQHRRGGVARAGSRGSRKRKRHTFCYSCGEDGHIRVQCINPSNLLLAKE
TKEILEGGEREAQTNSR SEQ ID NO: 17
GALTLLEDWCKGMDMDPRKALLIVGIPMECSEVEIQDTVKAGLQPLCAYRVLGRMFRREDNAKAVFIELADTV
NYTTLPSHIPGKGGSWEVVVKPRNPDDEFLSRLNYFLKDEGRSMTDVARALGCCSLPAESLDAEVMPQVRSPP
LEPPKESMWYRKLKVFSGTASPSPGEETFEDWLEQVTEIMPIWQVSEVEKRRRLLESLRGPALSIMRVLQANN
DSITVEQCLDALKQIFGDKEDFRASQFRFLQTSPKIGEKVSTFLLRLEPLLQKAVHKSPLSVRSTDMIRLKHL
LARVAMTPALRGKLELLDQRGCPPNFLELMKLIRDEEEWENTEAVMKNKEKPSGRGRGASGRQARAEASVSAP
QATVQARSFSDSSPQTIQGGLPPLVKRRRLLGSESTRGEDHGQATYPKAENQTPGREGPQAAGEELGNEAGAG
AMSHPKPWET SEQ ID NO: 18
GAVTMLQDWCRWMGVNARRGLLILGIPEDCDDAEFQESLEAALRPMGHFTVLGKAFREEDNATAALVELDREV
NYALVPREIPGTGGPWNVVFVPRCSGEEFLGLGRVFHFPEQEGQMVESVAGALGVGLRRVCWLRSIGQAVQPW
VEAVRCQSLGVFSGRDQPAPGEESFEVWLDHTTEMLHVWQGVSERERRRRLLEGLRGTALQLVHALLAENPAR
TAQDCLAALAQVFGDNESQATIRVKCLTAQQQSGERLSAFVLRLEVLLQKAMEKEALARASADRVRLRQMLTR
AHLTEPLDEALRKLRMAGRSPSFLEMLGLVRESEAWEASLARSVRAQTQEGAGARAGAQAVARASTKVEAVPG
GPGREPEGLLQAGGQEAEELLQEGLKPVLEECDN SEQ ID NO: 19
GAVTMLQDWCRWMGVNARRGLLILGIPEDCDDAEFQESLEAALRPMGHFTVLGKVFREEDNATAALVELDREV
NYALVPREIPGTGGPWNVVFVPRCSGEEFLGLGRVFHFPEQEGQMVESVAGALGVGLRRVCWLRSIGQAVQPW
VEAVRYQSLGVFSGRDQPAPGEESFEVWLDHTTEMLHVWQGVSERERRRRLLEGLRGTALQLVHALLAENPAR
TAQDCLAALAQVFGDNESQATIRVKCLTAQQQSGERLSAFVLRLEVLLQKAMEKEALARASADRVRLRQMLTR
AHLTEPLDEALRKLRMAGRSPSFLEMLGLVRESEAWEASLARSVRAQTQEGAGARAGAQAVARASTKVEAVPG
GPGREPEGLRQAGGQEAEELLQEGLKPVLEECDN SEQ ID NO: 20
GVEDLAASYIVLKLENElRQAQVQWLMEENAALQAQIPELQKSQAAKEYDLLRKSSEAKEPQKLPEHMNPPAA
WEAQKTPEFKEPQKPPEPQDLLPWEPPAAWELQEAPAAPESLAPPATRESQKPPMAHEIPTVLEGQGPANTQD
ATIAQEPKNSEPQDPPNIEKPQEAPEYQETAAQLEFLELPPPQEPLEPSNAQEFLELSAAQESLEGLIVVETS
AASEFPQAPIGLEATDFPLQYTLTFSGDSQKLPEFLVQLYSYMRVRGHLYPTEAALVSFVGNCFSGRAGWWFQ
LLLDIQSPLLEQCESFIPVLQDTFDNPENMKDANQCIHQLCQGEGHVATHFHLIAQELNWDESTLWIQFQEGL
ASSIQDELSHTSPATNLSDLITQCISLEEKPDPNPLGKSSSAEGDGPESPPAENQPMQAAINCPHISEAEWVR
WHKGRLCLYCGYPGHFARDCPVKPHQALQAGNIQACQ SEQ ID NO: 21
GVQPQTSKAESPALAASPNAQMDDVIDTLTSLRLTNSALRREASTLRAEKANLTNMLESVMAELTLLRTRARI
PGALQITPPISSITSNGTRPMTTPPTSLPEPFSGDPGRLAGFLMQMDRFMIFQASRFPGEAERVAFLVSRLTG
EAEKWAIPHMQPDSPLRNNYQGFLAELRRTYKSPLRHARRAQIRKTSASNRAVRERQMLCRQLASAGTGPCPV
HPASNGTSPAPALPARARNL SEQ ID NO: 22
GDGRVQLMKALLAGPLRPAARRWRNPIPFPETFDGDTDRLPEFIVQTSSYMFVDENTFSNDALKVTFLITRLT
GPALQWVIPYIRKESPLLNDYRGFLAEMKRVFGWEEDEDF SEQ ID NO: 23
GEGRVQLMKALLARPLRPAARRWRNPIPFPETFDGDTDRLPEFIVQTSSYMFVDENTFSNDALKVTFLITRLT
GPALQWVIPYIKKESPLLSDYRGFLAEMKRVFGWEEDEDF SEQ ID NO: 24
GPRGRCRQQGPRIPIWAAANYANAHPWQQMDKASPGVAYTPLVDPW1ERPCCGDTVCVRTTMEQKSTASGTCG
GKPAERGPLAGHMPSSRPHRVDFCWVPGSDPGTFDGSPWLLDRFLAQLGDYMSFHFEHYQDNISRVCEILRRL
TGRAQAWAAPYLDGDLPLPDDYELFCQDLKEVVQDPNSFAEYHAVVICPLPLASSQLPVAPQLPVVRQYLARF
LEGLALDMGTAPRSLPAAMATPAVSGSNSVSRSALFEQQLTKESTPGPKEPPVLPSSTCSSKPGPVEPASSQP
EEAAPTPVPRLSESANPPAQRPDPAHPGGPKPQKTEEEVLETEGDQEVSLGTPQEVVEAPETPGEPPLSPGF
SEQ ID NO: 25
GVDELVLLLHALLMRHRALSIENSQLMEQLRLLVCERASLLRQVRPPSCPVPFPETFNGESSRLPEFIVQTAS
YMLVNENRFCNDAMKVAFLISLLTGEAEEWVVPYIEMDSP1LGDYRAFLDEMKQCFGWDDDEDDDDEEEEDDY
SEQ ID NO: 26
GPVDLGQALGLLPSLAKAEDSQFSESDAALQEELSSPETARQLFRQFRYQVMSGPHETLKQLRKLCFQWLQPE
VHTKEQILEILMLEQFLTILPGEIQMWVRKQCPGSGEEAVTLVESLKGDPQRLWQWISIQVLGQDILSEKMES
PSCQVGEVEPHLEVVPQELGLENSSSGPGELLSHIVKEESDTEAELALAASQPARLEERLIRDQDLGASLLPA
APQEQWRQLDSTQKEQYWDLMLETYGKMVSGAGISHPKSDLTNSIEFGEELAGIYLHVNEKIPRPTCIGDRQE
NDKENLNLENHRDQELLHASCQASGEVPSQASLRGFFTEDEPGCFGEGENLPEALQNIQDEGTGEQLSPQERI
SEKQLGQHLPNPHSGEMSTMWLEEKRETSQKGQPRAPMAQKLPTCRECGKTFYRNSQLIFHQRTHIGETYFQC
TICKKAFLRSSDFVKHQRTHTGEKPCKCDYCGKGFSDFSGLRHHEKIHTGEKPYKCPICEKSFIQRSNFNRHQ
RVHTGEKPYKCSHCGKSFSWSSSLDKHQRSHLGKKPFQ SEQ ID NO: 27
GTLRLLEDWCRGMDMNPRKALLIAGISQSCSVAEIEEALQAGLAPLGEYRLLGRMFRRDENRKVALVGLTAET
SHALVPKEIPGKGGIWRVIFKPPDPDNTFLSRLNEFLAGEGMTVGELSRALGHENGSLDPEQGMIPEMWAPML
AQALEALQPALQCLKYKKLRVFSGRESPEPGEEEFGRWMFHTTQMIKAWQVPDVEKRRRLLESLRGPALDVIR
VLKINNPLITVDECLQALEEVFGVTDNPRELQVKYLTTYHKDEEKLSAYVLRLEPLLQKLVQRGAIERDAVNQ
ARLDQVIAGAVHKTIRRELNLPEDGPAPGFLQLLVLIKDYEAAEEEEALLQAILEGNFT SEQ ID
NO: 28
GTERRRDELSEEINNLREKVMKQSEENNNLQSQVQKLTEENTTLREQVEPTPEDEDDDIELRGAAAAAAPPPP
IEEECPEDLPEKFDGNPDMLAPFMAQCQIFMEKSTRDFSVDRVRVCFVTSMMTGRAARWASAKLERSHYLMHN
YPAFMMEMKHVFEDPQRREVAKRKIRRLRQGMGSVIDYSNAFQMIAQDLDWNEPALIDQYHEGLSDHIQEELS
HLEVAKSLSALIGQCIHIERRLARAAAARKPRSPPRALVLPHIASHHQVDPTEPVGGARMRLTQEEKERRRKL
NLCLYCGTGGHYADNCPAKASKSSPAGKLPGPAVEGPSATGPEIIRSPQDDASSPHLQVMLQIHLPGRHTLFV
RAMIDSGASGNFIDHEYVAQNGIPLRIKDWPILVEAIDGRPIASGPVVHETHDLIVDLGDHREVLSFDVTQSP
FFPVVLGVRWLSTHDPNITWSTRSIVFDSEYCRYHCRMYSPIPPSLPPPAPQPPLYYPVDGYRVYQPVRYYYV
QNVYTPVDEHVYPDHRLVDPHIEMIPGAHSIPSGHVYSLSEPEMAALRDFVARNVKDGLITPTIAPNGAQVLQ
VKRGWKLQVSYDCRAPNNFTIQNQYPRLSIPNLEDQAHLATYTEFVPQIPGYQTYPTYAAYPTYPVGFAWYPV
GRDGQGRSLYVPVMITWNPHWYRQPPVPQYPPPQPPPPPPPPPPPPSYSTL SEQ ID NO: 29
GGGGAGCTGGACCACCGGACCAGCGGCGGGCTCCACGCCTACCCCGGGCCGCGGGGCGGGCAGGTGGCCAAGC
CCAACGTGATCCTGCAGATCGGGAAGTGCCGGGCCGAGATGCTGGAGCACGTGCGGCGGACGCACCGGCACCT
GCTGGCCGAGGTGTCCAAGCAGGTGGAGCGCGAGCTGAAGGGGCTGCACCGGTCGGTCGGGAAGCTGGAGAGC
AACCTGGACGGCTACGTGCCCACGAGCGACTCGCAGCGCTGGAAGAAGTCCATCAAGGCCTGCCTGTGCCGCT
GCCAGGAGACCATCGCCAACCTGGAGCGCTGGGTCAAGCGCGAGATGCACGTGTGGCGCGAGGTGTTCTACCG
CCTGGAGCGCTGGGCCGACCGCCTGGAGTCCACGGGCGGCAAGTACCCGGTGGGCAGCGAGTCAGCCCGCCAC
ACCGTTTCCGTGGGCGTGGGGGGTCCCGAGAGCTACTGCCACGAGGCAGACGGCTACGACTACACCGTCAGCC
CCTACGCCATCACCCCGCCCCCAGCCGCTGGCGAGCTGCCCGGGCAGGAGCCCGCCGAGGCCCAGCAGTACCA
GCCGTGGGTCCCCGGCGAGGACGGGCAGCCCAGCCCCGGCGTGGACACGCAGATCTTCGAGGACCCTCGAGAG
TTCCTGAGCCACCTAGAGGAGTACTTGCGGCAGGTGGGCGGCTCTGAGGAGTACTGGCTGTCCCAGATCCAGA
ATCACATGAACGGGCCGGCCAAGAAGTGGTGGGAGTTCAAGCAGGGCTCCGTGAAGAACTGGGTGGAGTTCAA
GAAGGAGTTCCTGCAGTACAGCGAGGGCACGCTGTCCCGAGAGGCCATCCAGCGCGAGCTGGACCTGCCGCAG
AAGCAGGGCGAGCCGCTGGACCAGTTCCTGTGGCGCAAGCGGGACCTGTACCAGACGCTCTACGTGGACGCGG
ACGAGGAGGAGATCATCCAGTACGTGGTGGGCACCCTGCAGCCCAAGCTCAAGCGTTTCCTGCGCCACCCCCT
GCCCAAGACCCTGGAGCAGCTCATCCAGAGGGGCATGGAGGTGCAGGATGACCTGGAGCAGGCGGCCGAGCCG
GCCGGCCCCCACCTCCCGGTGGAGGATGAGGCGGAGACCCTCACGCCCGCCCCCAACAGCGAGTCCGTGGCCA
GTGACCGGACCCAGCCCGAG SEQ ID NO: 30
GGGGAATTGGATCAACGTACTACCGGTGGCCTTCACGCATACCCTGCACCACGCGGGGGCCCTGTCGCGAAGC
CAAATGTCATCCTGCAGATTGGGAAGTGCCGGGCTGAGATGCTGGAGCACGTCCGTCGGACGCATCGTCATCT
TCTTACTGAGGTGTCAAAACAGGTGGAGCGTGAACTCAAAGGCTTGCACCGCAGCGTTGGGAAACTTGAAAGC
AACTTAGATGGCTATGTGCCGACTGGCGACAGCCAGCGTTGGCGTAAGTCCATCAAAGCATGTTTGTGTCGTT
GCCAGGAAACGATTGCAAACCTGGAGCGTTGGGTCAAACGGGAGATGCATGTCTGGCGTGAAGTATTTTATCG
TTTAGAGCGTTGGGCCGATCGTTTAGAGAGCATGGGTGGTAAGTACCCTGTGGGGAGCAACCCTTCTCGGCAT
ACGACGTCAGTCGGTGTTGGCGGGCCGGAGTCCTACGGTCATGAAGCGGACACCTACGACTATACCGTAAGCC
CTTATGCTATTACCCCACCACCTGCGGCCGGCGAATTACCTGGCCAGGAAGCCGTTGAGGCTCAACAATACCC
TCCTTGGGGGCTGGGCGAGGATGGTCAACCTAGCCCAGGGGTAGACACGCAAATCTTTGAGGACCCACGGGAG
TTTCTTTCCCACCTGGAAGAATACCTGCGTCAGGTTGGTGGGAGCGAAGAATACTGGCTGTCACAAATTCAAA
ACCATATGAATGGTCCTGCAAAAAAATGGTGGGAATATAAACAGGGTTCCGTGAAAAACTGGGTTGAGTTTAA
AAAGGAGTTTCTTCAATATTCCGAGGGCGCCCTCAGTCGGGAGGCGGTCCAACGCGAGTTGGACTTGCCACAG
AAACAGGGGGAACCACTCGATCAATTCCTTTGGCGGAAACGTGACCTTTACCAGACATTGTACGTGGATGCAG
ATGAGGAAGAAATTATCCAATATGTTGTGGGGACCCTGCAGCCGAAACTGAAACGTTTCCTTCGCCCGCCGCT
GCCTAAAACGTTGGAACAACTTATTCAGAAAGGTATGGAGGTCGAGGATGGCTTAGAACAAGTCGCAGAGCCG
GCCTCGCCACACTTGCCTACAGAGGAGGAATCGGAGGCGCTGACCCCAGCACTTACATCAGAGTCAGTGGCAT
CAGACCGGACACAACCAGAG SEQ ID NO: 31
GGGGAGTTAGATCACCGTACAACGGGGGGGTTGCACGCATACCCTGCTCCACGTGGCGGGCCGGCAGCTAAGC
CAAACGTAATCCTGCAGATTGGGAAGTGCCGGGCAGAGATGTTGGAGCACGTCCGGCGGACCCACCGGCACCT
CCTGGCTGAAGTGTCTAAACAAGTAGAACGGGAACTCAAAGGTCTTCATCGTAGCGTCGGGAAATTGGAATCG
AATTTGGACGGGTATGTTCCTACAGGCGACTCACAGCGGTGGAAAAAGAGCATCAAGGCCTGCCTGAGTCGCT
GCCAGGAGACGATTGCTAACCTCGAACGCTGGGTTAAGCGGGAGATGCACGTTTGGCGCGAAGTCTTCTACCG
GCTGGAGCGTTGGGCTGATCGGCTCGAATCTGGTGGGGGTAAGTATCCAGTTGGGTCCGACCCTGCTCGCCAC
ACAGTCTCAGTTGGCGTAGGTGGGCCGGAGTCGTATTGCCAAGATGCGGACAACTATGATTATACAGTTTCCC
CATACGCGATCACACCACCGCCGGCAGCAGGGCAGCTGCCAGGTCAGGAAGAGGTTGAGGCCCAGCAGTATCC
ACCATGGGCCCCAGGGGAAGACGGCCAGCTTTCTCCTGGGGTGGACACTCAAGTTTTTGAAGATCCGCGTGAA
TTTCTGCGGCATTTAGAAGATTATCTCCGCCAGGTCGGGGGGTCTGAAGAGTATTGGTTAAGCCAAATTCAAA
ACCATATGAACGGCCCGGCCAAGAAGTGGTGGGAGTACAAGCAAGGGTCTGTGAAAAATTGGGTGGAGTTTAA
GAAAGAATTCTTGCAATATTCTGAGGGCACTCTTTCGCGTGAAGCCATCCAACGCGAACTCGACTTACCGCAG
AAACAAGGGGAACCTCTCGACCAATTTCTGTGGCGCAAACGCGACCTGTACCAGACTCTTTACGTCGATGCTG
AGGAGGAAGAAATTATTCAATACGTAGTTGGCACACTGCAGCCTAAGCTTAAACGGTTTTTACGTCCACCATT
GCCGAAGACGCTTGAACAACTCATCCAGAAGGGTATGGAGGTTCAAGATGGTCTGGAACAGGCAGCGGAACCA
GCGGCGGAGGAGGCAGAAGCCCTGACACCTGCGTTAACTAACGAGTCTGTCGCGAGCGACCGCACCCAGCCGG
AA SEQ ID NO: 32
GGGGAATTAGACCGCCTGAACCCAAGCTCAGGCCTGCATCCATCCTCTGGTTTGCATCCATACCCAGGTCTCC
GGGGCGGGGCAACCGCGAAGCCTAATGTCATTTTGCAAATTGGCAAATGCCGTGCGGAAATGCTTGAACACGT
CCGCAAAACTCACCGTCATCTCCTCACAGAAGTATCGCGCCAAGTAGAACGCGAGCTCAAAGGCCTTCACAAA
AGTGTTGGCAAGTTGGAATCAAATCTTGATGGGTACGTACCGTCAAGCGACTCCCAACGCTGGAAGAAAAGCA
TTAAGGCGTGCTTATCCCGTTGCCAAGAGACGATTGCGCATTTAGAACGCTGGGTTAAACGTGAAATGAATGT
ATGGCGTGAGGTGTTCTACCGTTTGGAACGTTGGGCGGACCGTCTGGAGGCTATGGGCGGTAAGTATCCTGCC
GGTGAGCAGGCCCGGCGTACAGTTTCAGTGGGCGTTGGGGGCCCTGAGACATGTTGTCCAGGGGATGAAAGTT
ATGATTGTCCGATTTCTCCGTATGCAGTTCCACCTTCCACCGGCGAGTCTCCGGAATCCTTAGACCAAGGGGA
TCAGCACTATCAGCAGTGGTTTGCCCTCCCGGAGGAGTCCCCTGTTAGCCCTGGGGTTGATACCCAGATCTTT
GAAGATCCTCGCGAGTTTTTACGTCATCTGGAGAAGTACCTGAAACAAGTCGGCGGGACAGAGGAAGACTGGC
TTTCTCAAATCCAGAATCACATGAATGGGCCGGCGAAGAAGTGGTGGGAGTACAAGCAAGGGAGTGTTAAGAA
TTGGCTTGAATTTAAGAAGGAATTTTTACAGTATTCGGAGGGCACACTGACGCGGGACGCGTTGAAACGTGAA
CTGGATCTCCCACAGAAACAAGGCGAACCACTTGATCAATTTTTATGGCGGAAGCGCGACTTATATCAGACAC
TCTACGTTGACGCCGATGAAGAGGAAATCATTCAGTACGTCGTGGGCACTCTTCAGCCGAAATTAAAACGCTT
TCTCCATCACCCACTCCCTAAGACGCTTGAGCAGCTTATCCAACGGGGCCAAGAAGTTCAGAATGGTCTGGAG
CCTACCGACGATCCTGCAGGCCAACGCACTCAATCGGAGGACAACGACGAAAGCCTTACCCCTGCCGTCACCA
ATGAGAGTACTGCAAGCGAGGGCACCCTGCCAGAG SEQ ID NO: 33
GGGCAGCTTGATAACGTTACAAACGCGGGCATCCACTCCTTCCAGGGGCATCGTGGCGTAGCGAATAAGCCAA
ATGTCATTCTGCAAATTGGTAAATGTCGTGCGGAAATGCTGGAGCACGTTCGCCGCACCCACCGCCATTTATT
ATCTGAAGTATCTAAGCAGGTAGAACGTGAGCTGAAAGGGCTGCAAAAGTCCGTGGGCAAGCTCGAGAATAAC
TTGGAGGATCATGTCCCTACAGATAACCAACGCTGGAAGAAGTCCATTAAAGCGTGCTTGGCTCGTTGTCAAG
AGACTATCGCGCATTTAGAGCGTTGGGTGAAACGCGAAATGAACGTCTGGAAGGAGGTGTTTTTCCGGCTGGA
AAAGTGGGCAGACCGGCTGGAGTCAATGGGTGGCAAGTACTGCCCGGGCGAACACGGGAAACAAACCGTCAGT
GTAGGCGTGGGGGGTCCTGAAATCCGGCCTTCGGAGGGGGAAATTTATGATTATGCTCTGGATATGAGCCAGA
TGTATGCACTCACCCCACCTCCAGGCGAAATGCCATCAATCCCACAAGCCCATGACAGCTATCAGTGGGTTAG
TGTCTCAGAAGATGCCCCGGCGAGCCCTGTCGAAACCCAGGTATTTGAGGACCCTCGGGAATTCCTGTCTCAC
CTGGAGGAATACCTGAAGCAGGTAGGCGGCACGGAGGAGTATTGGTTGTCCCAGATCCAGAATCACATGAATG
GTCCGGCAAAAAAATGGTGGGAATATAAACAGGACTCCGTTAAAAACTGGGTTGAGTTTAAAAAGGAATTCTT
GCAATACTCTGAAGGTACTTTAACTCGGGATGCTATTAAGCGTGAACTCGACTTGCCGCAAAAGGAAGGTGAA
CCTCTTGACCAATTCCTTTGGCGGAAGCGGGACCTCTATCAGACACTTTACGTGGACGCGGATGAGGAGGAGA
TCATTCAGTATGTGGTCGGTACCCTGCAGCCGAAGCTCAAGCGTTTCCTGAGCTATCCTCTCCCAAAGACTTT
AGAACAGCTCATCCAGCGCGGTAAAGAAGTGCAGGGTAACATGGATCACTCCGATGAGCCTTCGCCGCAGCGT
ACACCTGAAATTCAATCAGGTGACTCCGTAGAATCTATGCCACCTTCAACAACGGCATCTCCGGTTCCATCTA
ATGGTACCCAACCTGAGCCGCCGAGCCCGCCAGCCACCGTTATC SEQ ID NO: 34
GGGCAACTTGACAACGTAACAAACGCTGGGATTCACTCCTTTCAGGGCCACCGCGGTGTCGCCAACAAGCCAA
ACGTAATCTTGCAAATTGGCAAATGCCGTGCGGAGATGTTGGAACACGTTCGTCGTACACATCGTCACTTGCT
GTCGGAAGTCTCTAAACAAGTAGAACGTGAACTTAAAGGGCTTCAAAAGTCAGTCGGCAAATTGGAAAACAAC
CTTGAAGACCATGTACCAACCGACAATCAGCGTTGGAAAAAGTCTATCAAAGCTTGCCTGGCCCGTTGTCAAG
AGACGATTGCTCACCTGGAGCGGTGGGTAAAGCGCGAGATGAATGTGTGGAAAGAGGTCTTCTTCCGCTTGGA
AAAATGGGCCGACCGTTTGGAGTCCATGGGCGGTAAATATTGTCCGGGTGAACATGGTAAGCAAACAGTCTCT
GTGGGCGTTGGTGGGCCGGAGATTCGGCCTTCTGAAGGCGAGATTTACGATTATGCGCTCGACATGTCCCAGA
TGTATGCGCTTACACCACCACCGGGCGAGGTACCAAGCATTCCTCAAGCGCATGACAGTTATCAGTGGGTTAG
CGTATCCGAAGACGCTCCTGCCTCGCCGGTAGAGACCCAGGTTTTTGAAGATCCTCGTGAATTTTTAAGCCAC
TTGGAGGAGTATTTGAAGCAGGTAGGGGGGACAGAGGAATATTGGCTGTCTCAGATCCAGAACCACATGAATG
GCCCGGCTAAAAAGTGGTGGGAATACAAACAAGATTCGGTAAAGAATTGGGTAGAATTTAAAAAGGAGTTTTT
ACAGTACTCAGAGGGGACTCTCACGCGTGATGCGATCAAACGCGAGTTGGATCTTCCTCAAAAAGAGGGGGAG
CCACTCGATCAGTTCCTCTGGCGCAAGCGGGATCTCTACCAAACACTCTACGTAGACGCAGACGAAGAAGAGA
TCATCCAGTACGTGGTGGGTACGCTCCAGCCGAAACTCAAACGTTTCCTCAGCTACCCACTTCCTAAGACTCT
GGAACAACTGATTCAGCGGGGCAAAGAGGTCCAGGGTAACATGGACCATTCAGAGGAACCTAGTCCGCAACGT
ACACCTGAGATCCAATCTGGGGATTCTGTCGATTCGGTTCCACCTTCTACAACAGCGTCTCCGGTGCCGTCAA
ATGGGACCCAACCAGAG SEQ ID NO: 35
GGGCAGCTTGATAATGTAACCAATGCAGGTATCCACTCTTTCCAGGGTCACCGCGGTGTGGCAAACAAGCCAA
ATGTTATTCTGCAAATTGGTAAGTGTCGCGCTGAGATGTTAGAACACGTCCGGCGCACGCATCGGCATCTCCT
GTCAGAGGTTTCAAAGCAGGTAGAGCGTGAATTAAAGGGCCTCCAGAAGTCCGTAGGTAAACTCGAAAATAAT
CTTGAAGACCACGTTCCTACCGATAATCAACGGTGGAAAAAGTCAATCAAGGCGTGCTTAGCACGGTGTCAGG
AAACGATCGCGCACCTCGAACGTTGGGTGAAGCGCGAAATGAATGTCTGGAAAGAAGTGTTCTTCCGGCTTGA
GAAGTGGGCTGATCGGCTCGAATCCATGGGTGGCAAATATTGTCCAGGTGATCATGGCAAGCAAACGGTCTCC
GTCGGTGTTGGTGGTCCGGAAATCCGGCCGAGCGAGGGTGAAATCTATGACTACGCTCTTGATATGTCCCAGA
TGTATGCACTCACTCCTCCGCCGGGTGAGGTCCCGTCGATCCCGCAGGCGCATGACTCATACCAATGGGTGTC
GACTAGCGAAGACGCACCAGCCTCCCCTGTTGAAACTCAAGTATTCGAGGACCCGCGTGAGTTCCTGAGCCAT
TTAGAGGAGTACCTTAAGCAGGTTGGTGGTACCGAGGAATACTGGTTGAGCCAGATTCAGAATCACATGAACG
GGCCGGCTAAGAAATGGTGGGAATACAAGCAGGATTCAGTCAAGAATTGGGTCGAATTTAAGAAGGAGTTTTT
GCAGTACAGTGAGGGGACGCTCACACGCGACGCTATCAAACGGGAGCTGGACCTGCCACAAAAGGAGGGTGAA
CCGCTTGATCAGTTTCTTTGGCGCAAGCGTGATCTGTATCAAACCCTGTATGTGGACGCTGACGAAGAAGAGA
TCATTCAGTACGTGGTTGGGACTCTGCAACCAAAGCTGAAGCGTTTTCTTTCTTATCCTCTCCCTAAGACACT
GGAACAGTTAATCCAACGTGGCAAGGAGGTCCAGGGTAATATGGACCACTCTGAGGAACCGAGCCCGCAACGT
ACTCCTGAAATTCAGAGCGGGGATAGTGTCGACTCAGTTCCTCCAAGTACGACCGCATCCCCGGTCCCAAGTA
ACGGTACCCAACCAGAG SEQ ID NO: 36
GGGTCTTGGGGCTTGCAACGTCACGTGGCTGATGAACGTCGTGGCCTCGCTACGCCTACCTACGGCGCGGTTT
GTTCCATTCGGGAGAAAAAAGCCTCCCAACTGAGCGGCCAGAGCTGTTTGGAGAAAGAGTTGCTTGGTTGGAA
ATGTACGGAGGCAATCGTGGAAATGATGCAAGTCGATAACTTTAACCACGGTAACTTACATAGCTGCCAAGGC
CATCGGGGGATGGCAAATCACAAACCGAACGTAATCCTTCAAATCGGGAAATGTCGCGCAGAAATGTTAGACC
ACGTGCGTCGCACCCACCGCCATCTCTTGACGGAGGTTTCGAAGCAGGTAGAACGCGAATTGAAGTCTCTCCA
AAAGTCGGTTGGCAAGCTCGAGAATAATCTGGAAGACCACGTGCCATCGGCAGCGGAGAACCAACGTTGGAAG
AAATCAATTAAAGCCTGCCTGGCCCGGTGCCAAGAAACAATTGCTCACCTCGAACGCTGGGTTAAACGCGAAA
TCAACGTCTGGAAAGAAGTATTCTTTCGTCTGGAGAAGTGGGCGGACCGCCTTGAGTCGGGTGGGGGCAAGTA
TGGGCCTGGTGACCAAAGTCGTCAAACTGTAAGTGTCGGTGTTGGGGCCCCAGAAATCCAACCGCGGAAAGAA
GAAATCTATGACTACGCTCTCGACATGTCGCAGATGTATGCCTTAACACCACCGCCGATGGGTGAAGACCCAA
ACGTACCTCAATCCCACGATAGCTACCAGTGGATTACCATCTCAGACGATTCACCTCCGTCGCCAGTGGAAAC
TCAAATTTTCGAGGATCCACGCGAATTCCTTACCCATCTCGAGGATTATCTTAAGCAAGTGGGCGGGACTGAA
GAATATTGGTTGAGTCAGATTCAAAATCATATGAACGGTCCGGCCAAGAAATGGTGGGAGTACAAACAAGATT
CCGTGAAAAACTGGTTGGAATTCAAGAAGGAATTCCTTCAATACTCTGAGGGTACTTTGACACGTGACGCAAT
TAAACAAGAACTTGACTTACCGCAGAAGGACGGCGAGCCATTGGATCAATTTCTTTGGCGGAAGCGGGACCTG
TATCAGACGCTCTATATTGATGCAGAGGAGGAAGAAGTAATCCAATACGTTGTTGGCACACTCCAACCGAAAT
TAAAACGTTTCCTTTCCCACCCGTATCCGAAAACTTTGGAACAGTTAATCCAACGTGGGAAAGAGGTGGAAGG
CAACCTCGATAACTCTGAGGAGCCTAGCCCGCAACGGAGTCCAAAGCACCAATTGGGTGGTAGCGTCGAGAGC
CTCCCACCTTCGTCGACCGCAAGTCCTGTTGCGTCAGACGAGACTCACCCAGACGTGAGCGCACCTCCGGTAA
CGGTGATT SEQ ID NO: 37
GGGGACGGCGAGACTCAAGCTGAGAATCCATCTACCAGCTTGAACAACACTGACGAAGATATCTTGGAACAGC
TCAAGAAAATTGTCATGGATCAACAACACCTGTATCAGAAAGAATTAAAGGCATCTTTTGAACAACTCAGTCG
CAAAATGTTTTCCCAGATGGAACAAATGAATAGCAAGCAAACGGATCTGCTTTTAGAACATCAAAAACAGACT
GTCAAACATGTAGACAAGCGCGTGGAGTATTTGCGGGCGCAATTCGATGCATCGTTAGGCTGGCGGTTGAAAG
AGCAACACGCGGATATTACGACCAAAATCATTCCTGAGATCATCCAAACGGTGAAGGAAGATATTAGCCTGTG
TCTTTCTACGCTCTGCAGTATCGCTGAAGATATCCAGACATCACGGGCTACCACTGTCACAGGGCATGCTGCC
GTACAAACCCATCCTGTGGATCTTTTGGGTGAACACCATTTAGGGACCACGGGGCACCCACGCTTACAGTCGA
CCCGTGTAGGGAAACCAGACGACGTACCTGAGTCGCCGGTAAGCCTGTTTATGCAAGGTGAGGCGCGTTCCCG
GATCGTTGGCAAGAGTCCGATTAAACTGCAATTTCCGACGTTCGGCAAAGCAAACGATTCTTCCGACCCACTC
CAATATCTGGAGCGGTGTGAGGACTTTCTTGCTCTTAACCCTTTAACTGATGAGGAACTTATGGCTACTTTGC
GGAATGTGTTACATGGCACCTCTCGGGATTGGTGGGATGTCGCACGTCATAAAATCCAAACTTGGCGTGAGTT
TAATAAACACTTCCGGGCGGCTTTCCTCAGCGAGGATTATGAAGATGAGTTGGCTGAGCGCGTCCGTAACCGC
ATCCAAAAAGAAGATGAGTCTATCCGCGATTTCGCTTATATGTATCAGTCCTTGTGCAAGCGGTGGAACCCTG
CTATCTGCGAAGGTGATGTAGTAAAGCTCATCCTGAAGAACATCAATCCACAACTGCCGTCTCAGTTACGCTC
CCGGGTCACGACCGTGGATGAGCTTGTTCGCTTGGGCCAGCAGCTTGAAAAAGATCGTCAGAATCAGCTCCAA
TATGAGCTTCGGAAGAGTTCCGGCAAAATTATCCAAAAATCTAGTTCGTGCGAAACTTCAGCGCTCCCGAACA
CGAAGAGTACACCTAATCAACAAAACCCTGCTACCAGTAACCGTCCTCCACAGGTGTATTGCTGGCGGTGTAA
GGGTCACCATGCCCCTGCCTCTTGTCCGCAATGGAAAGCTGATAAGCACCGTGCGCAACCTTCGCGGAGTTCT
GGGCCACAAACTCTGACTAATCTCCAAGCTCAAGACATC SEQ ID NO: 38
GGGGAATTGGATCAACGTGCGGCAGGGGGCTTGCGCGCGTACCCGGCGCCGCGTGGTGGTCCAGTTGCCAAAC
CGAGCGTAATTCTTCAGATTGGTAAGTGCCGCGCTGAGATGCTGGAACACGTCCGCCGCACGCATCGCCATCT
TCTGACGGAGGTAAGTAAACAAGTGGAGCGCGAACTCAAGGGGTTACATCGGTCTGTCGGTAAGTTGGAGGGC
AATTTAGACGGCTATGTGCCTACCGGTGATTCCCAACGCTGGAAAAAAAGTATCAAGGCGTGTCTCTGCCGGT
GTCAGGAAACAATTGCAAATCTCGAGCGTTGGGTGAAACGTGAGATGCATGTTTGGCGTGAGGTATTCTATCG
TTTGGAACGGTGGGCAGACCGTTTGGAGTCTATGGGGGGCAAGTATCCGGTGGGCACTAACCCGTCGCGGCAC
ACAGTAAGTGTCGGGGTAGGGGGCCCGGAAGGCTATTCTCATGAAGCGGATACTTATGACTACACGGTGTCTC
CGTATGCTATCACGCCACCGCCTGCCGCGGGTGAGTTGCCTGGTCAAGAGGCTGTCGAGGCACAACAGTACCC
TCCATGGGGTCTGGGGGAGGACGGGCAACCAGGTCCGGGCGTGGACACGCAGATTTTTGAGGACCCTCGCGAA
TTTTTGAGCCACTTAGAGGAGTACCTGCGGCAAGTAGGGGGGAGTGAAGAGTACTGGTTATCGCAAATTCAAA
ATCATATGAATGGCCCTGCGAAGAAATGGTGGGAGTTCAAACAGGGGTCAGTCAAGAATTGGGTCGAGTTTAA
GAAAGAATTTTTGCAATACAGTGAGGGTACGTTGAGTCGCGAGGCCATCCAACGTGAACTGGACCTCCCTCAG
AAGCAGGGGGAGCCGTTAGATCAATTTTTATGGCGGAAACGTGACTTATACCAAACCCTCTACGTTGACGCTG
AGGAAGAAGAAATTATTCAATATGTTGTCGGTACGCTGCAGCCAAAGCTGAAGCGGTTCCTCCGTCCTCCACT
CCCTAAAACCTTAGAACAATTAATCCAAAAAGGCATGGAAGTTCAGGACGGGTTAGAACAAGCGGCCGAACCG
GCCTCTCCGCGTCTGCCGCCGGAAGAGGAGAGTGAGGCTCTTACGCCTGCGCTCACGAGCGAATCAGTAGCCT
CCGATCGGACACAGCCAGAG SEQ ID NO: 39
GGGCAGCTTGACAATGTGACGAACGCGGGGATTCACAGCTTTCAAGGGCACCGCGGCGTCGCCAACAAACCGA
ATGTCATTCTGCAAATCGGTAAATGTCGTGCTGAAATGCTTGAGCACGTTCGTCGTACCCATCGTCACTTGCT
TTCTGAAGTATCAAAACAAGTGGAGCGGGAACTCAAAGGCCTGCAAAAGTCAGTGGGTAAATTGGAGAATAAC
CTCGAAGACCATGTACCTACAGACAACCAGCGGTGGAAAAAATCTATCAAGGCATGCCTCGCTCGTTGCCAGG
AGACTATTGCCCATCTTGAGCGGTGGGTGAAACGTGAAATGAACGTATGGAAGGAAGTATTTTTTCGCTTAGA
GAAGTGGGCTGATCGTCTTGAATCGATGGGCGGCAAGTACTGTCCTGGGGAACACGGCAAACAAACTGTATCT
GTCGGCGTGGGGGGCCCGGAGATCCGGCCATCGGAAGGGGAAATTTATGATTATGCTCTCGACATGTCCCAAA
TGTATGCTCTCACACCAGGGCCAGGGGAAGTACCGTCAATTCCGCAAGCACACGACAGCTACCAATGGGTATC
TGTGAGCGAGGACGCGCCTGCCTCTCCGGTTGAGACGCAAATCTTTGAGGACCCACATGAATTTTTGTCTCAT
CTTGAAGAATATCTCAAACAGGTTGGCGGCACAGAAGAATACTGGTTATCTCAGATCCAGAATCACATGAACG
GCCCGGCTAAAAAGTGGTGGGAGTATAAGCAAGATTCCGTAAAGAACTGGGTCGAATTCAAGAAAGAGTTTCT
TCAATACTCTGAGGGTACTCTGACGCGCGATGCAATTAAGCGGGAGTTAGACCTTCCACAAAAAGAGGGGGAG
CCTCTTGACCAGTTCCTGTGGCGTAAGCGCGACCTCTATCAGACACTTTACGTCGACGCTGATGAAGAAGAGA
TTATTCAATATGTTGTGGGTACCCTGCAGCCAAAGCTTAAGCGTTTCCTTAGCTACCCACTTCCGAAAACTCT
GGAGCAGCTCATTCAACGCGGTAAGGAAGTGCAGGGCAACATGGACCACTCTGAAGAGCCTAGCCCGCAGCGC
ACTCCTGAAATCCAATCAGGTGACAGTGTGGAGTCAATGCCGCCGTCAACCACCGCTTCTCCGGTACCTAGCA
ACGGGACGCAACCAGAGCCTCCAAGCCCACCGGCTACAGTCATC SEQ ID NO: 40
GGGCAACTTGAGAATATTAACCAAGGTTCCCTGCACGCGTTTCAGGGTCATCGCGGCGTGGTCCATAACAACA
AGCCTAACGTTATTCTCCAGATCGGGAAGTGCCGCGCCGAAATGCTGGAGCATGTGCGGCGCACCCATCGCCA
TTTGCTCACTGAAGTATCAAAACAGGTGGAGCGTGAGTTGAAGGGGTTGCAGAAAAGTGTAGGCAAACTTGAA
AATAATTTAGAAGACCACGTACCAAGTGCGGCTGAGAACCAACGCTGGAAGAAGTCGATTAAAGCCTGCTTAG
CGCGTTGTCAGGAGACCATTGCGAACTTGGAACGCTGGGTTAAACGTGAGATGAATGTTTGGAAGGAGGTCTT
TTTCCGCTTAGAGCGCTGGGCAGATCGCCTCGAATCCGGGGGTGGCAAGTACTGCCATGCAGACCAGGGTCGC
CAAACTGTCAGCGTAGGTGTTGGTGGTCCTGAAGTGCGTCCGTCTGAAGGTGAAATTTACGATTACGCGTTGG
ATATGAGCCAAATGTACGCCTTGACTCCGCCGCCTATGGGTGATGTTCCAGTAATTCCTCAGCCGCATGACAG
TTATCAGTGGGTGACAGATCCGGAAGAAGCGCCACCAAGTCCGGTTGAGACACAAATTTTCGAGGACCCTCGG
GAGTTTCTGACCCATCTTGAGGATTATTTAAAACAAGTCGGCGGGACAGAGGAATATTGGCTCTCACAGATCC
AAAATCATATGAATGGGCCAGCGAAAAAGTGGTGGGAATATAAACAGGATAGTGTGAAGAACTGGCTTGAGTT
CAAAAAAGAATTCTTGCAGTACTCAGAAGGCACGTTAACGCGGGACGCTATTAAACAGGAACTTGACCTTCCA
CAAAAAGAAGGGGAACCGCTGGATCAATTCCTCTGGCGCAAACGCGATTTGTACCAAACTCTCTACGTCGAGG
CAGAAGAAGAGGAGGTCATCCAATATGTAGTTGGCACACTGCAACCAAAACTGAAGCGGTTTCTTTCTCATCC
GTACCCTAAAACCCTGGAGCAACTCATCCAGCGCGGGAAGGAAGTTGAGGGGAATTTGGACAATAGTGAAGAA
CCGTCTCCACAGCGGACCCCAGAACATCAGCTGGGGGACAGTGTGGAATCTTTGCCGCCTAGTACTACGGCTT
CGCCTGCCGGTTCGGATAAAACGCAACCTGAGATTAGCTTACCTCCAACTACAGTCATT SEQ ID
NO: 41
GGGCAATTAGATTCGGTAACCAATGCGGGCGTCCACACCTACCAGGGCCATCGGAGCGTCGCCAATAAACCTA
ACGTCATTCTTCAAATCGGGAAATGTCGGACTGAGATGCTGGAGCATGTCCGTCGGACTCATCGCCACCTGCT
CACAGAAGTGTCAAAGCAAGTGGAACGTGAACTCAAGGGCTTACAGAAGAGCGTGGGCAAACTGGAAAACAAT
CTTGAAGACCATGTCCCAACTGACAATCAGCGGTGGAAGAAGTCAATCAAGGCATGTCTCGCGCGTTGCCAAG
AGACCATTGCTCACCTTGAGCGGTGGGTGAAACGTGAAATGAACGTGTGGAAGGAGGTGTTCTTCCGGTTAGA
ACGCTGGGCCGACCGCCTTGAATCAATGGGTGGTAAATACTGCCCGACGGACTCTGCACGTCAGACAGTTAGC
GTTGGGGTGGGGGGCCCGGAAATTCGGCCTAGTGAAGGCGAAATCTATGACTACGCGCTCGATATGAGCCAAA
TGTACGCTCTTACGCCGTCACCGGGCGAATTGCCGTCCGTCCCTCAACCGCATGATTCATACCAGTGGGTCAC
TAGTCCGGAAGACGCTCCGGCGTCACCAGTTGAAACGCAGGTATTCGAGGATCCTCGGGAGTTCTTGTGTCAT
TTGGAAGAGTACCTGAAGCAGGTTGGCGGTACAGAGGAATATTGGCTGAGCCAGATTCAGAATCATATGAATG
GTCCTGCAAAAAAGTGGTGGGAATATAAACAAGACACGGTTAAGAATTGGGTGGAATTCAAGAAGGAGTTCTT
ACAATACAGTGAGGGTACACTTACCCGTGATGCGATTAAGCGGGAATTAGACCTCCCGCAAAAGGACGGTGAG
CCTCTGGATCAATTTTTATGGCGTAAGCGTGACCTCTATCAGACATTATACATTGATGCCGATGAAGAACAGA
TCATTCAGTACGTCGTGGGGACATTGCAACCTAAACTCAAGCGGTTCTTGTCCTATCCACTTCCAAAAACTCT
TGAACAATTAATCCAGAAAGGGAAGGAGGTGCAGGGTTCACTTGACCACAGCGAGGAGCCGAGTCCTCAACGT
GCGAGCGAGGCTCGGACGGGCGATAGTGTGGAAACCTTGCCGCCTTCTACCACTACATCACCAAATACGTCAT
CTGGTACACAGCCAGAGGCACCATCGCCTCCAGCGACGGTAATC SEQ ID NO: 42
GGGCAGTTAGACAGTGTGACTAACGCCGGGGTGCATACGTACCAGGGGCACCGCGGGGTCGCCAATAAGCCAA
ATGTAATTCTCCAGATTGGGAAGTGTCGTACAGAGATGTTGGAACATGTCCGTCGCACTCATCGCCACTTGCT
CACCGAGGTCTCCAAACAAGTAGAACGCGAACTCAAGGGGCTCCAGAAGAGTGTTGGGAAGTTGGAGAATAAC
CTCGAAGACCACGTTCCGACAGATAACCAACGGTGGAAAAAGTCTATTAAAGCCTGTCTCGCCCGTTGTCAAG
AGACAATCGCACACTTGGAACGCTGGGTCAAACGGGAGATGAATGTGTGGAAGGAAGTCTTCTTCCGTCTCGA
GCGGTGGGCGGATCGTTTAGAAAGTATGGGCGGTAAATATTGCCCAACTGACTCGGCTCGTCAAACGGTGTCG
GTTGGCGTAGGCGGCCCGGAAATTCGCCCTAGCGAGGGTGAGATCTATGACTATGCACTTGACATGAGTCAGA
TGTATGCGTTAACTCCGTCGCCAGGGGAGCTTCCAAGTATTCCACAGCCTCACGATAGTTATCAATGGGTAAC
TTCTCCTGAAGACGCCCCAGCATCCCCAGTTGAGACACAAGTATTCGAGGACCCTCGTGAGTTTCTCTGTCAC
CTCGAGGAGTACCTTAAACAGGTAGGCGGGACCGAAGAGTACTGGTTATCGCAAATCCAAAACCATATGAATG
GTCCTGCCAAAAAGTGGTGGGAGTATAAACAAGATACTGTGAAGAATTGGGTAGAGTTCAAGAAAGAGTTCTT
ACAGTACTCTGAGGGGACGTTAACTCGTGATGCGATCAAGCGCGAATTGGATTTACCTCAGAAGGACGGCGAG
CCACTCGACCAGTTCTTATGGCGCAAGCGTGACTTGTATCAAACCCTTTATATCGATGCTGACGAGGAACAAA
TTATCCAGTACGTAGTCGGTACGTTGCAACCAAAACTTAAACGCTTTCTGAGCTACCCATTACCTAAAACGTT
GGAGCAACTGATCCAGAAAGGTAAAGAGGTGCAAGGGAGCCTGGATCATAGTGAAGAACCGAGCCCTCAGCGG
GCTTCTGAAGCTCGGACCGGTGATAGCGTCGAATCTTTACCACCTAGTACCACAACCAGCCCGAATGCGTCAT
CTGGTACCCAACCTGAAGCGCCTTCCCCACCTGCTACAGTCATT SEQ ID NO: 43
GGGCAGCTCGAGAATGTCAACCATGGGAACCTCCATTCTTTTCAAGGTCATCGCGGCGGCGTCGCCAACAAGC
CAAACGTTATCTTGCAGATCGGTAAATGTCGTGCAGAGATGCTGGACCACGTCCGGCGGACCCACCGGCATTT
ACTGACAGAGGTATCGAAACAGGTTGAACGTGAGTTGAAGGGGTTACAGAAATCAGTAGGGAAATTAGAAAAT
AACTTAGAAGACCATGTCCCTTCAGCCGTTGAAAACCAGCGTTGGAAAAAATCGATCAAGGCCTGCCTTTCCC
GCTGCCAAGAGACCATTGCCCACCTTGAGCGTTGGGTGAAGCGCGAGATGAACGTATGGAAAGAGGTTTTCTT
CCGCTTAGAGCGGTGGGCAGATCGGTTGGAATCTGGGGGCGGGAAATATTGTCACGGTGATAATCATCGTCAA
ACAGTATCAGTCGGTGTTGGCGGCCCTGAGGTACGTCCATCTGAAGGCGAAATTTACGATTACGCTCTCGACA
TGTCGCAAATGTACGCTTTAACACCGCCTAGCCCAGGGGATGTGCCTGTAGTTAGCCAGCCGCACGACAGCTA
TCAGTGGGTTACGGTTCCGGAGGATACCCCTCCATCCCCGGTGGAGACGCAAATCTTCGAGGACCCACGGGAG
TTCTTGACCCACTTAGAGGATTACTTAAAGCAAGTGGGGGGTACAGAGGAATATTGGTTATCTCAGATCCAGA
ATCACATGAACGGGCCAGCCAAGAAGTGGTGGGAGTATAAGCAAGACTCAGTAAAAAATTGGCTCGAGTTTAA
GAAGGAATTCCTTCAGTATTCCGAGGGGACACTTACGCGCGACGCTATCAAGGAAGAACTTGACCTCCCGCAA
AAGGACGGGGAACCTCTTGATCAGTTCCTGTGGCGCAAGCGCGACTTGTACCAGACCCTGTACGTGGAGGCGG
ATGAGGAGGAGGTGATCCAGTATGTTGTGGGGACTTTACAACCTAAATTAAAGCGTTTTCTCTCACACCCTTA
CCCGAAAACGTTAGAGCAACTTATCCAACGGGGCAAAGAGGTGGAAGGGAACCTCGACAATTCAGAGGAACCA
ACACCTCAGCGTACTCCAGAACACCAACTGTGTGGTTCTGTAGAATCGCTGCCTCCTTCCTCTACCGTCAGTC
CAGTGGCTAGCGATGGTACTCAACCTGAGACTTCGCCATTGCCAGCGACTGTTATT SEQ ID NO:
44
GGGCCATTGACGTTGTTACAAGACTGGTGTCGTGGTGAACATTTAAACACCCGCCGGTGCATGTTGATCCTCG
GTATCCCAGAAGATTGCGGCGAGGATGAGTTCGAAGAGACACTTCAGGAGGCGTGTCGCCATTTAGGGCGGTA
CCGCGTGATCGGCCGCATGTTCCGTCGTGAGGAAAATGCCCAAGCGATCCTCTTGGAATTGGCGCAGGATATT
GACTATGCCTTACTCCCTCGGGAAATCCCTGGGAAAGGCGGGCCTTGGGAGGTAATTGTGAAGCCGCGTAATT
CCGACGGCGAATTCTTAAATCGGCTTAATCGCTTTCTTGAAGAGGAGCGCCGTACGGTCTCCGATATGAACCG
TGTTTTGGGCTCGGATACTAACTGTTCAGCTCCTCGTGTCACCATTAGTCCTGAATTCTGGACTTGGGCACAG
ACGCTGGGCGCAGCTGTCCAACCATTGCTCGAACAGATGCTCTACCGGGAGTTACGGGTCTTCAGTGGCAATA
CGATTTCCATCCCAGGTGCTCTCGCTTTTGACGCGTGGCTGGAGCATACCACGGAAATGCTTCAAATGTGGCA
GGTGCCTGAAGGGGAGAAACGGCGGCGCTTGATGGAGTGTTTGCGGGGGCCAGCCCTGCAAGTCGTTAGTGGG
TTACGTGCATCGAATGCCAGTATCACTGTCGAAGAGTGTCTTGCTGCACTGCAGCAGGTATTCGGTCCAGTGG
AAAGTCATAAGATTGCCCAAGTAAAGTTATGCAAAGCTTACCAGGAGGCTGGGGAAAAAGTAAGCAGCTTCGT
TTTGCGTTTGGAGCCACTGCTTCAGCGTGCTGTAGAAAACAACGTGGTCAGTCGCCGCAATGTCAACCAAACA
CGTCTTAAGCGTGTTCTGTCGGGCGCCACCCTTCCTGACAAGCTGCGTGATAAATTGAAGTTAATGAAACAGC
GCCGTAAACCGCCGGGTTTCTTGGCGTTGGTTAAACTGTTACGTGAAGAGGAGGAGTGGGAGGCCACCTTAGG
GCCAGACCGCGAGTCATTGGAGGGGTTAGAAGTGGCACCGCGCCCGCCAGCACGGATTACGGGTGTTGGCGCA
GTACCTCTTCCGGCATCCGGGAATTCATTTGATGCCCGTCCTTCGCAAGGGTACCGGCGCCGTCGGGGTCGTG
GTCAGCACCGTCGGGGCGGCGTTGCTCGTGCAGGCTCTCGTGGCTCTCGTAAGCGGAAACGGCACACCTTCTG
CTATTCCTGTGGTGAGGATGGCCATATTCGTGTCCAATGCATTAACCCTAGCAATCTCCTGTTGGCTAAGGAG
ACCAAAGAGATTTTGGAAGGGGGAGAACGTGAAGCGCAAACGAATTCACGT SEQ ID NO: 45
GGGGCTCTTACGCTCTTAGAAGACTGGTGTAAGGGTATGGACATGGACCCGCGGAAGGCTCTCCTGATTGTAG
GTATTCCGATGGAATGCAGTGAGGTGGAAATCCAGGATACAGTTAAAGCTGGTCTTCAACCTCTGTGCGCTTA
TCGTGTACTCGGCCGTATGTTCCGGCGGGAGGATAATGCGAAGGCTGTTTTCATTGAGCTGGCAGACACCGTG
AATTACACCACGTTACCGTCTCACATTCCGGGTAAAGGGGGTTCCTGGGAAGTCGTTGTTAAACCTCGGAACC
CTGACGACGAGTTCCTTTCTCGGCTTAACTACTTCTTGAAAGATGAGGGCCGCTCGATGACGGATGTCGCCCG
GGCACTGGGGTGCTGTAGCTTACCTGCGGAATCACTGGACGCGGAAGTAATGCCACAGGTCCGCTCCCCACCA
TTAGAACCTCCAAAAGAGAGTATGTGGTACCGTAAGTTAAAAGTGTTTAGTGGTACCGCGTCGCCTTCGCCGG
GGGAGGAGACATTTGAGGACTGGTTAGAGCAAGTCACCGAGATCATGCCTATCTGGCAAGTATCTGAAGTTGA
AAAGCGCCGTCGGTTACTGGAGTCACTCCGGGGCCCGGCACTCTCAATTATGCGCGTGTTACAAGCCAATAAC
GATAGCATTACCGTTGAACAGTGTTTGGATGCATTAAAGCAGATCTTTGGCGACAAGGAAGACTTCCGTGCCT
CTCAATTTCGTTTTCTTCAAACGTCCCCTAAAATTGGGGAGAAGGTGAGTACGTTCCTGCTGCGTTTAGAGCC
ACTCTTGCAAAAGGCCGTTCACAAGAGCCCACTTTCGGTACGTAGTACTGATATGATTCGGTTAAAGCACCTG
TTGGCACGCGTAGCCATGACCCCGGCACTGCGTGGTAAACTCGAATTACTCGACCAACGCGGGTGCCCACCTA
ATTTTCTTGAGCTGATGAAGCTGATCCGGGATGAGGAAGAGTGGGAGAATACTGAAGCTGTGATGAAAAATAA
AGAGAAACCTTCAGGTCGTGGCCGCGGTGCATCAGGCCGTCAAGCTCGCGCCGAGGCCAGTGTAAGTGCTCCG
CAAGCAACAGTCCAAGCACGTAGCTTCTCTGATTCTAGCCCGCAGACGATTCAGGGGGGCTTACCACCTCTTG
TCAAGCGTCGGCGCCTTTTGGGTTCGGAGAGCACACGTGGGGAAGACCACGGGCAAGCTACTTATCCGAAAGC
AGAGAATCAGACTCCAGGGCGTGAGGGCCCGCAGGCGGCTGGGGAGGAACTTGGTAATGAGGCCGGGGCCGGC
GCGATGTCCCACCCGAAACCGTGGGAAACC SEQ ID NO: 46
GGGGCTGTGACAATGCTCCAGGACTGGTGCCGTTGGATGGGCGTGAACGCTCGGCGGGGGCTGTTAATCTTAG
GTATCCCTGAAGACTGTGACGATGCAGAGTTCCAAGAGTCGTTAGAAGCTGCACTCCGTCCTATGGGTCACTT
TACTGTACTCGGTAAGGCCTTCCGCGAGGAAGACAACGCTACCGCTGCGCTGGTGGAATTAGATCGCGAGGTT
AATTACGCACTTGTTCCACGCGAAATTCCGGGCACCGGCGGGCCTTGGAACGTCGTGTTCGTTCCTCGGTGCT
CCGGCGAGGAATTCCTGGGGTTAGGCCGCGTGTTCCACTTTCCTGAACAGGAGGGCCAAATGGTAGAATCGGT
TGCGGGGGCACTGGGGGTAGGTCTGCGCCGCGTGTGTTGGTTACGCTCGATCGGGCAAGCTGTACAACCATGG
GTAGAAGCTGTTCGCTGCCAAAGCTTAGGGGTATTTAGTGGTCGTGATCAACCTGCACCTGGTGAAGAAAGCT
TCGAGGTCTGGTTGGATCATACGACCGAGATGTTGCATGTGTGGCAAGGCGTGTCGGAACGGGAACGGCGCCG
TCGTCTGCTGGAAGGGCTGCGTGGCACAGCCTTACAACTTGTACATGCCTTACTGGCAGAAAATCCGGCACGG
ACAGCACAAGATTGCTTGGCTGCATTAGCCCAAGTTTTTGGTGATAACGAAAGCCAGGCAACGATTCGTGTTA
AATGTTTGACAGCCCAACAGCAGAGTGGCGAACGCCTCTCTGCGTTCGTTCTCCGCTTAGAAGTACTTCTGCA
AAAGGCTATGGAGAAGGAAGCATTGGCGCGCGCGTCAGCGGATCGGGTGCGTCTTCGTCAGATGCTGACACGC
GCACATCTCACAGAGCCGTTGGATGAAGCCTTACGGAAATTGCGTATGGCAGGGCGTTCTCCGTCTTTTTTGG
AAATGCTCGGCTTAGTACGCGAGTCAGAGGCCTGGGAGGCAAGTCTGGCTCGGTCCGTCCGGGCGCAAACCCA
GGAGGGTGCAGGGGCCCGGGCGGGGGCCCAAGCAGTTGCGCGTGCCAGCACTAAGGTTGAAGCTGTACCTGGT
GGCCCTGGCCGGGAGCCAGAAGGTCTCCTCCAAGCCGGGGGCCAAGAAGCGGAAGAACTTCTCCAAGAGGGCT
TAAAGCCGGTTTTAGAGGAATGTGACAAT SEQ ID NO: 47
GGGGCGGTCACCATGTTGCAAGACTGGTGTCGGTGGATGGGCGTGAATGCTCGGCGGGGTTTATTGATCTTGG
GTATCCCAGAAGACTGTGACGACGCCGAGTTTCAGGAGTCGCTCGAGGCCGCCCTTCGTCCAATGGGGCATTT
TACGGTTCTGGGCAAGGTGTTCCGTGAAGAGGATAACGCTACAGCAGCTCTTGTGGAGCTTGACCGTGAGGTG
AATTATGCGTTAGTACCTCGCGAGATTCCAGGTACCGGTGGGCCATGGAACGTAGTCTTCGTCCCACGTTGCT
CGGGGGAGGAATTTCTGGGGCTTGGGCGCGTATTCCACTTTCCAGAACAGGAAGGGCAGATGGTCGAAAGCGT
AGCAGGCGCTCTTGGCGTTGGTCTCCGGCGCGTGTGCTGGTTACGCTCCATCGGCCAAGCAGTCCAACCATGG
GTTGAAGCCGTACGCTATCAATCTTTAGGTGTCTTCTCAGGCCGTGACCAGCCGGCGCCTGGTGAGGAATCCT
TCGAAGTCTGGCTCGATCATACAACTGAGATGCTGCATGTATGGCAAGGTGTCTCAGAGCGGGAACGGCGGCG
GCGGTTATTAGAGGGGCTCCGTGGGACTGCGCTCCAATTAGTACATGCGCTTTTGGCCGAAAATCCAGCCCGT
ACTGCCCAAGATTGTCTGGCAGCACTCGCCCAAGTATTCGGCGACAACGAATCGCAGGCAACAATCCGCGTAA
AGTGTCTTACAGCACAGCAGCAGTCAGGGGAACGTCTTAGTGCGTTCGTTCTGCGGCTGGAAGTGTTACTCCA
GAAAGCCATGGAAAAGGAGGCATTGGCTCGCGCGAGCGCTGACCGTGTACGTCTGCGGCAAATGCTTACTCGC
GCACATCTCACCGAGCCTCTCGATGAAGCACTGCGGAAACTGCGCATGGCAGGCCGCAGCCCGTCTTTCCTGG
AAATGTTAGGCTTAGTCCGGGAGTCCGAAGCCTGGGAGGCCAGTCTGGCACGGTCAGTGCGGGCACAAACGCA
AGAGGGTGCAGGGGCACGGGCGGGTGCACAAGCAGTTGCACGTGCCTCCACTAAAGTTGAGGCAGTGCCGGGT
GGGCCAGGCCGTGAACCGGAGGGTTTGCGCCAAGCCGGCGGGCAGGAAGCCGAAGAATTACTCCAAGAAGGTT
TAAAACCGGTTTTGGAGGAATGCGATAAC SEQ ID NO: 48
GGGGTGGAAGATTTGGCGGCATCTTACATCGTATTAAAGCTTGAGAACGAAATCCGGCAGGCGCAGGTCCAAT
GGTTAATGGAGGAAAACGCCGCCCTGCAGGCCCAGATCCCTGAACTTCAAAAGTCGCAAGCCGCGAAGGAGTA
TGATCTTCTGCGTAAATCTTCGGAGGCGAAGGAGCCGCAAAAACTGCCAGAACATATGAATCCACCGGCCGCT
TGGGAAGCACAAAAGACTCCAGAGTTTAAGGAACCACAGAAACCTCCTGAACCACAGGATTTGCTTCCTTGGG
AGCCGCCTGCTGCCTGGGAGTTGCAAGAAGCACCGGCTGCCCCTGAGTCACTGGCTCCGCCTGCAACCCGTGA
GTCTCAGAAACCACCTATGGCGCATGAAATCCCTACTGTATTGGAGGGGCAAGGGCCTGCCAACACACAAGAC
GCTACGATTGCTCAAGAACCAAAGAATAGCGAGCCGCAAGACCCTCCAAATATCGAGAAACCTCAGGAAGCTC
CGGAATATCAAGAAACAGCGGCACAGTTGGAGTTTTTAGAACTTCCTCCACCTCAGGAGCCACTCGAACCGAG
CAATGCGCAAGAATTTCTCGAGTTGTCGGCTGCCCAGGAGTCCTTAGAAGGCCTCATTGTAGTTGAAACGTCC
GCGGCTTCGGAGTTCCCACAGGCTCCTATCGGGCTTGAAGCCACCGACTTTCCGCTGCAGTACACGCTTACCT
TCTCTGGCGACAGCCAGAAGTTGCCAGAATTTTTGGTCCAACTCTACAGTTATATGCGGGTACGTGGGCACTT
ATACCCTACCGAGGCGGCGTTAGTGTCGTTTGTAGGCAATTGTTTCTCAGGGCGCGCGGGCTGGTGGTTTCAG
TTGCTTTTGGATATCCAGTCGCCTCTGTTAGAACAGTGTGAAAGTTTTATCCCGGTTCTCCAAGACACATTTG
ACAATCCGGAAAACATGAAGGACGCAAACCAATGCATCCACCAGCTTTGTCAGGGCGAGGGTCATGTGGCCAC
ACACTTCCACCTCATTGCACAAGAGCTTAATTGGGATGAAAGCACGCTGTGGATCCAGTTCCAGGAAGGCCTG
GCCTCATCCATCCAGGATGAACTTTCCCATACATCGCCTGCTACCAACCTGAGTGATCTGATTACTCAATGCA
TCTCATTAGAGGAAAAGCCTGACCCAAACCCGTTAGGGAAGTCCTCCTCGGCGGAGGGGGATGGCCCGGAAAG
TCCGCCAGCAGAAAACCAACCTATGCAAGCTGCGATCAATTGTCCTCACATTTCCGAAGCAGAGTGGGTTCGT
TGGCACAAAGGCCGGCTTTGTCTCTATTGCGGCTATCCGGGTCACTTCGCACGTGATTGCCCAGTGAAGCCAC
ACCAGGCGTTACAGGCAGGGAACATTCAGGCTTGCCAA SEQ ID NO: 49
GGGGTGCAGCCGCAGACTAGCAAAGCTGAATCGCCGGCTCTCGCTGCCTCACCGAACGCACAAATGGATGACG
TTATTGATACATTAACCTCCCTGCGTCTGACGAATTCGGCTCTGCGGCGGGAGGCTAGCACTCTTCGGGCCGA
GAAAGCAAATTTAACTAATATGCTCGAGTCAGTGATGGCCGAGTTAACGCTGTTACGGACCCGTGCGCGGATT
CCGGGGGCCCTGCAGATTACGCCACCAATTTCGTCTATTACTAGCAACGGTACTCGCCCGATGACGACTCCTC
CAACTAGTTTACCTGAACCGTTTTCTGGCGATCCTGGCCGGTTAGCTGGTTTCCTTATGCAGATGGACCGTTT
TATGATCTTTCAAGCTAGCCGGTTTCCAGGGGAGGCAGAGCGTGTTGCGTTCCTGGTGTCGCGCTTAACTGGC
GAAGCAGAAAAATGGGCCATTCCTCACATGCAACCAGACTCTCCTTTGCGTAACAACTATCAAGGCTTCTTAG
CAGAGTTACGGCGGACCTATAAGAGCCCGTTGCGTCACGCCCGGCGGGCGCAAATCCGGAAGACATCGGCCTC
GAACCGGGCAGTCCGTGAACGCCAAATGCTTTGCCGGCAACTTGCATCAGCAGGTACAGGCCCATGCCCGGTA
CACCCTGCTAGTAACGGGACTTCCCCGGCACCGGCATTACCAGCACGGGCGCGTAACTTA SEQ ID
NO: 50
GGGGACGGTCGGGTACAGTTGATGAAGGCTTTATTGGCTGGCCCTTTACGTCCGGCGGCACGCCGTTGGCGGA
ATCCTATTCCATTTCCAGAGACTTTTGATGGGGATACTGATCGCCTCCCGGAGTTTATCGTCCAAACTTCGTC
CTACATGTTCGTTGACGAAAATACTTTCTCTAACGACGCTCTGAAAGTGACATTTCTCATTACCCGGCTGACA
GGTCCAGCCTTGCAATGGGTCATTCCGTACATTCGTAAAGAAAGCCCGCTTCTTAACGACTATCGGGGTTTCC
TGGCCGAGATGAAGCGGGTTTTTGGGTGGGAAGAGGACGAGGACTTT SEQ ID NO: 51
GGGGAAGGTCGGGTGCAACTTATGAAAGCGTTGCTTGCCCGCCCGCTTCGTCCAGCAGCACGTCGCTGGCGGA
ATCCAATTCCTTTCCCGGAGACTTTTGACGGGGACACCGATCGGCTCCCAGAGTTCATTGTGCAGACGTCAAG
CTATATGTTCGTGGATGAGAACACGTTCTCTAACGACGCGTTGAAAGTGACTTTCTTAATTACGCGTTTGACT
GGCCCGGCTTTACAATGGGTGATTCCATACATTAAGAAAGAGTCACCGCTTCTCAGTGATTATCGCGGTTTTT
TAGCCGAGATGAAGCGGGTCTTCGGGTGGGAAGAAGACGAAGACTTT SEQ ID NO: 52
GGGCCGCGTGGGCGTTGCCGTCAACAAGGTCCTCGGATTCCGATTTGGGCAGCGGCCAACTATGCCAACGCCC
ACCCGTGGCAACAAATGGATAAGGCTTCGCCAGGCGTTGCTTACACACCTTTGGTTGATCCTTGGATTGAGCG
GCCTTGTTGCGGTGACACGGTTTGTGTGCGCACCACAATGGAACAGAAGAGCACAGCGTCAGGCACTTGTGGT
GGTAAGCCTGCTGAGCGTGGTCCTCTCGCGGGGCATATGCCGAGCTCACGCCCACATCGGGTTGATTTCTGTT
GGGTTCCTGGTAGCGACCCAGGCACATTCGACGGCAGTCCATGGCTCTTAGATCGCTTTTTGGCGCAACTTGG
TGATTACATGAGTTTTCACTTTGAACACTACCAGGACAATATCAGCCGTGTCTGCGAGATTCTTCGTCGGTTA
ACGGGCCGCGCTCAGGCATGGGCTGCTCCTTACCTGGACGGGGACCTTCCACTGCCAGACGACTACGAATTGT
TTTGTCAAGACCTTAAGGAGGTAGTACAGGACCCTAACAGTTTCGCCGAGTATCACGCCGTGGTGACTTGTCC
ACTCCCTCTTGCTTCGTCCCAACTTCCTGTAGCTCCTCAGCTTCCGGTGGTACGCCAATACCTTGCGCGCTTC
TTGGAGGGCCTTGCTTTGGATATGGGTACGGCGCCTCGGTCACTCCCGGCCGCTATGGCCACACCGGCAGTCT
CCGGCTCGAACTCCGTTTCTCGTTCTGCCTTATTTGAACAACAACTCACAAAGGAATCCACTCCAGGCCCGAA
AGAGCCACCTGTTCTCCCTAGCTCGACTTGCTCTAGCAAACCGGGTCCTGTCGAACCAGCCAGTTCACAACCT
GAAGAGGCTGCTCCTACCCCGGTGCCGCGTTTGTCAGAGTCGGCTAACCCACCGGCTCAGCGTCCAGACCCTG
CTCACCCTGGTGGTCCTAAACCACAAAAAACCGAAGAGGAAGTTTTAGAAACTGAGGGGGACCAGGAAGTTAG
CCTGGGGACGCCGCAGGAGGTCGTAGAAGCGCCGGAAACACCAGGTGAACCACCGCTCAGCCCTGGGTTC
SEQ ID NO: 53
GGGGTTGATGAATTGGTGCTCTTGTTGCACGCGCTGTTAATGCGCCATCGGGCGCTTTCCATTGAAAATTCTC
AGTTGATGGAGCAACTTCGCTTGTTGGTCTGCGAACGGGCGAGCCTTCTTCGTCAGGTACGTCCGCCGAGCTG
TCCAGTGCCATTTCCTGAGACTTTTAACGGGGAGTCATCACGGTTACCTGAGTTCATCGTCCAAACCGCAAGC
TATATGTTAGTTAATGAAAATCGCTTTTGCAATGACGCAATGAAAGTCGCTTTTTTGATTAGCCTTCTTACTG
GTGAAGCAGAAGAATGGGTCGTCCCATACATTGAGATGGATTCACCAATTCTTGGGGACTACCGTGCGTTCTT
GGATGAGATGAAGCAGTGTTTTGGGTGGGACGATGATGAAGATGACGACGATGAGGAAGAGGAGGATGACTAT
SEQ ID NO: 54
GGGCCTGTGGATTTAGGTCAGGCTTTGGGGTTGTTGCCATCCCTCGCTAAGGCCGAAGATTCCCAATTTAGCG
AAAGCGATGCAGCTTTACAGGAGGAATTGTCTTCTCCGGAAACCGCACGGCAACTTTTTCGTCAATTTCGCTA
TCAAGTCATGTCGGGGCCTCATGAAACACTGAAACAGTTACGGAAGTTATGTTTTCAGTGGCTGCAACCTGAA
GTCCATACAAAGGAACAAATCCTCGAAATTCTGATGCTGGAACAGTTCTTGACCATTCTGCCTGGTGAAATTC
AGATGTGGGTCCGCAAGCAGTGCCCTGGTAGTGGGGAGGAGGCGGTTACGTTAGTAGAATCCCTGAAAGGTGA
TCCACAACGGCTCTGGCAATGGATCTCCATCCAAGTCCTGGGTCAGGATATCCTGTCTGAGAAAATGGAGTCA
CCTTCTTGCCAGGTGGGCGAAGTGGAGCCACACCTGGAAGTTGTACCTCAGGAACTGGGGTTAGAGAATTCAT
CTTCAGGGCCGGGGGAACTTCTTTCGCACATCGTGAAAGAGGAGTCTGACACTGAAGCAGAGTTGGCGTTAGC
GGCATCCCAGCCAGCTCGTTTGGAAGAACGGCTGATTCGGGATCAGGACCTTGGGGCGTCCCTCCTCCCGGCA
GCACCGCAGGAGCAATGGCGTCAATTAGACAGCACTCAAAAAGAACAATATTGGGACCTGATGCTGGAGACCT
ACGGCAAAATGGTATCCGGCGCGGGTATCTCACACCCGAAGTCCGATTTAACGAACTCAATTGAGTTCGGTGA
AGAGTTGGCAGGTATTTATTTACATGTAAACGAAAAGATTCCGCGGCCTACCTGCATTGGTGACCGCCAAGAA
AACGACAAAGAAAACCTTAATTTGGAAAACCATCGTGACCAGGAATTATTACATGCCAGCTGCCAGGCCTCGG
GCGAAGTGCCATCCCAGGCATCGTTACGTGGCTTCTTTACCGAGGACGAACCTGGTTGCTTCGGCGAAGGGGA
GAACCTTCCTGAGGCACTTCAGAATATCCAGGATGAGGGGACTGGCGAACAGCTGAGCCCGCAAGAACGCATT
AGTGAAAAACAGTTGGGTCAACATTTGCCAAATCCGCACTCGGGGGAGATGTCGACGATGTGGCTTGAAGAAA
AACGGGAGACCAGCCAGAAAGGCCAACCACGTGCACCAATGGCGCAGAAATTGCCAACGTGCCGCGAATGTGG
CAAAACGTTTTATCGCAATAGTCAACTTATCTTTCACCAACGCACACACACCGGTGAGACATATTTTCAATGC
ACCATCTGCAAAAAGGCGTTTCTCCGGTCATCTGATTTCGTGAAACATCAGCGGACTCATACTGGCGAAAAAC
CTTGTAAATGTGACTATTGTGGCAAGGGCTTTAGTGATTTTAGCGGGCTTCGGCATCACGAGAAGATCCATAC
CGGCGAGAAGCCATACAAGTGTCCAATCTGTGAGAAATCTTTCATCCAGCGCAGTAATTTTAACCGCCACCAA
CGGGTTCACACCGGTGAAAAGCCTTATAAATGCTCGCATTGTGGCAAGAGCTTCAGCTGGAGCTCCTCGCTCG
ATAAGCATCAACGTTCACATCTGGGGAAGAAGCCGTTCCAA SEQ ID NO: 55
GGGACTCTCCGCTTACTTGAGGATTGGTGTCGGGGGATGGACATGAACCCACGTAAGGCCCTTCTTATCGCCG
GGATTTCCCAGTCATGTTCAGTCGCCGAGATTGAAGAGGCGCTCCAAGCCGGGCTTGCTCCTTTAGGCGAGTA
TCGTCTCCTTGGGCGGATGTTTCGCCGCGATGAAAATCGCAAAGTAGCGTTGGTTGGTCTCACAGCTGAAACT
AGCCATGCGCTTGTACCTAAAGAAATTCCTGGTAAAGGCGGGATCTGGCGGGTTATTTTTAAACCACCGGACC
CGGACAATACGTTTCTTTCTCGTTTGAATGAGTTCCTCGCGGGCGAGGGGATGACGGTGGGGGAACTTAGTCG
TGCTCTTGGTCACGAAAATGGGTCATTAGACCCTGAACAGGGTATGATTCCGGAAATGTGGGCGCCGATGCTG
GCACAGGCTCTGGAGGCTCTCCAACCGGCTTTACAGTGCCTTAAGTACAAGAAGCTGCGCGTTTTTTCAGGGC
GCGAGTCTCCAGAGCCGGGTGAGGAGGAATTCGGCCGTTGGATGTTCCATACCACCCAGATGATCAAAGCGTG
GCAGGTGCCGGATGTCGAGAAACGCCGCCGGCTGTTGGAATCACTCCGCGGGCCGGCACTTGACGTTATTCGG
GTTCTGAAAATTAACAACCCGTTAATTACGGTAGATGAATGTTTGCAAGCACTTGAAGAGGTCTTTGGGGTGA
CTGACAATCCTCGGGAATTGCAAGTAAAATACTTAACGACCTACCATAAGGACGAGGAGAAATTATCAGCCTA
CGTACTGCGGCTGGAACCGCTGCTGCAGAAGCTCGTCCAGCGGGGGGCTATTGAACGGGACGCTGTTAATCAG
GCTCGCCTGGATCAGGTAATCGCTGGGGCGGTACATAAAACTATCCGCCGTGAGCTGAACCTGCCTGAAGACG
GGCCGGCGCCAGGCTTTCTTCAACTCCTCGTTTTGATTAAGGATTACGAGGCAGCTGAAGAGGAGGAAGCATT
ACTTCAGGCCATTCTTGAAGGGAACTTTACT SEQ ID NO: 56
GGGACAGAACGGCGTCGCGACGAATTAAGTGAAGAAATTAATAATCTTCGTGAAAAGGTTATGAAACAGAGTG
AGGAAAACAACAATCTTCAATCCCAAGTCCAGAAACTCACTGAGGAGAATACTACACTCCGTGAGCAAGTTGA
ACCTACACCTGAAGATGAAGATGACGACATTGAGTTGCGGGGCGCAGCAGCCGCAGCCGCGCCTCCGCCGCCG
ATCGAGGAGGAATGCCCGGAGGATTTACCGGAAAAATTTGATGGTAATCCGGACATGTTAGCGCCATTCATGG
CCCAGTGCCAAATTTTTATGGAAAAGTCTACGCGCGATTTTAGTGTAGATCGCGTACGTGTATGTTTTGTGAC
GAGCATGATGACTGGTCGCGCAGCCCGTTGGGCGTCAGCGAAATTGGAGCGGTCGCACTACCTGATGCATAAT
TACCCGGCGTTCATGATGGAGATGAAACACGTGTTTGAAGACCCGCAGCGGCGGGAGGTGGCCAAACGCAAGA
TCCGGCGGTTGCGGCAGGGCATGGGCAGCGTAATTGATTATAGTAATGCGTTTCAAATGATTGCGCAGGATCT
GGATTGGAATGAACCTGCTCTCATTGATCAATATCATGAAGGGCTTAGTGACCATATTCAAGAGGAACTCTCT
CACCTGGAAGTGGCTAAATCTCTCTCCGCCCTTATTGGCCAATGCATTCATATTGAGCGCCGTCTTGCACGTG
CTGCTGCCGCTCGGAAACCGCGTAGTCCACCACGGGCTTTAGTGCTCCCACATATCGCGTCACACCATCAAGT
AGATCCTACTGAGCCAGTGGGGGGTGCACGCATGCGCTTAACCCAAGAAGAAAAGGAACGTCGTCGTAAGCTG
AATTTATGCCTGTACTGCGGCACTGGTGGCCATTATGCCGATAACTGTCCTGCCAAAGCCAGTAAGTCAAGCC
CGGCTGGGAAACTTCCAGGTCCTGCCGTCGAGGGCCCTTCTGCTACCGGCCCAGAGATTATCCGCTCCCCGCA
AGACGATGCGTCGTCGCCTCATCTCCAGGTAATGCTCCAAATCCACCTCCCTGGCCGGCACACACTCTTTGTC
CGGGCGATGATTGACTCTGGGGCGTCTGGTAATTTTATTGATCACGAGTATGTTGCTCAAAATGGTATCCCTC
TCCGGATCAAAGACTGGCCTATTCTGGTTGAAGCCATCGATGGCCGTCCGATCGCGAGCGGTCCTGTGGTTCA
TGAAACGCATGACCTCATCGTTGATCTGGGTGACCACCGTGAAGTATTATCCTTTGATGTGACTCAGTCACCG
TTTTTTCCAGTTGTTTTGGGCGTCCGTTGGCTTTCGACTCACGATCCTAACATCACGTGGTCGACACGGTCGA
TTGTCTTCGATTCGGAATATTGTCGTTATCATTGCCGCATGTATTCACCAATTCCGCCGTCTCTCCCGCCGCC
TGCGCCGCAACCTCCTCTGTATTACCCGGTGGACGGTTACCGTGTTTACCAGCCAGTTCGCTACTACTACGTA
CAAAACGTGTACACGCCTGTTGATGAACACGTGTACCCAGATCACCGCCTGGTCGACCCTCATATTGAGATGA
TCCCGGGTGCGCACTCGATCCCATCGGGCCATGTTTATTCCTTGTCTGAGCCAGAAATGGCCGCCTTACGGGA
TTTTGTGGCCCGGAATGTCAAAGACGGCCTGATTACCCCGACAATTGCACCAAACGGTGCTCAGGTGTTGCAG
GTGAAGCGGGGCTGGAAGTTGCAAGTCAGCTATGATTGTCGTGCGCCAAACAACTTCACTATTCAGAACCAAT
ATCCACGTCTCAGCATCCCTAATCTCGAGGACCAGGCACATCTTGCAACATATACTGAATTTGTACCTCAGAT
TCCTGGCTATCAGACTTATCCTACGTATGCTGCCTACCCAACATACCCGGTAGGTTTCGCATGGTACCCAGTA
GGCCGGGACGGGCAGGGCCGCTCTTTATATGTTCCTGTCATGATTACATGGAACCCGCATTGGTACCGCCAGC
CTCCGGTCCCACAGTACCCACCTCCTCAACCTCCACCACCTCCGCCGCCTCCTCCACCGCCACCTTCTTACTC
GACATTA
Sequence CWU 1
1
851396PRTHomo sapiens 1Gly Glu Leu Asp His Arg Thr Ser Gly Gly Leu
His Ala Tyr Pro Gly1 5 10 15Pro Arg Gly Gly Gln Val Ala Lys Pro Asn
Val Ile Leu Gln Ile Gly 20 25 30Lys Cys Arg Ala Glu Met Leu Glu His
Val Arg Arg Thr His Arg His 35 40 45Leu Leu Ala Glu Val Ser Lys Gln
Val Glu Arg Glu Leu Lys Gly Leu 50 55 60His Arg Ser Val Gly Lys Leu
Glu Ser Asn Leu Asp Gly Tyr Val Pro65 70 75 80Thr Ser Asp Ser Gln
Arg Trp Lys Lys Ser Ile Lys Ala Cys Leu Cys 85 90 95Arg Cys Gln Glu
Thr Ile Ala Asn Leu Glu Arg Trp Val Lys Arg Glu 100 105 110Met His
Val Trp Arg Glu Val Phe Tyr Arg Leu Glu Arg Trp Ala Asp 115 120
125Arg Leu Glu Ser Thr Gly Gly Lys Tyr Pro Val Gly Ser Glu Ser Ala
130 135 140Arg His Thr Val Ser Val Gly Val Gly Gly Pro Glu Ser Tyr
Cys His145 150 155 160Glu Ala Asp Gly Tyr Asp Tyr Thr Val Ser Pro
Tyr Ala Ile Thr Pro 165 170 175Pro Pro Ala Ala Gly Glu Leu Pro Gly
Gln Glu Pro Ala Glu Ala Gln 180 185 190Gln Tyr Gln Pro Trp Val Pro
Gly Glu Asp Gly Gln Pro Ser Pro Gly 195 200 205Val Asp Thr Gln Ile
Phe Glu Asp Pro Arg Glu Phe Leu Ser His Leu 210 215 220Glu Glu Tyr
Leu Arg Gln Val Gly Gly Ser Glu Glu Tyr Trp Leu Ser225 230 235
240Gln Ile Gln Asn His Met Asn Gly Pro Ala Lys Lys Trp Trp Glu Phe
245 250 255Lys Gln Gly Ser Val Lys Asn Trp Val Glu Phe Lys Lys Glu
Phe Leu 260 265 270Gln Tyr Ser Glu Gly Thr Leu Ser Arg Glu Ala Ile
Gln Arg Glu Leu 275 280 285Asp Leu Pro Gln Lys Gln Gly Glu Pro Leu
Asp Gln Phe Leu Trp Arg 290 295 300Lys Arg Asp Leu Tyr Gln Thr Leu
Tyr Val Asp Ala Asp Glu Glu Glu305 310 315 320Ile Ile Gln Tyr Val
Val Gly Thr Leu Gln Pro Lys Leu Lys Arg Phe 325 330 335Leu Arg His
Pro Leu Pro Lys Thr Leu Glu Gln Leu Ile Gln Arg Gly 340 345 350Met
Glu Val Gln Asp Asp Leu Glu Gln Ala Ala Glu Pro Ala Gly Pro 355 360
365His Leu Pro Val Glu Asp Glu Ala Glu Thr Leu Thr Pro Ala Pro Asn
370 375 380Ser Glu Ser Val Ala Ser Asp Arg Thr Gln Pro Glu385 390
3952396PRTOrcinus orca 2Gly Glu Leu Asp Gln Arg Thr Thr Gly Gly Leu
His Ala Tyr Pro Ala1 5 10 15Pro Arg Gly Gly Pro Val Ala Lys Pro Asn
Val Ile Leu Gln Ile Gly 20 25 30Lys Cys Arg Ala Glu Met Leu Glu His
Val Arg Arg Thr His Arg His 35 40 45Leu Leu Thr Glu Val Ser Lys Gln
Val Glu Arg Glu Leu Lys Gly Leu 50 55 60His Arg Ser Val Gly Lys Leu
Glu Ser Asn Leu Asp Gly Tyr Val Pro65 70 75 80Thr Gly Asp Ser Gln
Arg Trp Arg Lys Ser Ile Lys Ala Cys Leu Cys 85 90 95Arg Cys Gln Glu
Thr Ile Ala Asn Leu Glu Arg Trp Val Lys Arg Glu 100 105 110Met His
Val Trp Arg Glu Val Phe Tyr Arg Leu Glu Arg Trp Ala Asp 115 120
125Arg Leu Glu Ser Met Gly Gly Lys Tyr Pro Val Gly Ser Asn Pro Ser
130 135 140Arg His Thr Thr Ser Val Gly Val Gly Gly Pro Glu Ser Tyr
Gly His145 150 155 160Glu Ala Asp Thr Tyr Asp Tyr Thr Val Ser Pro
Tyr Ala Ile Thr Pro 165 170 175Pro Pro Ala Ala Gly Glu Leu Pro Gly
Gln Glu Ala Val Glu Ala Gln 180 185 190Gln Tyr Pro Pro Trp Gly Leu
Gly Glu Asp Gly Gln Pro Ser Pro Gly 195 200 205Val Asp Thr Gln Ile
Phe Glu Asp Pro Arg Glu Phe Leu Ser His Leu 210 215 220Glu Glu Tyr
Leu Arg Gln Val Gly Gly Ser Glu Glu Tyr Trp Leu Ser225 230 235
240Gln Ile Gln Asn His Met Asn Gly Pro Ala Lys Lys Trp Trp Glu Tyr
245 250 255Lys Gln Gly Ser Val Lys Asn Trp Val Glu Phe Lys Lys Glu
Phe Leu 260 265 270Gln Tyr Ser Glu Gly Ala Leu Ser Arg Glu Ala Val
Gln Arg Glu Leu 275 280 285Asp Leu Pro Gln Lys Gln Gly Glu Pro Leu
Asp Gln Phe Leu Trp Arg 290 295 300Lys Arg Asp Leu Tyr Gln Thr Leu
Tyr Val Asp Ala Asp Glu Glu Glu305 310 315 320Ile Ile Gln Tyr Val
Val Gly Thr Leu Gln Pro Lys Leu Lys Arg Phe 325 330 335Leu Arg Pro
Pro Leu Pro Lys Thr Leu Glu Gln Leu Ile Gln Lys Gly 340 345 350Met
Glu Val Glu Asp Gly Leu Glu Gln Val Ala Glu Pro Ala Ser Pro 355 360
365His Leu Pro Thr Glu Glu Glu Ser Glu Ala Leu Thr Pro Ala Leu Thr
370 375 380Ser Glu Ser Val Ala Ser Asp Arg Thr Gln Pro Glu385 390
3953390PRTOdocoileus virginianus texanus 3Gly Glu Leu Asp His Arg
Thr Thr Gly Gly Leu His Ala Tyr Pro Ala1 5 10 15Pro Arg Gly Gly Pro
Ala Ala Lys Pro Asn Val Ile Leu Gln Ile Gly 20 25 30Lys Cys Arg Ala
Glu Met Leu Glu His Val Arg Arg Thr His Arg His 35 40 45Leu Leu Ala
Glu Val Ser Lys Gln Val Glu Arg Glu Leu Lys Gly Leu 50 55 60His Arg
Ser Val Gly Lys Leu Glu Ser Asn Leu Asp Gly Tyr Val Pro65 70 75
80Thr Gly Asp Ser Gln Arg Trp Lys Lys Ser Ile Lys Ala Cys Leu Ser
85 90 95Arg Cys Gln Glu Thr Ile Ala Asn Leu Glu Arg Trp Val Lys Arg
Glu 100 105 110Met His Val Trp Arg Glu Val Phe Tyr Arg Leu Glu Arg
Trp Ala Asp 115 120 125Arg Leu Glu Ser Gly Gly Gly Lys Tyr Pro Val
Gly Ser Asp Pro Ala 130 135 140Arg His Thr Val Ser Val Gly Val Gly
Gly Pro Glu Ser Tyr Cys Gln145 150 155 160Asp Ala Asp Asn Tyr Asp
Tyr Thr Val Ser Pro Tyr Ala Ile Thr Pro 165 170 175Pro Pro Ala Ala
Gly Gln Leu Pro Gly Gln Glu Glu Val Glu Ala Gln 180 185 190Gln Tyr
Pro Pro Trp Ala Pro Gly Glu Asp Gly Gln Leu Ser Pro Gly 195 200
205Val Asp Thr Gln Val Phe Glu Asp Pro Arg Glu Phe Leu Arg His Leu
210 215 220Glu Asp Tyr Leu Arg Gln Val Gly Gly Ser Glu Glu Tyr Trp
Leu Ser225 230 235 240Gln Ile Gln Asn His Met Asn Gly Pro Ala Lys
Lys Trp Trp Glu Tyr 245 250 255Lys Gln Gly Ser Val Lys Asn Trp Val
Glu Phe Lys Lys Glu Phe Leu 260 265 270Gln Tyr Ser Glu Gly Thr Leu
Ser Arg Glu Ala Ile Gln Arg Glu Leu 275 280 285Asp Leu Pro Gln Lys
Gln Gly Glu Pro Leu Asp Gln Phe Leu Trp Arg 290 295 300Lys Arg Asp
Leu Tyr Gln Thr Leu Tyr Val Asp Ala Glu Glu Glu Glu305 310 315
320Ile Ile Gln Tyr Val Val Gly Thr Leu Gln Pro Lys Leu Lys Arg Phe
325 330 335Leu Arg Pro Pro Leu Pro Lys Thr Leu Glu Gln Leu Ile Gln
Lys Gly 340 345 350Met Glu Val Gln Asp Gly Leu Glu Gln Ala Ala Glu
Pro Ala Ala Glu 355 360 365Glu Ala Glu Ala Leu Thr Pro Ala Leu Thr
Asn Glu Ser Val Ala Ser 370 375 380Asp Arg Thr Gln Pro Glu385
3904401PRTOrnithorhynchus anatinus 4Gly Glu Leu Asp Arg Leu Asn Pro
Ser Ser Gly Leu His Pro Ser Ser1 5 10 15Gly Leu His Pro Tyr Pro Gly
Leu Arg Gly Gly Ala Thr Ala Lys Pro 20 25 30Asn Val Ile Leu Gln Ile
Gly Lys Cys Arg Ala Glu Met Leu Glu His 35 40 45Val Arg Lys Thr His
Arg His Leu Leu Thr Glu Val Ser Arg Gln Val 50 55 60Glu Arg Glu Leu
Lys Gly Leu His Lys Ser Val Gly Lys Leu Glu Ser65 70 75 80Asn Leu
Asp Gly Tyr Val Pro Ser Ser Asp Ser Gln Arg Trp Lys Lys 85 90 95Ser
Ile Lys Ala Cys Leu Ser Arg Cys Gln Glu Thr Ile Ala His Leu 100 105
110Glu Arg Trp Val Lys Arg Glu Met Asn Val Trp Arg Glu Val Phe Tyr
115 120 125Arg Leu Glu Arg Trp Ala Asp Arg Leu Glu Ala Met Gly Gly
Lys Tyr 130 135 140Pro Ala Gly Glu Gln Ala Arg Arg Thr Val Ser Val
Gly Val Gly Gly145 150 155 160Pro Glu Thr Cys Cys Pro Gly Asp Glu
Ser Tyr Asp Cys Pro Ile Ser 165 170 175Pro Tyr Ala Val Pro Pro Ser
Thr Gly Glu Ser Pro Glu Ser Leu Asp 180 185 190Gln Gly Asp Gln His
Tyr Gln Gln Trp Phe Ala Leu Pro Glu Glu Ser 195 200 205Pro Val Ser
Pro Gly Val Asp Thr Gln Ile Phe Glu Asp Pro Arg Glu 210 215 220Phe
Leu Arg His Leu Glu Lys Tyr Leu Lys Gln Val Gly Gly Thr Glu225 230
235 240Glu Asp Trp Leu Ser Gln Ile Gln Asn His Met Asn Gly Pro Ala
Lys 245 250 255Lys Trp Trp Glu Tyr Lys Gln Gly Ser Val Lys Asn Trp
Leu Glu Phe 260 265 270Lys Lys Glu Phe Leu Gln Tyr Ser Glu Gly Thr
Leu Thr Arg Asp Ala 275 280 285Leu Lys Arg Glu Leu Asp Leu Pro Gln
Lys Gln Gly Glu Pro Leu Asp 290 295 300Gln Phe Leu Trp Arg Lys Arg
Asp Leu Tyr Gln Thr Leu Tyr Val Asp305 310 315 320Ala Asp Glu Glu
Glu Ile Ile Gln Tyr Val Val Gly Thr Leu Gln Pro 325 330 335Lys Leu
Lys Arg Phe Leu His His Pro Leu Pro Lys Thr Leu Glu Gln 340 345
350Leu Ile Gln Arg Gly Gln Glu Val Gln Asn Gly Leu Glu Pro Thr Asp
355 360 365Asp Pro Ala Gly Gln Arg Thr Gln Ser Glu Asp Asn Asp Glu
Ser Leu 370 375 380Thr Pro Ala Val Thr Asn Glu Ser Thr Ala Ser Glu
Gly Thr Leu Pro385 390 395 400Glu5404PRTAnser cygnoides domesticus
5Gly Gln Leu Asp Asn Val Thr Asn Ala Gly Ile His Ser Phe Gln Gly1 5
10 15His Arg Gly Val Ala Asn Lys Pro Asn Val Ile Leu Gln Ile Gly
Lys 20 25 30Cys Arg Ala Glu Met Leu Glu His Val Arg Arg Thr His Arg
His Leu 35 40 45Leu Ser Glu Val Ser Lys Gln Val Glu Arg Glu Leu Lys
Gly Leu Gln 50 55 60Lys Ser Val Gly Lys Leu Glu Asn Asn Leu Glu Asp
His Val Pro Thr65 70 75 80Asp Asn Gln Arg Trp Lys Lys Ser Ile Lys
Ala Cys Leu Ala Arg Cys 85 90 95Gln Glu Thr Ile Ala His Leu Glu Arg
Trp Val Lys Arg Glu Met Asn 100 105 110Val Trp Lys Glu Val Phe Phe
Arg Leu Glu Lys Trp Ala Asp Arg Leu 115 120 125Glu Ser Met Gly Gly
Lys Tyr Cys Pro Gly Glu His Gly Lys Gln Thr 130 135 140Val Ser Val
Gly Val Gly Gly Pro Glu Ile Arg Pro Ser Glu Gly Glu145 150 155
160Ile Tyr Asp Tyr Ala Leu Asp Met Ser Gln Met Tyr Ala Leu Thr Pro
165 170 175Pro Pro Gly Glu Met Pro Ser Ile Pro Gln Ala His Asp Ser
Tyr Gln 180 185 190Trp Val Ser Val Ser Glu Asp Ala Pro Ala Ser Pro
Val Glu Thr Gln 195 200 205Val Phe Glu Asp Pro Arg Glu Phe Leu Ser
His Leu Glu Glu Tyr Leu 210 215 220Lys Gln Val Gly Gly Thr Glu Glu
Tyr Trp Leu Ser Gln Ile Gln Asn225 230 235 240His Met Asn Gly Pro
Ala Lys Lys Trp Trp Glu Tyr Lys Gln Asp Ser 245 250 255Val Lys Asn
Trp Val Glu Phe Lys Lys Glu Phe Leu Gln Tyr Ser Glu 260 265 270Gly
Thr Leu Thr Arg Asp Ala Ile Lys Arg Glu Leu Asp Leu Pro Gln 275 280
285Lys Glu Gly Glu Pro Leu Asp Gln Phe Leu Trp Arg Lys Arg Asp Leu
290 295 300Tyr Gln Thr Leu Tyr Val Asp Ala Asp Glu Glu Glu Ile Ile
Gln Tyr305 310 315 320Val Val Gly Thr Leu Gln Pro Lys Leu Lys Arg
Phe Leu Ser Tyr Pro 325 330 335Leu Pro Lys Thr Leu Glu Gln Leu Ile
Gln Arg Gly Lys Glu Val Gln 340 345 350Gly Asn Met Asp His Ser Asp
Glu Pro Ser Pro Gln Arg Thr Pro Glu 355 360 365Ile Gln Ser Gly Asp
Ser Val Glu Ser Met Pro Pro Ser Thr Thr Ala 370 375 380Ser Pro Val
Pro Ser Asn Gly Thr Gln Pro Glu Pro Pro Ser Pro Pro385 390 395
400Ala Thr Val Ile6395PRTPelecanus crispus 6Gly Gln Leu Asp Asn Val
Thr Asn Ala Gly Ile His Ser Phe Gln Gly1 5 10 15His Arg Gly Val Ala
Asn Lys Pro Asn Val Ile Leu Gln Ile Gly Lys 20 25 30Cys Arg Ala Glu
Met Leu Glu His Val Arg Arg Thr His Arg His Leu 35 40 45Leu Ser Glu
Val Ser Lys Gln Val Glu Arg Glu Leu Lys Gly Leu Gln 50 55 60Lys Ser
Val Gly Lys Leu Glu Asn Asn Leu Glu Asp His Val Pro Thr65 70 75
80Asp Asn Gln Arg Trp Lys Lys Ser Ile Lys Ala Cys Leu Ala Arg Cys
85 90 95Gln Glu Thr Ile Ala His Leu Glu Arg Trp Val Lys Arg Glu Met
Asn 100 105 110Val Trp Lys Glu Val Phe Phe Arg Leu Glu Lys Trp Ala
Asp Arg Leu 115 120 125Glu Ser Met Gly Gly Lys Tyr Cys Pro Gly Glu
His Gly Lys Gln Thr 130 135 140Val Ser Val Gly Val Gly Gly Pro Glu
Ile Arg Pro Ser Glu Gly Glu145 150 155 160Ile Tyr Asp Tyr Ala Leu
Asp Met Ser Gln Met Tyr Ala Leu Thr Pro 165 170 175Pro Pro Gly Glu
Val Pro Ser Ile Pro Gln Ala His Asp Ser Tyr Gln 180 185 190Trp Val
Ser Val Ser Glu Asp Ala Pro Ala Ser Pro Val Glu Thr Gln 195 200
205Val Phe Glu Asp Pro Arg Glu Phe Leu Ser His Leu Glu Glu Tyr Leu
210 215 220Lys Gln Val Gly Gly Thr Glu Glu Tyr Trp Leu Ser Gln Ile
Gln Asn225 230 235 240His Met Asn Gly Pro Ala Lys Lys Trp Trp Glu
Tyr Lys Gln Asp Ser 245 250 255Val Lys Asn Trp Val Glu Phe Lys Lys
Glu Phe Leu Gln Tyr Ser Glu 260 265 270Gly Thr Leu Thr Arg Asp Ala
Ile Lys Arg Glu Leu Asp Leu Pro Gln 275 280 285Lys Glu Gly Glu Pro
Leu Asp Gln Phe Leu Trp Arg Lys Arg Asp Leu 290 295 300Tyr Gln Thr
Leu Tyr Val Asp Ala Asp Glu Glu Glu Ile Ile Gln Tyr305 310 315
320Val Val Gly Thr Leu Gln Pro Lys Leu Lys Arg Phe Leu Ser Tyr Pro
325 330 335Leu Pro Lys Thr Leu Glu Gln Leu Ile Gln Arg Gly Lys Glu
Val Gln 340 345 350Gly Asn Met Asp His Ser Glu Glu Pro Ser Pro Gln
Arg Thr Pro Glu 355 360 365Ile Gln Ser Gly Asp Ser Val Asp Ser Val
Pro Pro Ser Thr Thr Ala 370 375 380Ser Pro Val Pro Ser Asn Gly Thr
Gln Pro Glu385 390 3957395PRTHaliaeetus albicilla 7Gly Gln Leu Asp
Asn Val Thr Asn Ala Gly Ile His Ser Phe Gln Gly1 5 10 15His Arg Gly
Val Ala Asn Lys Pro Asn Val Ile Leu Gln Ile Gly Lys 20 25 30Cys Arg
Ala Glu Met Leu Glu His Val Arg Arg Thr His Arg His Leu 35 40 45Leu
Ser Glu Val Ser Lys Gln Val Glu Arg Glu Leu Lys Gly Leu Gln 50 55
60Lys Ser Val Gly Lys Leu Glu Asn Asn Leu Glu Asp His Val Pro
Thr65
70 75 80Asp Asn Gln Arg Trp Lys Lys Ser Ile Lys Ala Cys Leu Ala Arg
Cys 85 90 95Gln Glu Thr Ile Ala His Leu Glu Arg Trp Val Lys Arg Glu
Met Asn 100 105 110Val Trp Lys Glu Val Phe Phe Arg Leu Glu Lys Trp
Ala Asp Arg Leu 115 120 125Glu Ser Met Gly Gly Lys Tyr Cys Pro Gly
Asp His Gly Lys Gln Thr 130 135 140Val Ser Val Gly Val Gly Gly Pro
Glu Ile Arg Pro Ser Glu Gly Glu145 150 155 160Ile Tyr Asp Tyr Ala
Leu Asp Met Ser Gln Met Tyr Ala Leu Thr Pro 165 170 175Pro Pro Gly
Glu Val Pro Ser Ile Pro Gln Ala His Asp Ser Tyr Gln 180 185 190Trp
Val Ser Thr Ser Glu Asp Ala Pro Ala Ser Pro Val Glu Thr Gln 195 200
205Val Phe Glu Asp Pro Arg Glu Phe Leu Ser His Leu Glu Glu Tyr Leu
210 215 220Lys Gln Val Gly Gly Thr Glu Glu Tyr Trp Leu Ser Gln Ile
Gln Asn225 230 235 240His Met Asn Gly Pro Ala Lys Lys Trp Trp Glu
Tyr Lys Gln Asp Ser 245 250 255Val Lys Asn Trp Val Glu Phe Lys Lys
Glu Phe Leu Gln Tyr Ser Glu 260 265 270Gly Thr Leu Thr Arg Asp Ala
Ile Lys Arg Glu Leu Asp Leu Pro Gln 275 280 285Lys Glu Gly Glu Pro
Leu Asp Gln Phe Leu Trp Arg Lys Arg Asp Leu 290 295 300Tyr Gln Thr
Leu Tyr Val Asp Ala Asp Glu Glu Glu Ile Ile Gln Tyr305 310 315
320Val Val Gly Thr Leu Gln Pro Lys Leu Lys Arg Phe Leu Ser Tyr Pro
325 330 335Leu Pro Lys Thr Leu Glu Gln Leu Ile Gln Arg Gly Lys Glu
Val Gln 340 345 350Gly Asn Met Asp His Ser Glu Glu Pro Ser Pro Gln
Arg Thr Pro Glu 355 360 365Ile Gln Ser Gly Asp Ser Val Asp Ser Val
Pro Pro Ser Thr Thr Ala 370 375 380Ser Pro Val Pro Ser Asn Gly Thr
Gln Pro Glu385 390 3958465PRTOphiophagus hannah 8Gly Ser Trp Gly
Leu Gln Arg His Val Ala Asp Glu Arg Arg Gly Leu1 5 10 15Ala Thr Pro
Thr Tyr Gly Ala Val Cys Ser Ile Arg Glu Lys Lys Ala 20 25 30Ser Gln
Leu Ser Gly Gln Ser Cys Leu Glu Lys Glu Leu Leu Gly Trp 35 40 45Lys
Cys Thr Glu Ala Ile Val Glu Met Met Gln Val Asp Asn Phe Asn 50 55
60His Gly Asn Leu His Ser Cys Gln Gly His Arg Gly Met Ala Asn His65
70 75 80Lys Pro Asn Val Ile Leu Gln Ile Gly Lys Cys Arg Ala Glu Met
Leu 85 90 95Asp His Val Arg Arg Thr His Arg His Leu Leu Thr Glu Val
Ser Lys 100 105 110Gln Val Glu Arg Glu Leu Lys Ser Leu Gln Lys Ser
Val Gly Lys Leu 115 120 125Glu Asn Asn Leu Glu Asp His Val Pro Ser
Ala Ala Glu Asn Gln Arg 130 135 140Trp Lys Lys Ser Ile Lys Ala Cys
Leu Ala Arg Cys Gln Glu Thr Ile145 150 155 160Ala His Leu Glu Arg
Trp Val Lys Arg Glu Ile Asn Val Trp Lys Glu 165 170 175Val Phe Phe
Arg Leu Glu Lys Trp Ala Asp Arg Leu Glu Ser Gly Gly 180 185 190Gly
Lys Tyr Gly Pro Gly Asp Gln Ser Arg Gln Thr Val Ser Val Gly 195 200
205Val Gly Ala Pro Glu Ile Gln Pro Arg Lys Glu Glu Ile Tyr Asp Tyr
210 215 220Ala Leu Asp Met Ser Gln Met Tyr Ala Leu Thr Pro Pro Pro
Met Gly225 230 235 240Glu Asp Pro Asn Val Pro Gln Ser His Asp Ser
Tyr Gln Trp Ile Thr 245 250 255Ile Ser Asp Asp Ser Pro Pro Ser Pro
Val Glu Thr Gln Ile Phe Glu 260 265 270Asp Pro Arg Glu Phe Leu Thr
His Leu Glu Asp Tyr Leu Lys Gln Val 275 280 285Gly Gly Thr Glu Glu
Tyr Trp Leu Ser Gln Ile Gln Asn His Met Asn 290 295 300Gly Pro Ala
Lys Lys Trp Trp Glu Tyr Lys Gln Asp Ser Val Lys Asn305 310 315
320Trp Leu Glu Phe Lys Lys Glu Phe Leu Gln Tyr Ser Glu Gly Thr Leu
325 330 335Thr Arg Asp Ala Ile Lys Gln Glu Leu Asp Leu Pro Gln Lys
Asp Gly 340 345 350Glu Pro Leu Asp Gln Phe Leu Trp Arg Lys Arg Asp
Leu Tyr Gln Thr 355 360 365Leu Tyr Ile Asp Ala Glu Glu Glu Glu Val
Ile Gln Tyr Val Val Gly 370 375 380Thr Leu Gln Pro Lys Leu Lys Arg
Phe Leu Ser His Pro Tyr Pro Lys385 390 395 400Thr Leu Glu Gln Leu
Ile Gln Arg Gly Lys Glu Val Glu Gly Asn Leu 405 410 415Asp Asn Ser
Glu Glu Pro Ser Pro Gln Arg Ser Pro Lys His Gln Leu 420 425 430Gly
Gly Ser Val Glu Ser Leu Pro Pro Ser Ser Thr Ala Ser Pro Val 435 440
445Ala Ser Asp Glu Thr His Pro Asp Val Ser Ala Pro Pro Val Thr Val
450 455 460Ile4659451PRTAustrofundulus limnaeus 9Gly Asp Gly Glu
Thr Gln Ala Glu Asn Pro Ser Thr Ser Leu Asn Asn1 5 10 15Thr Asp Glu
Asp Ile Leu Glu Gln Leu Lys Lys Ile Val Met Asp Gln 20 25 30Gln His
Leu Tyr Gln Lys Glu Leu Lys Ala Ser Phe Glu Gln Leu Ser 35 40 45Arg
Lys Met Phe Ser Gln Met Glu Gln Met Asn Ser Lys Gln Thr Asp 50 55
60Leu Leu Leu Glu His Gln Lys Gln Thr Val Lys His Val Asp Lys Arg65
70 75 80Val Glu Tyr Leu Arg Ala Gln Phe Asp Ala Ser Leu Gly Trp Arg
Leu 85 90 95Lys Glu Gln His Ala Asp Ile Thr Thr Lys Ile Ile Pro Glu
Ile Ile 100 105 110Gln Thr Val Lys Glu Asp Ile Ser Leu Cys Leu Ser
Thr Leu Cys Ser 115 120 125Ile Ala Glu Asp Ile Gln Thr Ser Arg Ala
Thr Thr Val Thr Gly His 130 135 140Ala Ala Val Gln Thr His Pro Val
Asp Leu Leu Gly Glu His His Leu145 150 155 160Gly Thr Thr Gly His
Pro Arg Leu Gln Ser Thr Arg Val Gly Lys Pro 165 170 175Asp Asp Val
Pro Glu Ser Pro Val Ser Leu Phe Met Gln Gly Glu Ala 180 185 190Arg
Ser Arg Ile Val Gly Lys Ser Pro Ile Lys Leu Gln Phe Pro Thr 195 200
205Phe Gly Lys Ala Asn Asp Ser Ser Asp Pro Leu Gln Tyr Leu Glu Arg
210 215 220Cys Glu Asp Phe Leu Ala Leu Asn Pro Leu Thr Asp Glu Glu
Leu Met225 230 235 240Ala Thr Leu Arg Asn Val Leu His Gly Thr Ser
Arg Asp Trp Trp Asp 245 250 255Val Ala Arg His Lys Ile Gln Thr Trp
Arg Glu Phe Asn Lys His Phe 260 265 270Arg Ala Ala Phe Leu Ser Glu
Asp Tyr Glu Asp Glu Leu Ala Glu Arg 275 280 285Val Arg Asn Arg Ile
Gln Lys Glu Asp Glu Ser Ile Arg Asp Phe Ala 290 295 300Tyr Met Tyr
Gln Ser Leu Cys Lys Arg Trp Asn Pro Ala Ile Cys Glu305 310 315
320Gly Asp Val Val Lys Leu Ile Leu Lys Asn Ile Asn Pro Gln Leu Pro
325 330 335Ser Gln Leu Arg Ser Arg Val Thr Thr Val Asp Glu Leu Val
Arg Leu 340 345 350Gly Gln Gln Leu Glu Lys Asp Arg Gln Asn Gln Leu
Gln Tyr Glu Leu 355 360 365Arg Lys Ser Ser Gly Lys Ile Ile Gln Lys
Ser Ser Ser Cys Glu Thr 370 375 380Ser Ala Leu Pro Asn Thr Lys Ser
Thr Pro Asn Gln Gln Asn Pro Ala385 390 395 400Thr Ser Asn Arg Pro
Pro Gln Val Tyr Cys Trp Arg Cys Lys Gly His 405 410 415His Ala Pro
Ala Ser Cys Pro Gln Trp Lys Ala Asp Lys His Arg Ala 420 425 430Gln
Pro Ser Arg Ser Ser Gly Pro Gln Thr Leu Thr Asn Leu Gln Ala 435 440
445Gln Asp Ile 45010396PRTPhyseter catodon 10Gly Glu Leu Asp Gln
Arg Ala Ala Gly Gly Leu Arg Ala Tyr Pro Ala1 5 10 15Pro Arg Gly Gly
Pro Val Ala Lys Pro Ser Val Ile Leu Gln Ile Gly 20 25 30Lys Cys Arg
Ala Glu Met Leu Glu His Val Arg Arg Thr His Arg His 35 40 45Leu Leu
Thr Glu Val Ser Lys Gln Val Glu Arg Glu Leu Lys Gly Leu 50 55 60His
Arg Ser Val Gly Lys Leu Glu Gly Asn Leu Asp Gly Tyr Val Pro65 70 75
80Thr Gly Asp Ser Gln Arg Trp Lys Lys Ser Ile Lys Ala Cys Leu Cys
85 90 95Arg Cys Gln Glu Thr Ile Ala Asn Leu Glu Arg Trp Val Lys Arg
Glu 100 105 110Met His Val Trp Arg Glu Val Phe Tyr Arg Leu Glu Arg
Trp Ala Asp 115 120 125Arg Leu Glu Ser Met Gly Gly Lys Tyr Pro Val
Gly Thr Asn Pro Ser 130 135 140Arg His Thr Val Ser Val Gly Val Gly
Gly Pro Glu Gly Tyr Ser His145 150 155 160Glu Ala Asp Thr Tyr Asp
Tyr Thr Val Ser Pro Tyr Ala Ile Thr Pro 165 170 175Pro Pro Ala Ala
Gly Glu Leu Pro Gly Gln Glu Ala Val Glu Ala Gln 180 185 190Gln Tyr
Pro Pro Trp Gly Leu Gly Glu Asp Gly Gln Pro Gly Pro Gly 195 200
205Val Asp Thr Gln Ile Phe Glu Asp Pro Arg Glu Phe Leu Ser His Leu
210 215 220Glu Glu Tyr Leu Arg Gln Val Gly Gly Ser Glu Glu Tyr Trp
Leu Ser225 230 235 240Gln Ile Gln Asn His Met Asn Gly Pro Ala Lys
Lys Trp Trp Glu Phe 245 250 255Lys Gln Gly Ser Val Lys Asn Trp Val
Glu Phe Lys Lys Glu Phe Leu 260 265 270Gln Tyr Ser Glu Gly Thr Leu
Ser Arg Glu Ala Ile Gln Arg Glu Leu 275 280 285Asp Leu Pro Gln Lys
Gln Gly Glu Pro Leu Asp Gln Phe Leu Trp Arg 290 295 300Lys Arg Asp
Leu Tyr Gln Thr Leu Tyr Val Asp Ala Glu Glu Glu Glu305 310 315
320Ile Ile Gln Tyr Val Val Gly Thr Leu Gln Pro Lys Leu Lys Arg Phe
325 330 335Leu Arg Pro Pro Leu Pro Lys Thr Leu Glu Gln Leu Ile Gln
Lys Gly 340 345 350Met Glu Val Gln Asp Gly Leu Glu Gln Ala Ala Glu
Pro Ala Ser Pro 355 360 365Arg Leu Pro Pro Glu Glu Glu Ser Glu Ala
Leu Thr Pro Ala Leu Thr 370 375 380Ser Glu Ser Val Ala Ser Asp Arg
Thr Gln Pro Glu385 390 39511404PRTMeleagris gallopavo 11Gly Gln Leu
Asp Asn Val Thr Asn Ala Gly Ile His Ser Phe Gln Gly1 5 10 15His Arg
Gly Val Ala Asn Lys Pro Asn Val Ile Leu Gln Ile Gly Lys 20 25 30Cys
Arg Ala Glu Met Leu Glu His Val Arg Arg Thr His Arg His Leu 35 40
45Leu Ser Glu Val Ser Lys Gln Val Glu Arg Glu Leu Lys Gly Leu Gln
50 55 60Lys Ser Val Gly Lys Leu Glu Asn Asn Leu Glu Asp His Val Pro
Thr65 70 75 80Asp Asn Gln Arg Trp Lys Lys Ser Ile Lys Ala Cys Leu
Ala Arg Cys 85 90 95Gln Glu Thr Ile Ala His Leu Glu Arg Trp Val Lys
Arg Glu Met Asn 100 105 110Val Trp Lys Glu Val Phe Phe Arg Leu Glu
Lys Trp Ala Asp Arg Leu 115 120 125Glu Ser Met Gly Gly Lys Tyr Cys
Pro Gly Glu His Gly Lys Gln Thr 130 135 140Val Ser Val Gly Val Gly
Gly Pro Glu Ile Arg Pro Ser Glu Gly Glu145 150 155 160Ile Tyr Asp
Tyr Ala Leu Asp Met Ser Gln Met Tyr Ala Leu Thr Pro 165 170 175Gly
Pro Gly Glu Val Pro Ser Ile Pro Gln Ala His Asp Ser Tyr Gln 180 185
190Trp Val Ser Val Ser Glu Asp Ala Pro Ala Ser Pro Val Glu Thr Gln
195 200 205Ile Phe Glu Asp Pro His Glu Phe Leu Ser His Leu Glu Glu
Tyr Leu 210 215 220Lys Gln Val Gly Gly Thr Glu Glu Tyr Trp Leu Ser
Gln Ile Gln Asn225 230 235 240His Met Asn Gly Pro Ala Lys Lys Trp
Trp Glu Tyr Lys Gln Asp Ser 245 250 255Val Lys Asn Trp Val Glu Phe
Lys Lys Glu Phe Leu Gln Tyr Ser Glu 260 265 270Gly Thr Leu Thr Arg
Asp Ala Ile Lys Arg Glu Leu Asp Leu Pro Gln 275 280 285Lys Glu Gly
Glu Pro Leu Asp Gln Phe Leu Trp Arg Lys Arg Asp Leu 290 295 300Tyr
Gln Thr Leu Tyr Val Asp Ala Asp Glu Glu Glu Ile Ile Gln Tyr305 310
315 320Val Val Gly Thr Leu Gln Pro Lys Leu Lys Arg Phe Leu Ser Tyr
Pro 325 330 335Leu Pro Lys Thr Leu Glu Gln Leu Ile Gln Arg Gly Lys
Glu Val Gln 340 345 350Gly Asn Met Asp His Ser Glu Glu Pro Ser Pro
Gln Arg Thr Pro Glu 355 360 365Ile Gln Ser Gly Asp Ser Val Glu Ser
Met Pro Pro Ser Thr Thr Ala 370 375 380Ser Pro Val Pro Ser Asn Gly
Thr Gln Pro Glu Pro Pro Ser Pro Pro385 390 395 400Ala Thr Val
Ile12409PRTPogona vitticeps 12Gly Gln Leu Glu Asn Ile Asn Gln Gly
Ser Leu His Ala Phe Gln Gly1 5 10 15His Arg Gly Val Val His Asn Asn
Lys Pro Asn Val Ile Leu Gln Ile 20 25 30Gly Lys Cys Arg Ala Glu Met
Leu Glu His Val Arg Arg Thr His Arg 35 40 45His Leu Leu Thr Glu Val
Ser Lys Gln Val Glu Arg Glu Leu Lys Gly 50 55 60Leu Gln Lys Ser Val
Gly Lys Leu Glu Asn Asn Leu Glu Asp His Val65 70 75 80Pro Ser Ala
Ala Glu Asn Gln Arg Trp Lys Lys Ser Ile Lys Ala Cys 85 90 95Leu Ala
Arg Cys Gln Glu Thr Ile Ala Asn Leu Glu Arg Trp Val Lys 100 105
110Arg Glu Met Asn Val Trp Lys Glu Val Phe Phe Arg Leu Glu Arg Trp
115 120 125Ala Asp Arg Leu Glu Ser Gly Gly Gly Lys Tyr Cys His Ala
Asp Gln 130 135 140Gly Arg Gln Thr Val Ser Val Gly Val Gly Gly Pro
Glu Val Arg Pro145 150 155 160Ser Glu Gly Glu Ile Tyr Asp Tyr Ala
Leu Asp Met Ser Gln Met Tyr 165 170 175Ala Leu Thr Pro Pro Pro Met
Gly Asp Val Pro Val Ile Pro Gln Pro 180 185 190His Asp Ser Tyr Gln
Trp Val Thr Asp Pro Glu Glu Ala Pro Pro Ser 195 200 205Pro Val Glu
Thr Gln Ile Phe Glu Asp Pro Arg Glu Phe Leu Thr His 210 215 220Leu
Glu Asp Tyr Leu Lys Gln Val Gly Gly Thr Glu Glu Tyr Trp Leu225 230
235 240Ser Gln Ile Gln Asn His Met Asn Gly Pro Ala Lys Lys Trp Trp
Glu 245 250 255Tyr Lys Gln Asp Ser Val Lys Asn Trp Leu Glu Phe Lys
Lys Glu Phe 260 265 270Leu Gln Tyr Ser Glu Gly Thr Leu Thr Arg Asp
Ala Ile Lys Gln Glu 275 280 285Leu Asp Leu Pro Gln Lys Glu Gly Glu
Pro Leu Asp Gln Phe Leu Trp 290 295 300Arg Lys Arg Asp Leu Tyr Gln
Thr Leu Tyr Val Glu Ala Glu Glu Glu305 310 315 320Glu Val Ile Gln
Tyr Val Val Gly Thr Leu Gln Pro Lys Leu Lys Arg 325 330 335Phe Leu
Ser His Pro Tyr Pro Lys Thr Leu Glu Gln Leu Ile Gln Arg 340 345
350Gly Lys Glu Val Glu Gly Asn Leu Asp Asn Ser Glu Glu Pro Ser Pro
355 360 365Gln Arg Thr Pro Glu His Gln Leu Gly Asp Ser Val Glu Ser
Leu Pro 370 375 380Pro Ser Thr Thr Ala Ser Pro Ala Gly Ser Asp Lys
Thr Gln Pro Glu385 390 395 400Ile Ser Leu Pro Pro Thr Thr Val Ile
40513404PRTAlligator sinensis 13Gly Gln Leu Asp Ser Val Thr Asn Ala
Gly Val His Thr Tyr Gln Gly1 5 10
15His Arg Ser Val Ala Asn Lys Pro Asn Val Ile Leu Gln Ile Gly Lys
20 25 30Cys Arg Thr Glu Met Leu Glu His Val Arg Arg Thr His Arg His
Leu 35 40 45Leu Thr Glu Val Ser Lys Gln Val Glu Arg Glu Leu Lys Gly
Leu Gln 50 55 60Lys Ser Val Gly Lys Leu Glu Asn Asn Leu Glu Asp His
Val Pro Thr65 70 75 80Asp Asn Gln Arg Trp Lys Lys Ser Ile Lys Ala
Cys Leu Ala Arg Cys 85 90 95Gln Glu Thr Ile Ala His Leu Glu Arg Trp
Val Lys Arg Glu Met Asn 100 105 110Val Trp Lys Glu Val Phe Phe Arg
Leu Glu Arg Trp Ala Asp Arg Leu 115 120 125Glu Ser Met Gly Gly Lys
Tyr Cys Pro Thr Asp Ser Ala Arg Gln Thr 130 135 140Val Ser Val Gly
Val Gly Gly Pro Glu Ile Arg Pro Ser Glu Gly Glu145 150 155 160Ile
Tyr Asp Tyr Ala Leu Asp Met Ser Gln Met Tyr Ala Leu Thr Pro 165 170
175Ser Pro Gly Glu Leu Pro Ser Val Pro Gln Pro His Asp Ser Tyr Gln
180 185 190Trp Val Thr Ser Pro Glu Asp Ala Pro Ala Ser Pro Val Glu
Thr Gln 195 200 205Val Phe Glu Asp Pro Arg Glu Phe Leu Cys His Leu
Glu Glu Tyr Leu 210 215 220Lys Gln Val Gly Gly Thr Glu Glu Tyr Trp
Leu Ser Gln Ile Gln Asn225 230 235 240His Met Asn Gly Pro Ala Lys
Lys Trp Trp Glu Tyr Lys Gln Asp Thr 245 250 255Val Lys Asn Trp Val
Glu Phe Lys Lys Glu Phe Leu Gln Tyr Ser Glu 260 265 270Gly Thr Leu
Thr Arg Asp Ala Ile Lys Arg Glu Leu Asp Leu Pro Gln 275 280 285Lys
Asp Gly Glu Pro Leu Asp Gln Phe Leu Trp Arg Lys Arg Asp Leu 290 295
300Tyr Gln Thr Leu Tyr Ile Asp Ala Asp Glu Glu Gln Ile Ile Gln
Tyr305 310 315 320Val Val Gly Thr Leu Gln Pro Lys Leu Lys Arg Phe
Leu Ser Tyr Pro 325 330 335Leu Pro Lys Thr Leu Glu Gln Leu Ile Gln
Lys Gly Lys Glu Val Gln 340 345 350Gly Ser Leu Asp His Ser Glu Glu
Pro Ser Pro Gln Arg Ala Ser Glu 355 360 365Ala Arg Thr Gly Asp Ser
Val Glu Thr Leu Pro Pro Ser Thr Thr Thr 370 375 380Ser Pro Asn Thr
Ser Ser Gly Thr Gln Pro Glu Ala Pro Ser Pro Pro385 390 395 400Ala
Thr Val Ile14404PRTAlligator mississippiensis 14Gly Gln Leu Asp Ser
Val Thr Asn Ala Gly Val His Thr Tyr Gln Gly1 5 10 15His Arg Gly Val
Ala Asn Lys Pro Asn Val Ile Leu Gln Ile Gly Lys 20 25 30Cys Arg Thr
Glu Met Leu Glu His Val Arg Arg Thr His Arg His Leu 35 40 45Leu Thr
Glu Val Ser Lys Gln Val Glu Arg Glu Leu Lys Gly Leu Gln 50 55 60Lys
Ser Val Gly Lys Leu Glu Asn Asn Leu Glu Asp His Val Pro Thr65 70 75
80Asp Asn Gln Arg Trp Lys Lys Ser Ile Lys Ala Cys Leu Ala Arg Cys
85 90 95Gln Glu Thr Ile Ala His Leu Glu Arg Trp Val Lys Arg Glu Met
Asn 100 105 110Val Trp Lys Glu Val Phe Phe Arg Leu Glu Arg Trp Ala
Asp Arg Leu 115 120 125Glu Ser Met Gly Gly Lys Tyr Cys Pro Thr Asp
Ser Ala Arg Gln Thr 130 135 140Val Ser Val Gly Val Gly Gly Pro Glu
Ile Arg Pro Ser Glu Gly Glu145 150 155 160Ile Tyr Asp Tyr Ala Leu
Asp Met Ser Gln Met Tyr Ala Leu Thr Pro 165 170 175Ser Pro Gly Glu
Leu Pro Ser Ile Pro Gln Pro His Asp Ser Tyr Gln 180 185 190Trp Val
Thr Ser Pro Glu Asp Ala Pro Ala Ser Pro Val Glu Thr Gln 195 200
205Val Phe Glu Asp Pro Arg Glu Phe Leu Cys His Leu Glu Glu Tyr Leu
210 215 220Lys Gln Val Gly Gly Thr Glu Glu Tyr Trp Leu Ser Gln Ile
Gln Asn225 230 235 240His Met Asn Gly Pro Ala Lys Lys Trp Trp Glu
Tyr Lys Gln Asp Thr 245 250 255Val Lys Asn Trp Val Glu Phe Lys Lys
Glu Phe Leu Gln Tyr Ser Glu 260 265 270Gly Thr Leu Thr Arg Asp Ala
Ile Lys Arg Glu Leu Asp Leu Pro Gln 275 280 285Lys Asp Gly Glu Pro
Leu Asp Gln Phe Leu Trp Arg Lys Arg Asp Leu 290 295 300Tyr Gln Thr
Leu Tyr Ile Asp Ala Asp Glu Glu Gln Ile Ile Gln Tyr305 310 315
320Val Val Gly Thr Leu Gln Pro Lys Leu Lys Arg Phe Leu Ser Tyr Pro
325 330 335Leu Pro Lys Thr Leu Glu Gln Leu Ile Gln Lys Gly Lys Glu
Val Gln 340 345 350Gly Ser Leu Asp His Ser Glu Glu Pro Ser Pro Gln
Arg Ala Ser Glu 355 360 365Ala Arg Thr Gly Asp Ser Val Glu Ser Leu
Pro Pro Ser Thr Thr Thr 370 375 380Ser Pro Asn Ala Ser Ser Gly Thr
Gln Pro Glu Ala Pro Ser Pro Pro385 390 395 400Ala Thr Val
Ile15408PRTGekko japonicus 15Gly Gln Leu Glu Asn Val Asn His Gly
Asn Leu His Ser Phe Gln Gly1 5 10 15His Arg Gly Gly Val Ala Asn Lys
Pro Asn Val Ile Leu Gln Ile Gly 20 25 30Lys Cys Arg Ala Glu Met Leu
Asp His Val Arg Arg Thr His Arg His 35 40 45Leu Leu Thr Glu Val Ser
Lys Gln Val Glu Arg Glu Leu Lys Gly Leu 50 55 60Gln Lys Ser Val Gly
Lys Leu Glu Asn Asn Leu Glu Asp His Val Pro65 70 75 80Ser Ala Val
Glu Asn Gln Arg Trp Lys Lys Ser Ile Lys Ala Cys Leu 85 90 95Ser Arg
Cys Gln Glu Thr Ile Ala His Leu Glu Arg Trp Val Lys Arg 100 105
110Glu Met Asn Val Trp Lys Glu Val Phe Phe Arg Leu Glu Arg Trp Ala
115 120 125Asp Arg Leu Glu Ser Gly Gly Gly Lys Tyr Cys His Gly Asp
Asn His 130 135 140Arg Gln Thr Val Ser Val Gly Val Gly Gly Pro Glu
Val Arg Pro Ser145 150 155 160Glu Gly Glu Ile Tyr Asp Tyr Ala Leu
Asp Met Ser Gln Met Tyr Ala 165 170 175Leu Thr Pro Pro Ser Pro Gly
Asp Val Pro Val Val Ser Gln Pro His 180 185 190Asp Ser Tyr Gln Trp
Val Thr Val Pro Glu Asp Thr Pro Pro Ser Pro 195 200 205Val Glu Thr
Gln Ile Phe Glu Asp Pro Arg Glu Phe Leu Thr His Leu 210 215 220Glu
Asp Tyr Leu Lys Gln Val Gly Gly Thr Glu Glu Tyr Trp Leu Ser225 230
235 240Gln Ile Gln Asn His Met Asn Gly Pro Ala Lys Lys Trp Trp Glu
Tyr 245 250 255Lys Gln Asp Ser Val Lys Asn Trp Leu Glu Phe Lys Lys
Glu Phe Leu 260 265 270Gln Tyr Ser Glu Gly Thr Leu Thr Arg Asp Ala
Ile Lys Glu Glu Leu 275 280 285Asp Leu Pro Gln Lys Asp Gly Glu Pro
Leu Asp Gln Phe Leu Trp Arg 290 295 300Lys Arg Asp Leu Tyr Gln Thr
Leu Tyr Val Glu Ala Asp Glu Glu Glu305 310 315 320Val Ile Gln Tyr
Val Val Gly Thr Leu Gln Pro Lys Leu Lys Arg Phe 325 330 335Leu Ser
His Pro Tyr Pro Lys Thr Leu Glu Gln Leu Ile Gln Arg Gly 340 345
350Lys Glu Val Glu Gly Asn Leu Asp Asn Ser Glu Glu Pro Thr Pro Gln
355 360 365Arg Thr Pro Glu His Gln Leu Cys Gly Ser Val Glu Ser Leu
Pro Pro 370 375 380Ser Ser Thr Val Ser Pro Val Ala Ser Asp Gly Thr
Gln Pro Glu Thr385 390 395 400Ser Pro Leu Pro Ala Thr Val Ile
40516455PRTHomo sapiens 16Gly Pro Leu Thr Leu Leu Gln Asp Trp Cys
Arg Gly Glu His Leu Asn1 5 10 15Thr Arg Arg Cys Met Leu Ile Leu Gly
Ile Pro Glu Asp Cys Gly Glu 20 25 30Asp Glu Phe Glu Glu Thr Leu Gln
Glu Ala Cys Arg His Leu Gly Arg 35 40 45Tyr Arg Val Ile Gly Arg Met
Phe Arg Arg Glu Glu Asn Ala Gln Ala 50 55 60Ile Leu Leu Glu Leu Ala
Gln Asp Ile Asp Tyr Ala Leu Leu Pro Arg65 70 75 80Glu Ile Pro Gly
Lys Gly Gly Pro Trp Glu Val Ile Val Lys Pro Arg 85 90 95Asn Ser Asp
Gly Glu Phe Leu Asn Arg Leu Asn Arg Phe Leu Glu Glu 100 105 110Glu
Arg Arg Thr Val Ser Asp Met Asn Arg Val Leu Gly Ser Asp Thr 115 120
125Asn Cys Ser Ala Pro Arg Val Thr Ile Ser Pro Glu Phe Trp Thr Trp
130 135 140Ala Gln Thr Leu Gly Ala Ala Val Gln Pro Leu Leu Glu Gln
Met Leu145 150 155 160Tyr Arg Glu Leu Arg Val Phe Ser Gly Asn Thr
Ile Ser Ile Pro Gly 165 170 175Ala Leu Ala Phe Asp Ala Trp Leu Glu
His Thr Thr Glu Met Leu Gln 180 185 190Met Trp Gln Val Pro Glu Gly
Glu Lys Arg Arg Arg Leu Met Glu Cys 195 200 205Leu Arg Gly Pro Ala
Leu Gln Val Val Ser Gly Leu Arg Ala Ser Asn 210 215 220Ala Ser Ile
Thr Val Glu Glu Cys Leu Ala Ala Leu Gln Gln Val Phe225 230 235
240Gly Pro Val Glu Ser His Lys Ile Ala Gln Val Lys Leu Cys Lys Ala
245 250 255Tyr Gln Glu Ala Gly Glu Lys Val Ser Ser Phe Val Leu Arg
Leu Glu 260 265 270Pro Leu Leu Gln Arg Ala Val Glu Asn Asn Val Val
Ser Arg Arg Asn 275 280 285Val Asn Gln Thr Arg Leu Lys Arg Val Leu
Ser Gly Ala Thr Leu Pro 290 295 300Asp Lys Leu Arg Asp Lys Leu Lys
Leu Met Lys Gln Arg Arg Lys Pro305 310 315 320Pro Gly Phe Leu Ala
Leu Val Lys Leu Leu Arg Glu Glu Glu Glu Trp 325 330 335Glu Ala Thr
Leu Gly Pro Asp Arg Glu Ser Leu Glu Gly Leu Glu Val 340 345 350Ala
Pro Arg Pro Pro Ala Arg Ile Thr Gly Val Gly Ala Val Pro Leu 355 360
365Pro Ala Ser Gly Asn Ser Phe Asp Ala Arg Pro Ser Gln Gly Tyr Arg
370 375 380Arg Arg Arg Gly Arg Gly Gln His Arg Arg Gly Gly Val Ala
Arg Ala385 390 395 400Gly Ser Arg Gly Ser Arg Lys Arg Lys Arg His
Thr Phe Cys Tyr Ser 405 410 415Cys Gly Glu Asp Gly His Ile Arg Val
Gln Cys Ile Asn Pro Ser Asn 420 425 430Leu Leu Leu Ala Lys Glu Thr
Lys Glu Ile Leu Glu Gly Gly Glu Arg 435 440 445Glu Ala Gln Thr Asn
Ser Arg 450 45517448PRTHomo sapiens 17Gly Ala Leu Thr Leu Leu Glu
Asp Trp Cys Lys Gly Met Asp Met Asp1 5 10 15Pro Arg Lys Ala Leu Leu
Ile Val Gly Ile Pro Met Glu Cys Ser Glu 20 25 30Val Glu Ile Gln Asp
Thr Val Lys Ala Gly Leu Gln Pro Leu Cys Ala 35 40 45Tyr Arg Val Leu
Gly Arg Met Phe Arg Arg Glu Asp Asn Ala Lys Ala 50 55 60Val Phe Ile
Glu Leu Ala Asp Thr Val Asn Tyr Thr Thr Leu Pro Ser65 70 75 80His
Ile Pro Gly Lys Gly Gly Ser Trp Glu Val Val Val Lys Pro Arg 85 90
95Asn Pro Asp Asp Glu Phe Leu Ser Arg Leu Asn Tyr Phe Leu Lys Asp
100 105 110Glu Gly Arg Ser Met Thr Asp Val Ala Arg Ala Leu Gly Cys
Cys Ser 115 120 125Leu Pro Ala Glu Ser Leu Asp Ala Glu Val Met Pro
Gln Val Arg Ser 130 135 140Pro Pro Leu Glu Pro Pro Lys Glu Ser Met
Trp Tyr Arg Lys Leu Lys145 150 155 160Val Phe Ser Gly Thr Ala Ser
Pro Ser Pro Gly Glu Glu Thr Phe Glu 165 170 175Asp Trp Leu Glu Gln
Val Thr Glu Ile Met Pro Ile Trp Gln Val Ser 180 185 190Glu Val Glu
Lys Arg Arg Arg Leu Leu Glu Ser Leu Arg Gly Pro Ala 195 200 205Leu
Ser Ile Met Arg Val Leu Gln Ala Asn Asn Asp Ser Ile Thr Val 210 215
220Glu Gln Cys Leu Asp Ala Leu Lys Gln Ile Phe Gly Asp Lys Glu
Asp225 230 235 240Phe Arg Ala Ser Gln Phe Arg Phe Leu Gln Thr Ser
Pro Lys Ile Gly 245 250 255Glu Lys Val Ser Thr Phe Leu Leu Arg Leu
Glu Pro Leu Leu Gln Lys 260 265 270Ala Val His Lys Ser Pro Leu Ser
Val Arg Ser Thr Asp Met Ile Arg 275 280 285Leu Lys His Leu Leu Ala
Arg Val Ala Met Thr Pro Ala Leu Arg Gly 290 295 300Lys Leu Glu Leu
Leu Asp Gln Arg Gly Cys Pro Pro Asn Phe Leu Glu305 310 315 320Leu
Met Lys Leu Ile Arg Asp Glu Glu Glu Trp Glu Asn Thr Glu Ala 325 330
335Val Met Lys Asn Lys Glu Lys Pro Ser Gly Arg Gly Arg Gly Ala Ser
340 345 350Gly Arg Gln Ala Arg Ala Glu Ala Ser Val Ser Ala Pro Gln
Ala Thr 355 360 365Val Gln Ala Arg Ser Phe Ser Asp Ser Ser Pro Gln
Thr Ile Gln Gly 370 375 380Gly Leu Pro Pro Leu Val Lys Arg Arg Arg
Leu Leu Gly Ser Glu Ser385 390 395 400Thr Arg Gly Glu Asp His Gly
Gln Ala Thr Tyr Pro Lys Ala Glu Asn 405 410 415Gln Thr Pro Gly Arg
Glu Gly Pro Gln Ala Ala Gly Glu Glu Leu Gly 420 425 430Asn Glu Ala
Gly Ala Gly Ala Met Ser His Pro Lys Pro Trp Glu Thr 435 440
44518399PRTHomo sapiens 18Gly Ala Val Thr Met Leu Gln Asp Trp Cys
Arg Trp Met Gly Val Asn1 5 10 15Ala Arg Arg Gly Leu Leu Ile Leu Gly
Ile Pro Glu Asp Cys Asp Asp 20 25 30Ala Glu Phe Gln Glu Ser Leu Glu
Ala Ala Leu Arg Pro Met Gly His 35 40 45Phe Thr Val Leu Gly Lys Ala
Phe Arg Glu Glu Asp Asn Ala Thr Ala 50 55 60Ala Leu Val Glu Leu Asp
Arg Glu Val Asn Tyr Ala Leu Val Pro Arg65 70 75 80Glu Ile Pro Gly
Thr Gly Gly Pro Trp Asn Val Val Phe Val Pro Arg 85 90 95Cys Ser Gly
Glu Glu Phe Leu Gly Leu Gly Arg Val Phe His Phe Pro 100 105 110Glu
Gln Glu Gly Gln Met Val Glu Ser Val Ala Gly Ala Leu Gly Val 115 120
125Gly Leu Arg Arg Val Cys Trp Leu Arg Ser Ile Gly Gln Ala Val Gln
130 135 140Pro Trp Val Glu Ala Val Arg Cys Gln Ser Leu Gly Val Phe
Ser Gly145 150 155 160Arg Asp Gln Pro Ala Pro Gly Glu Glu Ser Phe
Glu Val Trp Leu Asp 165 170 175His Thr Thr Glu Met Leu His Val Trp
Gln Gly Val Ser Glu Arg Glu 180 185 190Arg Arg Arg Arg Leu Leu Glu
Gly Leu Arg Gly Thr Ala Leu Gln Leu 195 200 205Val His Ala Leu Leu
Ala Glu Asn Pro Ala Arg Thr Ala Gln Asp Cys 210 215 220Leu Ala Ala
Leu Ala Gln Val Phe Gly Asp Asn Glu Ser Gln Ala Thr225 230 235
240Ile Arg Val Lys Cys Leu Thr Ala Gln Gln Gln Ser Gly Glu Arg Leu
245 250 255Ser Ala Phe Val Leu Arg Leu Glu Val Leu Leu Gln Lys Ala
Met Glu 260 265 270Lys Glu Ala Leu Ala Arg Ala Ser Ala Asp Arg Val
Arg Leu Arg Gln 275 280 285Met Leu Thr Arg Ala His Leu Thr Glu Pro
Leu Asp Glu Ala Leu Arg 290 295 300Lys Leu Arg Met Ala Gly Arg Ser
Pro Ser Phe Leu Glu Met Leu Gly305 310 315 320Leu Val Arg Glu Ser
Glu Ala Trp Glu Ala Ser Leu Ala Arg Ser Val 325 330 335Arg Ala Gln
Thr Gln Glu Gly Ala Gly Ala Arg Ala Gly Ala Gln Ala 340 345 350Val
Ala Arg Ala Ser Thr Lys Val Glu Ala Val Pro Gly Gly Pro Gly
355 360 365Arg Glu Pro Glu Gly Leu Leu Gln Ala Gly Gly Gln Glu Ala
Glu Glu 370 375 380Leu Leu Gln Glu Gly Leu Lys Pro Val Leu Glu Glu
Cys Asp Asn385 390 39519399PRTHomo sapiens 19Gly Ala Val Thr Met
Leu Gln Asp Trp Cys Arg Trp Met Gly Val Asn1 5 10 15Ala Arg Arg Gly
Leu Leu Ile Leu Gly Ile Pro Glu Asp Cys Asp Asp 20 25 30Ala Glu Phe
Gln Glu Ser Leu Glu Ala Ala Leu Arg Pro Met Gly His 35 40 45Phe Thr
Val Leu Gly Lys Val Phe Arg Glu Glu Asp Asn Ala Thr Ala 50 55 60Ala
Leu Val Glu Leu Asp Arg Glu Val Asn Tyr Ala Leu Val Pro Arg65 70 75
80Glu Ile Pro Gly Thr Gly Gly Pro Trp Asn Val Val Phe Val Pro Arg
85 90 95Cys Ser Gly Glu Glu Phe Leu Gly Leu Gly Arg Val Phe His Phe
Pro 100 105 110Glu Gln Glu Gly Gln Met Val Glu Ser Val Ala Gly Ala
Leu Gly Val 115 120 125Gly Leu Arg Arg Val Cys Trp Leu Arg Ser Ile
Gly Gln Ala Val Gln 130 135 140Pro Trp Val Glu Ala Val Arg Tyr Gln
Ser Leu Gly Val Phe Ser Gly145 150 155 160Arg Asp Gln Pro Ala Pro
Gly Glu Glu Ser Phe Glu Val Trp Leu Asp 165 170 175His Thr Thr Glu
Met Leu His Val Trp Gln Gly Val Ser Glu Arg Glu 180 185 190Arg Arg
Arg Arg Leu Leu Glu Gly Leu Arg Gly Thr Ala Leu Gln Leu 195 200
205Val His Ala Leu Leu Ala Glu Asn Pro Ala Arg Thr Ala Gln Asp Cys
210 215 220Leu Ala Ala Leu Ala Gln Val Phe Gly Asp Asn Glu Ser Gln
Ala Thr225 230 235 240Ile Arg Val Lys Cys Leu Thr Ala Gln Gln Gln
Ser Gly Glu Arg Leu 245 250 255Ser Ala Phe Val Leu Arg Leu Glu Val
Leu Leu Gln Lys Ala Met Glu 260 265 270Lys Glu Ala Leu Ala Arg Ala
Ser Ala Asp Arg Val Arg Leu Arg Gln 275 280 285Met Leu Thr Arg Ala
His Leu Thr Glu Pro Leu Asp Glu Ala Leu Arg 290 295 300Lys Leu Arg
Met Ala Gly Arg Ser Pro Ser Phe Leu Glu Met Leu Gly305 310 315
320Leu Val Arg Glu Ser Glu Ala Trp Glu Ala Ser Leu Ala Arg Ser Val
325 330 335Arg Ala Gln Thr Gln Glu Gly Ala Gly Ala Arg Ala Gly Ala
Gln Ala 340 345 350Val Ala Arg Ala Ser Thr Lys Val Glu Ala Val Pro
Gly Gly Pro Gly 355 360 365Arg Glu Pro Glu Gly Leu Arg Gln Ala Gly
Gly Gln Glu Ala Glu Glu 370 375 380Leu Leu Gln Glu Gly Leu Lys Pro
Val Leu Glu Glu Cys Asp Asn385 390 39520475PRTHomo sapiens 20Gly
Val Glu Asp Leu Ala Ala Ser Tyr Ile Val Leu Lys Leu Glu Asn1 5 10
15Glu Ile Arg Gln Ala Gln Val Gln Trp Leu Met Glu Glu Asn Ala Ala
20 25 30Leu Gln Ala Gln Ile Pro Glu Leu Gln Lys Ser Gln Ala Ala Lys
Glu 35 40 45Tyr Asp Leu Leu Arg Lys Ser Ser Glu Ala Lys Glu Pro Gln
Lys Leu 50 55 60Pro Glu His Met Asn Pro Pro Ala Ala Trp Glu Ala Gln
Lys Thr Pro65 70 75 80Glu Phe Lys Glu Pro Gln Lys Pro Pro Glu Pro
Gln Asp Leu Leu Pro 85 90 95Trp Glu Pro Pro Ala Ala Trp Glu Leu Gln
Glu Ala Pro Ala Ala Pro 100 105 110Glu Ser Leu Ala Pro Pro Ala Thr
Arg Glu Ser Gln Lys Pro Pro Met 115 120 125Ala His Glu Ile Pro Thr
Val Leu Glu Gly Gln Gly Pro Ala Asn Thr 130 135 140Gln Asp Ala Thr
Ile Ala Gln Glu Pro Lys Asn Ser Glu Pro Gln Asp145 150 155 160Pro
Pro Asn Ile Glu Lys Pro Gln Glu Ala Pro Glu Tyr Gln Glu Thr 165 170
175Ala Ala Gln Leu Glu Phe Leu Glu Leu Pro Pro Pro Gln Glu Pro Leu
180 185 190Glu Pro Ser Asn Ala Gln Glu Phe Leu Glu Leu Ser Ala Ala
Gln Glu 195 200 205Ser Leu Glu Gly Leu Ile Val Val Glu Thr Ser Ala
Ala Ser Glu Phe 210 215 220Pro Gln Ala Pro Ile Gly Leu Glu Ala Thr
Asp Phe Pro Leu Gln Tyr225 230 235 240Thr Leu Thr Phe Ser Gly Asp
Ser Gln Lys Leu Pro Glu Phe Leu Val 245 250 255Gln Leu Tyr Ser Tyr
Met Arg Val Arg Gly His Leu Tyr Pro Thr Glu 260 265 270Ala Ala Leu
Val Ser Phe Val Gly Asn Cys Phe Ser Gly Arg Ala Gly 275 280 285Trp
Trp Phe Gln Leu Leu Leu Asp Ile Gln Ser Pro Leu Leu Glu Gln 290 295
300Cys Glu Ser Phe Ile Pro Val Leu Gln Asp Thr Phe Asp Asn Pro
Glu305 310 315 320Asn Met Lys Asp Ala Asn Gln Cys Ile His Gln Leu
Cys Gln Gly Glu 325 330 335Gly His Val Ala Thr His Phe His Leu Ile
Ala Gln Glu Leu Asn Trp 340 345 350Asp Glu Ser Thr Leu Trp Ile Gln
Phe Gln Glu Gly Leu Ala Ser Ser 355 360 365Ile Gln Asp Glu Leu Ser
His Thr Ser Pro Ala Thr Asn Leu Ser Asp 370 375 380Leu Ile Thr Gln
Cys Ile Ser Leu Glu Glu Lys Pro Asp Pro Asn Pro385 390 395 400Leu
Gly Lys Ser Ser Ser Ala Glu Gly Asp Gly Pro Glu Ser Pro Pro 405 410
415Ala Glu Asn Gln Pro Met Gln Ala Ala Ile Asn Cys Pro His Ile Ser
420 425 430Glu Ala Glu Trp Val Arg Trp His Lys Gly Arg Leu Cys Leu
Tyr Cys 435 440 445Gly Tyr Pro Gly His Phe Ala Arg Asp Cys Pro Val
Lys Pro His Gln 450 455 460Ala Leu Gln Ala Gly Asn Ile Gln Ala Cys
Gln465 470 47521239PRTHomo sapiens 21Gly Val Gln Pro Gln Thr Ser
Lys Ala Glu Ser Pro Ala Leu Ala Ala1 5 10 15Ser Pro Asn Ala Gln Met
Asp Asp Val Ile Asp Thr Leu Thr Ser Leu 20 25 30Arg Leu Thr Asn Ser
Ala Leu Arg Arg Glu Ala Ser Thr Leu Arg Ala 35 40 45Glu Lys Ala Asn
Leu Thr Asn Met Leu Glu Ser Val Met Ala Glu Leu 50 55 60Thr Leu Leu
Arg Thr Arg Ala Arg Ile Pro Gly Ala Leu Gln Ile Thr65 70 75 80Pro
Pro Ile Ser Ser Ile Thr Ser Asn Gly Thr Arg Pro Met Thr Thr 85 90
95Pro Pro Thr Ser Leu Pro Glu Pro Phe Ser Gly Asp Pro Gly Arg Leu
100 105 110Ala Gly Phe Leu Met Gln Met Asp Arg Phe Met Ile Phe Gln
Ala Ser 115 120 125Arg Phe Pro Gly Glu Ala Glu Arg Val Ala Phe Leu
Val Ser Arg Leu 130 135 140Thr Gly Glu Ala Glu Lys Trp Ala Ile Pro
His Met Gln Pro Asp Ser145 150 155 160Pro Leu Arg Asn Asn Tyr Gln
Gly Phe Leu Ala Glu Leu Arg Arg Thr 165 170 175Tyr Lys Ser Pro Leu
Arg His Ala Arg Arg Ala Gln Ile Arg Lys Thr 180 185 190Ser Ala Ser
Asn Arg Ala Val Arg Glu Arg Gln Met Leu Cys Arg Gln 195 200 205Leu
Ala Ser Ala Gly Thr Gly Pro Cys Pro Val His Pro Ala Ser Asn 210 215
220Gly Thr Ser Pro Ala Pro Ala Leu Pro Ala Arg Ala Arg Asn Leu225
230 23522113PRTHomo sapiens 22Gly Asp Gly Arg Val Gln Leu Met Lys
Ala Leu Leu Ala Gly Pro Leu1 5 10 15Arg Pro Ala Ala Arg Arg Trp Arg
Asn Pro Ile Pro Phe Pro Glu Thr 20 25 30Phe Asp Gly Asp Thr Asp Arg
Leu Pro Glu Phe Ile Val Gln Thr Ser 35 40 45Ser Tyr Met Phe Val Asp
Glu Asn Thr Phe Ser Asn Asp Ala Leu Lys 50 55 60Val Thr Phe Leu Ile
Thr Arg Leu Thr Gly Pro Ala Leu Gln Trp Val65 70 75 80Ile Pro Tyr
Ile Arg Lys Glu Ser Pro Leu Leu Asn Asp Tyr Arg Gly 85 90 95Phe Leu
Ala Glu Met Lys Arg Val Phe Gly Trp Glu Glu Asp Glu Asp 100 105
110Phe23113PRTHomo sapiens 23Gly Glu Gly Arg Val Gln Leu Met Lys
Ala Leu Leu Ala Arg Pro Leu1 5 10 15Arg Pro Ala Ala Arg Arg Trp Arg
Asn Pro Ile Pro Phe Pro Glu Thr 20 25 30Phe Asp Gly Asp Thr Asp Arg
Leu Pro Glu Phe Ile Val Gln Thr Ser 35 40 45Ser Tyr Met Phe Val Asp
Glu Asn Thr Phe Ser Asn Asp Ala Leu Lys 50 55 60Val Thr Phe Leu Ile
Thr Arg Leu Thr Gly Pro Ala Leu Gln Trp Val65 70 75 80Ile Pro Tyr
Ile Lys Lys Glu Ser Pro Leu Leu Ser Asp Tyr Arg Gly 85 90 95Phe Leu
Ala Glu Met Lys Arg Val Phe Gly Trp Glu Glu Asp Glu Asp 100 105
110Phe24364PRTHomo sapiens 24Gly Pro Arg Gly Arg Cys Arg Gln Gln
Gly Pro Arg Ile Pro Ile Trp1 5 10 15Ala Ala Ala Asn Tyr Ala Asn Ala
His Pro Trp Gln Gln Met Asp Lys 20 25 30Ala Ser Pro Gly Val Ala Tyr
Thr Pro Leu Val Asp Pro Trp Ile Glu 35 40 45Arg Pro Cys Cys Gly Asp
Thr Val Cys Val Arg Thr Thr Met Glu Gln 50 55 60Lys Ser Thr Ala Ser
Gly Thr Cys Gly Gly Lys Pro Ala Glu Arg Gly65 70 75 80Pro Leu Ala
Gly His Met Pro Ser Ser Arg Pro His Arg Val Asp Phe 85 90 95Cys Trp
Val Pro Gly Ser Asp Pro Gly Thr Phe Asp Gly Ser Pro Trp 100 105
110Leu Leu Asp Arg Phe Leu Ala Gln Leu Gly Asp Tyr Met Ser Phe His
115 120 125Phe Glu His Tyr Gln Asp Asn Ile Ser Arg Val Cys Glu Ile
Leu Arg 130 135 140Arg Leu Thr Gly Arg Ala Gln Ala Trp Ala Ala Pro
Tyr Leu Asp Gly145 150 155 160Asp Leu Pro Leu Pro Asp Asp Tyr Glu
Leu Phe Cys Gln Asp Leu Lys 165 170 175Glu Val Val Gln Asp Pro Asn
Ser Phe Ala Glu Tyr His Ala Val Val 180 185 190Thr Cys Pro Leu Pro
Leu Ala Ser Ser Gln Leu Pro Val Ala Pro Gln 195 200 205Leu Pro Val
Val Arg Gln Tyr Leu Ala Arg Phe Leu Glu Gly Leu Ala 210 215 220Leu
Asp Met Gly Thr Ala Pro Arg Ser Leu Pro Ala Ala Met Ala Thr225 230
235 240Pro Ala Val Ser Gly Ser Asn Ser Val Ser Arg Ser Ala Leu Phe
Glu 245 250 255Gln Gln Leu Thr Lys Glu Ser Thr Pro Gly Pro Lys Glu
Pro Pro Val 260 265 270Leu Pro Ser Ser Thr Cys Ser Ser Lys Pro Gly
Pro Val Glu Pro Ala 275 280 285Ser Ser Gln Pro Glu Glu Ala Ala Pro
Thr Pro Val Pro Arg Leu Ser 290 295 300Glu Ser Ala Asn Pro Pro Ala
Gln Arg Pro Asp Pro Ala His Pro Gly305 310 315 320Gly Pro Lys Pro
Gln Lys Thr Glu Glu Glu Val Leu Glu Thr Glu Gly 325 330 335Asp Gln
Glu Val Ser Leu Gly Thr Pro Gln Glu Val Val Glu Ala Pro 340 345
350Glu Thr Pro Gly Glu Pro Pro Leu Ser Pro Gly Phe 355
36025146PRTHomo sapiens 25Gly Val Asp Glu Leu Val Leu Leu Leu His
Ala Leu Leu Met Arg His1 5 10 15Arg Ala Leu Ser Ile Glu Asn Ser Gln
Leu Met Glu Gln Leu Arg Leu 20 25 30Leu Val Cys Glu Arg Ala Ser Leu
Leu Arg Gln Val Arg Pro Pro Ser 35 40 45Cys Pro Val Pro Phe Pro Glu
Thr Phe Asn Gly Glu Ser Ser Arg Leu 50 55 60Pro Glu Phe Ile Val Gln
Thr Ala Ser Tyr Met Leu Val Asn Glu Asn65 70 75 80Arg Phe Cys Asn
Asp Ala Met Lys Val Ala Phe Leu Ile Ser Leu Leu 85 90 95Thr Gly Glu
Ala Glu Glu Trp Val Val Pro Tyr Ile Glu Met Asp Ser 100 105 110Pro
Ile Leu Gly Asp Tyr Arg Ala Phe Leu Asp Glu Met Lys Gln Cys 115 120
125Phe Gly Trp Asp Asp Asp Glu Asp Asp Asp Asp Glu Glu Glu Glu Asp
130 135 140Asp Tyr14526549PRTHomo sapiens 26Gly Pro Val Asp Leu Gly
Gln Ala Leu Gly Leu Leu Pro Ser Leu Ala1 5 10 15Lys Ala Glu Asp Ser
Gln Phe Ser Glu Ser Asp Ala Ala Leu Gln Glu 20 25 30Glu Leu Ser Ser
Pro Glu Thr Ala Arg Gln Leu Phe Arg Gln Phe Arg 35 40 45Tyr Gln Val
Met Ser Gly Pro His Glu Thr Leu Lys Gln Leu Arg Lys 50 55 60Leu Cys
Phe Gln Trp Leu Gln Pro Glu Val His Thr Lys Glu Gln Ile65 70 75
80Leu Glu Ile Leu Met Leu Glu Gln Phe Leu Thr Ile Leu Pro Gly Glu
85 90 95Ile Gln Met Trp Val Arg Lys Gln Cys Pro Gly Ser Gly Glu Glu
Ala 100 105 110Val Thr Leu Val Glu Ser Leu Lys Gly Asp Pro Gln Arg
Leu Trp Gln 115 120 125Trp Ile Ser Ile Gln Val Leu Gly Gln Asp Ile
Leu Ser Glu Lys Met 130 135 140Glu Ser Pro Ser Cys Gln Val Gly Glu
Val Glu Pro His Leu Glu Val145 150 155 160Val Pro Gln Glu Leu Gly
Leu Glu Asn Ser Ser Ser Gly Pro Gly Glu 165 170 175Leu Leu Ser His
Ile Val Lys Glu Glu Ser Asp Thr Glu Ala Glu Leu 180 185 190Ala Leu
Ala Ala Ser Gln Pro Ala Arg Leu Glu Glu Arg Leu Ile Arg 195 200
205Asp Gln Asp Leu Gly Ala Ser Leu Leu Pro Ala Ala Pro Gln Glu Gln
210 215 220Trp Arg Gln Leu Asp Ser Thr Gln Lys Glu Gln Tyr Trp Asp
Leu Met225 230 235 240Leu Glu Thr Tyr Gly Lys Met Val Ser Gly Ala
Gly Ile Ser His Pro 245 250 255Lys Ser Asp Leu Thr Asn Ser Ile Glu
Phe Gly Glu Glu Leu Ala Gly 260 265 270Ile Tyr Leu His Val Asn Glu
Lys Ile Pro Arg Pro Thr Cys Ile Gly 275 280 285Asp Arg Gln Glu Asn
Asp Lys Glu Asn Leu Asn Leu Glu Asn His Arg 290 295 300Asp Gln Glu
Leu Leu His Ala Ser Cys Gln Ala Ser Gly Glu Val Pro305 310 315
320Ser Gln Ala Ser Leu Arg Gly Phe Phe Thr Glu Asp Glu Pro Gly Cys
325 330 335Phe Gly Glu Gly Glu Asn Leu Pro Glu Ala Leu Gln Asn Ile
Gln Asp 340 345 350Glu Gly Thr Gly Glu Gln Leu Ser Pro Gln Glu Arg
Ile Ser Glu Lys 355 360 365Gln Leu Gly Gln His Leu Pro Asn Pro His
Ser Gly Glu Met Ser Thr 370 375 380Met Trp Leu Glu Glu Lys Arg Glu
Thr Ser Gln Lys Gly Gln Pro Arg385 390 395 400Ala Pro Met Ala Gln
Lys Leu Pro Thr Cys Arg Glu Cys Gly Lys Thr 405 410 415Phe Tyr Arg
Asn Ser Gln Leu Ile Phe His Gln Arg Thr His Thr Gly 420 425 430Glu
Thr Tyr Phe Gln Cys Thr Ile Cys Lys Lys Ala Phe Leu Arg Ser 435 440
445Ser Asp Phe Val Lys His Gln Arg Thr His Thr Gly Glu Lys Pro Cys
450 455 460Lys Cys Asp Tyr Cys Gly Lys Gly Phe Ser Asp Phe Ser Gly
Leu Arg465 470 475 480His His Glu Lys Ile His Thr Gly Glu Lys Pro
Tyr Lys Cys Pro Ile 485 490 495Cys Glu Lys Ser Phe Ile Gln Arg Ser
Asn Phe Asn Arg His Gln Arg 500 505 510Val His Thr Gly Glu Lys Pro
Tyr Lys Cys Ser His Cys Gly Lys Ser 515 520 525Phe Ser Trp Ser Ser
Ser Leu Asp Lys His Gln Arg Ser His Leu Gly 530 535 540Lys Lys Pro
Phe Gln54527351PRTHomo sapiens 27Gly Thr Leu Arg Leu Leu Glu Asp
Trp Cys Arg Gly Met Asp Met Asn1 5 10 15Pro Arg Lys Ala Leu Leu Ile
Ala Gly Ile Ser Gln Ser Cys Ser Val 20 25
30Ala Glu Ile Glu Glu Ala Leu Gln Ala Gly Leu Ala Pro Leu Gly Glu
35 40 45Tyr Arg Leu Leu Gly Arg Met Phe Arg Arg Asp Glu Asn Arg Lys
Val 50 55 60Ala Leu Val Gly Leu Thr Ala Glu Thr Ser His Ala Leu Val
Pro Lys65 70 75 80Glu Ile Pro Gly Lys Gly Gly Ile Trp Arg Val Ile
Phe Lys Pro Pro 85 90 95Asp Pro Asp Asn Thr Phe Leu Ser Arg Leu Asn
Glu Phe Leu Ala Gly 100 105 110Glu Gly Met Thr Val Gly Glu Leu Ser
Arg Ala Leu Gly His Glu Asn 115 120 125Gly Ser Leu Asp Pro Glu Gln
Gly Met Ile Pro Glu Met Trp Ala Pro 130 135 140Met Leu Ala Gln Ala
Leu Glu Ala Leu Gln Pro Ala Leu Gln Cys Leu145 150 155 160Lys Tyr
Lys Lys Leu Arg Val Phe Ser Gly Arg Glu Ser Pro Glu Pro 165 170
175Gly Glu Glu Glu Phe Gly Arg Trp Met Phe His Thr Thr Gln Met Ile
180 185 190Lys Ala Trp Gln Val Pro Asp Val Glu Lys Arg Arg Arg Leu
Leu Glu 195 200 205Ser Leu Arg Gly Pro Ala Leu Asp Val Ile Arg Val
Leu Lys Ile Asn 210 215 220Asn Pro Leu Ile Thr Val Asp Glu Cys Leu
Gln Ala Leu Glu Glu Val225 230 235 240Phe Gly Val Thr Asp Asn Pro
Arg Glu Leu Gln Val Lys Tyr Leu Thr 245 250 255Thr Tyr His Lys Asp
Glu Glu Lys Leu Ser Ala Tyr Val Leu Arg Leu 260 265 270Glu Pro Leu
Leu Gln Lys Leu Val Gln Arg Gly Ala Ile Glu Arg Asp 275 280 285Ala
Val Asn Gln Ala Arg Leu Asp Gln Val Ile Ala Gly Ala Val His 290 295
300Lys Thr Ile Arg Arg Glu Leu Asn Leu Pro Glu Asp Gly Pro Ala
Pro305 310 315 320Gly Phe Leu Gln Leu Leu Val Leu Ile Lys Asp Tyr
Glu Ala Ala Glu 325 330 335Glu Glu Glu Ala Leu Leu Gln Ala Ile Leu
Glu Gly Asn Phe Thr 340 345 35028708PRTHomo sapiens 28Gly Thr Glu
Arg Arg Arg Asp Glu Leu Ser Glu Glu Ile Asn Asn Leu1 5 10 15Arg Glu
Lys Val Met Lys Gln Ser Glu Glu Asn Asn Asn Leu Gln Ser 20 25 30Gln
Val Gln Lys Leu Thr Glu Glu Asn Thr Thr Leu Arg Glu Gln Val 35 40
45Glu Pro Thr Pro Glu Asp Glu Asp Asp Asp Ile Glu Leu Arg Gly Ala
50 55 60Ala Ala Ala Ala Ala Pro Pro Pro Pro Ile Glu Glu Glu Cys Pro
Glu65 70 75 80Asp Leu Pro Glu Lys Phe Asp Gly Asn Pro Asp Met Leu
Ala Pro Phe 85 90 95Met Ala Gln Cys Gln Ile Phe Met Glu Lys Ser Thr
Arg Asp Phe Ser 100 105 110Val Asp Arg Val Arg Val Cys Phe Val Thr
Ser Met Met Thr Gly Arg 115 120 125Ala Ala Arg Trp Ala Ser Ala Lys
Leu Glu Arg Ser His Tyr Leu Met 130 135 140His Asn Tyr Pro Ala Phe
Met Met Glu Met Lys His Val Phe Glu Asp145 150 155 160Pro Gln Arg
Arg Glu Val Ala Lys Arg Lys Ile Arg Arg Leu Arg Gln 165 170 175Gly
Met Gly Ser Val Ile Asp Tyr Ser Asn Ala Phe Gln Met Ile Ala 180 185
190Gln Asp Leu Asp Trp Asn Glu Pro Ala Leu Ile Asp Gln Tyr His Glu
195 200 205Gly Leu Ser Asp His Ile Gln Glu Glu Leu Ser His Leu Glu
Val Ala 210 215 220Lys Ser Leu Ser Ala Leu Ile Gly Gln Cys Ile His
Ile Glu Arg Arg225 230 235 240Leu Ala Arg Ala Ala Ala Ala Arg Lys
Pro Arg Ser Pro Pro Arg Ala 245 250 255Leu Val Leu Pro His Ile Ala
Ser His His Gln Val Asp Pro Thr Glu 260 265 270Pro Val Gly Gly Ala
Arg Met Arg Leu Thr Gln Glu Glu Lys Glu Arg 275 280 285Arg Arg Lys
Leu Asn Leu Cys Leu Tyr Cys Gly Thr Gly Gly His Tyr 290 295 300Ala
Asp Asn Cys Pro Ala Lys Ala Ser Lys Ser Ser Pro Ala Gly Lys305 310
315 320Leu Pro Gly Pro Ala Val Glu Gly Pro Ser Ala Thr Gly Pro Glu
Ile 325 330 335Ile Arg Ser Pro Gln Asp Asp Ala Ser Ser Pro His Leu
Gln Val Met 340 345 350Leu Gln Ile His Leu Pro Gly Arg His Thr Leu
Phe Val Arg Ala Met 355 360 365Ile Asp Ser Gly Ala Ser Gly Asn Phe
Ile Asp His Glu Tyr Val Ala 370 375 380Gln Asn Gly Ile Pro Leu Arg
Ile Lys Asp Trp Pro Ile Leu Val Glu385 390 395 400Ala Ile Asp Gly
Arg Pro Ile Ala Ser Gly Pro Val Val His Glu Thr 405 410 415His Asp
Leu Ile Val Asp Leu Gly Asp His Arg Glu Val Leu Ser Phe 420 425
430Asp Val Thr Gln Ser Pro Phe Phe Pro Val Val Leu Gly Val Arg Trp
435 440 445Leu Ser Thr His Asp Pro Asn Ile Thr Trp Ser Thr Arg Ser
Ile Val 450 455 460Phe Asp Ser Glu Tyr Cys Arg Tyr His Cys Arg Met
Tyr Ser Pro Ile465 470 475 480Pro Pro Ser Leu Pro Pro Pro Ala Pro
Gln Pro Pro Leu Tyr Tyr Pro 485 490 495Val Asp Gly Tyr Arg Val Tyr
Gln Pro Val Arg Tyr Tyr Tyr Val Gln 500 505 510Asn Val Tyr Thr Pro
Val Asp Glu His Val Tyr Pro Asp His Arg Leu 515 520 525Val Asp Pro
His Ile Glu Met Ile Pro Gly Ala His Ser Ile Pro Ser 530 535 540Gly
His Val Tyr Ser Leu Ser Glu Pro Glu Met Ala Ala Leu Arg Asp545 550
555 560Phe Val Ala Arg Asn Val Lys Asp Gly Leu Ile Thr Pro Thr Ile
Ala 565 570 575Pro Asn Gly Ala Gln Val Leu Gln Val Lys Arg Gly Trp
Lys Leu Gln 580 585 590Val Ser Tyr Asp Cys Arg Ala Pro Asn Asn Phe
Thr Ile Gln Asn Gln 595 600 605Tyr Pro Arg Leu Ser Ile Pro Asn Leu
Glu Asp Gln Ala His Leu Ala 610 615 620Thr Tyr Thr Glu Phe Val Pro
Gln Ile Pro Gly Tyr Gln Thr Tyr Pro625 630 635 640Thr Tyr Ala Ala
Tyr Pro Thr Tyr Pro Val Gly Phe Ala Trp Tyr Pro 645 650 655Val Gly
Arg Asp Gly Gln Gly Arg Ser Leu Tyr Val Pro Val Met Ile 660 665
670Thr Trp Asn Pro His Trp Tyr Arg Gln Pro Pro Val Pro Gln Tyr Pro
675 680 685Pro Pro Gln Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro
Pro Ser 690 695 700Tyr Ser Thr Leu705291188DNAHomo sapiens
29ggggagctgg accaccggac cagcggcggg ctccacgcct accccgggcc gcggggcggg
60caggtggcca agcccaacgt gatcctgcag atcgggaagt gccgggccga gatgctggag
120cacgtgcggc ggacgcaccg gcacctgctg gccgaggtgt ccaagcaggt
ggagcgcgag 180ctgaaggggc tgcaccggtc ggtcgggaag ctggagagca
acctggacgg ctacgtgccc 240acgagcgact cgcagcgctg gaagaagtcc
atcaaggcct gcctgtgccg ctgccaggag 300accatcgcca acctggagcg
ctgggtcaag cgcgagatgc acgtgtggcg cgaggtgttc 360taccgcctgg
agcgctgggc cgaccgcctg gagtccacgg gcggcaagta cccggtgggc
420agcgagtcag cccgccacac cgtttccgtg ggcgtggggg gtcccgagag
ctactgccac 480gaggcagacg gctacgacta caccgtcagc ccctacgcca
tcaccccgcc cccagccgct 540ggcgagctgc ccgggcagga gcccgccgag
gcccagcagt accagccgtg ggtccccggc 600gaggacgggc agcccagccc
cggcgtggac acgcagatct tcgaggaccc tcgagagttc 660ctgagccacc
tagaggagta cttgcggcag gtgggcggct ctgaggagta ctggctgtcc
720cagatccaga atcacatgaa cgggccggcc aagaagtggt gggagttcaa
gcagggctcc 780gtgaagaact gggtggagtt caagaaggag ttcctgcagt
acagcgaggg cacgctgtcc 840cgagaggcca tccagcgcga gctggacctg
ccgcagaagc agggcgagcc gctggaccag 900ttcctgtggc gcaagcggga
cctgtaccag acgctctacg tggacgcgga cgaggaggag 960atcatccagt
acgtggtggg caccctgcag cccaagctca agcgtttcct gcgccacccc
1020ctgcccaaga ccctggagca gctcatccag aggggcatgg aggtgcagga
tgacctggag 1080caggcggccg agccggccgg cccccacctc ccggtggagg
atgaggcgga gaccctcacg 1140cccgccccca acagcgagtc cgtggccagt
gaccggaccc agcccgag 1188301188DNAOrcinus orca 30ggggaattgg
atcaacgtac taccggtggc cttcacgcat accctgcacc acgcgggggc 60cctgtcgcga
agccaaatgt catcctgcag attgggaagt gccgggctga gatgctggag
120cacgtccgtc ggacgcatcg tcatcttctt actgaggtgt caaaacaggt
ggagcgtgaa 180ctcaaaggct tgcaccgcag cgttgggaaa cttgaaagca
acttagatgg ctatgtgccg 240actggcgaca gccagcgttg gcgtaagtcc
atcaaagcat gtttgtgtcg ttgccaggaa 300acgattgcaa acctggagcg
ttgggtcaaa cgggagatgc atgtctggcg tgaagtattt 360tatcgtttag
agcgttgggc cgatcgttta gagagcatgg gtggtaagta ccctgtgggg
420agcaaccctt ctcggcatac gacgtcagtc ggtgttggcg ggccggagtc
ctacggtcat 480gaagcggaca cctacgacta taccgtaagc ccttatgcta
ttaccccacc acctgcggcc 540ggcgaattac ctggccagga agccgttgag
gctcaacaat accctccttg ggggctgggc 600gaggatggtc aacctagccc
aggggtagac acgcaaatct ttgaggaccc acgggagttt 660ctttcccacc
tggaagaata cctgcgtcag gttggtggga gcgaagaata ctggctgtca
720caaattcaaa accatatgaa tggtcctgca aaaaaatggt gggaatataa
acagggttcc 780gtgaaaaact gggttgagtt taaaaaggag tttcttcaat
attccgaggg cgccctcagt 840cgggaggcgg tccaacgcga gttggacttg
ccacagaaac agggggaacc actcgatcaa 900ttcctttggc ggaaacgtga
cctttaccag acattgtacg tggatgcaga tgaggaagaa 960attatccaat
atgttgtggg gaccctgcag ccgaaactga aacgtttcct tcgcccgccg
1020ctgcctaaaa cgttggaaca acttattcag aaaggtatgg aggtcgagga
tggcttagaa 1080caagtcgcag agccggcctc gccacacttg cctacagagg
aggaatcgga ggcgctgacc 1140ccagcactta catcagagtc agtggcatca
gaccggacac aaccagag 1188311170DNAOdocoileus virginianus texanus
31ggggagttag atcaccgtac aacggggggg ttgcacgcat accctgctcc acgtggcggg
60ccggcagcta agccaaacgt aatcctgcag attgggaagt gccgggcaga gatgttggag
120cacgtccggc ggacccaccg gcacctcctg gctgaagtgt ctaaacaagt
agaacgggaa 180ctcaaaggtc ttcatcgtag cgtcgggaaa ttggaatcga
atttggacgg gtatgttcct 240acaggcgact cacagcggtg gaaaaagagc
atcaaggcct gcctgagtcg ctgccaggag 300acgattgcta acctcgaacg
ctgggttaag cgggagatgc acgtttggcg cgaagtcttc 360taccggctgg
agcgttgggc tgatcggctc gaatctggtg ggggtaagta tccagttggg
420tccgaccctg ctcgccacac agtctcagtt ggcgtaggtg ggccggagtc
gtattgccaa 480gatgcggaca actatgatta tacagtttcc ccatacgcga
tcacaccacc gccggcagca 540gggcagctgc caggtcagga agaggttgag
gcccagcagt atccaccatg ggccccaggg 600gaagacggcc agctttctcc
tggggtggac actcaagttt ttgaagatcc gcgtgaattt 660ctgcggcatt
tagaagatta tctccgccag gtcggggggt ctgaagagta ttggttaagc
720caaattcaaa accatatgaa cggcccggcc aagaagtggt gggagtacaa
gcaagggtct 780gtgaaaaatt gggtggagtt taagaaagaa ttcttgcaat
attctgaggg cactctttcg 840cgtgaagcca tccaacgcga actcgactta
ccgcagaaac aaggggaacc tctcgaccaa 900tttctgtggc gcaaacgcga
cctgtaccag actctttacg tcgatgctga ggaggaagaa 960attattcaat
acgtagttgg cacactgcag cctaagctta aacggttttt acgtccacca
1020ttgccgaaga cgcttgaaca actcatccag aagggtatgg aggttcaaga
tggtctggaa 1080caggcagcgg aaccagcggc ggaggaggca gaagccctga
cacctgcgtt aactaacgag 1140tctgtcgcga gcgaccgcac ccagccggaa
1170321203DNAOrnithorhynchus anatinus 32ggggaattag accgcctgaa
cccaagctca ggcctgcatc catcctctgg tttgcatcca 60tacccaggtc tccggggcgg
ggcaaccgcg aagcctaatg tcattttgca aattggcaaa 120tgccgtgcgg
aaatgcttga acacgtccgc aaaactcacc gtcatctcct cacagaagta
180tcgcgccaag tagaacgcga gctcaaaggc cttcacaaaa gtgttggcaa
gttggaatca 240aatcttgatg ggtacgtacc gtcaagcgac tcccaacgct
ggaagaaaag cattaaggcg 300tgcttatccc gttgccaaga gacgattgcg
catttagaac gctgggttaa acgtgaaatg 360aatgtatggc gtgaggtgtt
ctaccgtttg gaacgttggg cggaccgtct ggaggctatg 420ggcggtaagt
atcctgccgg tgagcaggcc cggcgtacag tttcagtggg cgttgggggc
480cctgagacat gttgtccagg ggatgaaagt tatgattgtc cgatttctcc
gtatgcagtt 540ccaccttcca ccggcgagtc tccggaatcc ttagaccaag
gggatcagca ctatcagcag 600tggtttgccc tcccggagga gtcccctgtt
agccctgggg ttgataccca gatctttgaa 660gatcctcgcg agtttttacg
tcatctggag aagtacctga aacaagtcgg cgggacagag 720gaagactggc
tttctcaaat ccagaatcac atgaatgggc cggcgaagaa gtggtgggag
780tacaagcaag ggagtgttaa gaattggctt gaatttaaga aggaattttt
acagtattcg 840gagggcacac tgacgcggga cgcgttgaaa cgtgaactgg
atctcccaca gaaacaaggc 900gaaccacttg atcaattttt atggcggaag
cgcgacttat atcagacact ctacgttgac 960gccgatgaag aggaaatcat
tcagtacgtc gtgggcactc ttcagccgaa attaaaacgc 1020tttctccatc
acccactccc taagacgctt gagcagctta tccaacgggg ccaagaagtt
1080cagaatggtc tggagcctac cgacgatcct gcaggccaac gcactcaatc
ggaggacaac 1140gacgaaagcc ttacccctgc cgtcaccaat gagagtactg
caagcgaggg caccctgcca 1200gag 1203331212DNAAnser cygnoides
domesticus 33gggcagcttg ataacgttac aaacgcgggc atccactcct tccaggggca
tcgtggcgta 60gcgaataagc caaatgtcat tctgcaaatt ggtaaatgtc gtgcggaaat
gctggagcac 120gttcgccgca cccaccgcca tttattatct gaagtatcta
agcaggtaga acgtgagctg 180aaagggctgc aaaagtccgt gggcaagctc
gagaataact tggaggatca tgtccctaca 240gataaccaac gctggaagaa
gtccattaaa gcgtgcttgg ctcgttgtca agagactatc 300gcgcatttag
agcgttgggt gaaacgcgaa atgaacgtct ggaaggaggt gtttttccgg
360ctggaaaagt gggcagaccg gctggagtca atgggtggca agtactgccc
gggcgaacac 420gggaaacaaa ccgtcagtgt aggcgtgggg ggtcctgaaa
tccggccttc ggagggggaa 480atttatgatt atgctctgga tatgagccag
atgtatgcac tcaccccacc tccaggcgaa 540atgccatcaa tcccacaagc
ccatgacagc tatcagtggg ttagtgtctc agaagatgcc 600ccggcgagcc
ctgtcgaaac ccaggtattt gaggaccctc gggaattcct gtctcacctg
660gaggaatacc tgaagcaggt aggcggcacg gaggagtatt ggttgtccca
gatccagaat 720cacatgaatg gtccggcaaa aaaatggtgg gaatataaac
aggactccgt taaaaactgg 780gttgagttta aaaaggaatt cttgcaatac
tctgaaggta ctttaactcg ggatgctatt 840aagcgtgaac tcgacttgcc
gcaaaaggaa ggtgaacctc ttgaccaatt cctttggcgg 900aagcgggacc
tctatcagac actttacgtg gacgcggatg aggaggagat cattcagtat
960gtggtcggta ccctgcagcc gaagctcaag cgtttcctga gctatcctct
cccaaagact 1020ttagaacagc tcatccagcg cggtaaagaa gtgcagggta
acatggatca ctccgatgag 1080ccttcgccgc agcgtacacc tgaaattcaa
tcaggtgact ccgtagaatc tatgccacct 1140tcaacaacgg catctccggt
tccatctaat ggtacccaac ctgagccgcc gagcccgcca 1200gccaccgtta tc
1212341185DNAPelecanus crispus 34gggcaacttg acaacgtaac aaacgctggg
attcactcct ttcagggcca ccgcggtgtc 60gccaacaagc caaacgtaat cttgcaaatt
ggcaaatgcc gtgcggagat gttggaacac 120gttcgtcgta cacatcgtca
cttgctgtcg gaagtctcta aacaagtaga acgtgaactt 180aaagggcttc
aaaagtcagt cggcaaattg gaaaacaacc ttgaagacca tgtaccaacc
240gacaatcagc gttggaaaaa gtctatcaaa gcttgcctgg cccgttgtca
agagacgatt 300gctcacctgg agcggtgggt aaagcgcgag atgaatgtgt
ggaaagaggt cttcttccgc 360ttggaaaaat gggccgaccg tttggagtcc
atgggcggta aatattgtcc gggtgaacat 420ggtaagcaaa cagtctctgt
gggcgttggt gggccggaga ttcggccttc tgaaggcgag 480atttacgatt
atgcgctcga catgtcccag atgtatgcgc ttacaccacc accgggcgag
540gtaccaagca ttcctcaagc gcatgacagt tatcagtggg ttagcgtatc
cgaagacgct 600cctgcctcgc cggtagagac ccaggttttt gaagatcctc
gtgaattttt aagccacttg 660gaggagtatt tgaagcaggt aggggggaca
gaggaatatt ggctgtctca gatccagaac 720cacatgaatg gcccggctaa
aaagtggtgg gaatacaaac aagattcggt aaagaattgg 780gtagaattta
aaaaggagtt tttacagtac tcagagggga ctctcacgcg tgatgcgatc
840aaacgcgagt tggatcttcc tcaaaaagag ggggagccac tcgatcagtt
cctctggcgc 900aagcgggatc tctaccaaac actctacgta gacgcagacg
aagaagagat catccagtac 960gtggtgggta cgctccagcc gaaactcaaa
cgtttcctca gctacccact tcctaagact 1020ctggaacaac tgattcagcg
gggcaaagag gtccagggta acatggacca ttcagaggaa 1080cctagtccgc
aacgtacacc tgagatccaa tctggggatt ctgtcgattc ggttccacct
1140tctacaacag cgtctccggt gccgtcaaat gggacccaac cagag
1185351185DNAHaliaeetus albicilla 35gggcagcttg ataatgtaac
caatgcaggt atccactctt tccagggtca ccgcggtgtg 60gcaaacaagc caaatgttat
tctgcaaatt ggtaagtgtc gcgctgagat gttagaacac 120gtccggcgca
cgcatcggca tctcctgtca gaggtttcaa agcaggtaga gcgtgaatta
180aagggcctcc agaagtccgt aggtaaactc gaaaataatc ttgaagacca
cgttcctacc 240gataatcaac ggtggaaaaa gtcaatcaag gcgtgcttag
cacggtgtca ggaaacgatc 300gcgcacctcg aacgttgggt gaagcgcgaa
atgaatgtct ggaaagaagt gttcttccgg 360cttgagaagt gggctgatcg
gctcgaatcc atgggtggca aatattgtcc aggtgatcat 420ggcaagcaaa
cggtctccgt cggtgttggt ggtccggaaa tccggccgag cgagggtgaa
480atctatgact acgctcttga tatgtcccag atgtatgcac tcactcctcc
gccgggtgag 540gtcccgtcga tcccgcaggc gcatgactca taccaatggg
tgtcgactag cgaagacgca 600ccagcctccc ctgttgaaac tcaagtattc
gaggacccgc gtgagttcct gagccattta 660gaggagtacc ttaagcaggt
tggtggtacc gaggaatact ggttgagcca gattcagaat 720cacatgaacg
ggccggctaa gaaatggtgg gaatacaagc aggattcagt caagaattgg
780gtcgaattta agaaggagtt tttgcagtac agtgagggga cgctcacacg
cgacgctatc 840aaacgggagc tggacctgcc acaaaaggag ggtgaaccgc
ttgatcagtt tctttggcgc 900aagcgtgatc tgtatcaaac cctgtatgtg
gacgctgacg aagaagagat cattcagtac 960gtggttggga ctctgcaacc
aaagctgaag cgttttcttt cttatcctct ccctaagaca 1020ctggaacagt
taatccaacg tggcaaggag gtccagggta atatggacca ctctgaggaa
1080ccgagcccgc aacgtactcc tgaaattcag agcggggata gtgtcgactc
agttcctcca 1140agtacgaccg catccccggt cccaagtaac ggtacccaac cagag
1185361395DNAOphiophagus hannah 36gggtcttggg gcttgcaacg
tcacgtggct
gatgaacgtc gtggcctcgc tacgcctacc 60tacggcgcgg tttgttccat tcgggagaaa
aaagcctccc aactgagcgg ccagagctgt 120ttggagaaag agttgcttgg
ttggaaatgt acggaggcaa tcgtggaaat gatgcaagtc 180gataacttta
accacggtaa cttacatagc tgccaaggcc atcgggggat ggcaaatcac
240aaaccgaacg taatccttca aatcgggaaa tgtcgcgcag aaatgttaga
ccacgtgcgt 300cgcacccacc gccatctctt gacggaggtt tcgaagcagg
tagaacgcga attgaagtct 360ctccaaaagt cggttggcaa gctcgagaat
aatctggaag accacgtgcc atcggcagcg 420gagaaccaac gttggaagaa
atcaattaaa gcctgcctgg cccggtgcca agaaacaatt 480gctcacctcg
aacgctgggt taaacgcgaa atcaacgtct ggaaagaagt attctttcgt
540ctggagaagt gggcggaccg ccttgagtcg ggtgggggca agtatgggcc
tggtgaccaa 600agtcgtcaaa ctgtaagtgt cggtgttggg gccccagaaa
tccaaccgcg gaaagaagaa 660atctatgact acgctctcga catgtcgcag
atgtatgcct taacaccacc gccgatgggt 720gaagacccaa acgtacctca
atcccacgat agctaccagt ggattaccat ctcagacgat 780tcacctccgt
cgccagtgga aactcaaatt ttcgaggatc cacgcgaatt ccttacccat
840ctcgaggatt atcttaagca agtgggcggg actgaagaat attggttgag
tcagattcaa 900aatcatatga acggtccggc caagaaatgg tgggagtaca
aacaagattc cgtgaaaaac 960tggttggaat tcaagaagga attccttcaa
tactctgagg gtactttgac acgtgacgca 1020attaaacaag aacttgactt
accgcagaag gacggcgagc cattggatca atttctttgg 1080cggaagcggg
acctgtatca gacgctctat attgatgcag aggaggaaga agtaatccaa
1140tacgttgttg gcacactcca accgaaatta aaacgtttcc tttcccaccc
gtatccgaaa 1200actttggaac agttaatcca acgtgggaaa gaggtggaag
gcaacctcga taactctgag 1260gagcctagcc cgcaacggag tccaaagcac
caattgggtg gtagcgtcga gagcctccca 1320ccttcgtcga ccgcaagtcc
tgttgcgtca gacgagactc acccagacgt gagcgcacct 1380ccggtaacgg tgatt
1395371353DNAAustrofundulus limnaeus 37ggggacggcg agactcaagc
tgagaatcca tctaccagct tgaacaacac tgacgaagat 60atcttggaac agctcaagaa
aattgtcatg gatcaacaac acctgtatca gaaagaatta 120aaggcatctt
ttgaacaact cagtcgcaaa atgttttccc agatggaaca aatgaatagc
180aagcaaacgg atctgctttt agaacatcaa aaacagactg tcaaacatgt
agacaagcgc 240gtggagtatt tgcgggcgca attcgatgca tcgttaggct
ggcggttgaa agagcaacac 300gcggatatta cgaccaaaat cattcctgag
atcatccaaa cggtgaagga agatattagc 360ctgtgtcttt ctacgctctg
cagtatcgct gaagatatcc agacatcacg ggctaccact 420gtcacagggc
atgctgccgt acaaacccat cctgtggatc ttttgggtga acaccattta
480gggaccacgg ggcacccacg cttacagtcg acccgtgtag ggaaaccaga
cgacgtacct 540gagtcgccgg taagcctgtt tatgcaaggt gaggcgcgtt
cccggatcgt tggcaagagt 600ccgattaaac tgcaatttcc gacgttcggc
aaagcaaacg attcttccga cccactccaa 660tatctggagc ggtgtgagga
ctttcttgct cttaaccctt taactgatga ggaacttatg 720gctactttgc
ggaatgtgtt acatggcacc tctcgggatt ggtgggatgt cgcacgtcat
780aaaatccaaa cttggcgtga gtttaataaa cacttccggg cggctttcct
cagcgaggat 840tatgaagatg agttggctga gcgcgtccgt aaccgcatcc
aaaaagaaga tgagtctatc 900cgcgatttcg cttatatgta tcagtccttg
tgcaagcggt ggaaccctgc tatctgcgaa 960ggtgatgtag taaagctcat
cctgaagaac atcaatccac aactgccgtc tcagttacgc 1020tcccgggtca
cgaccgtgga tgagcttgtt cgcttgggcc agcagcttga aaaagatcgt
1080cagaatcagc tccaatatga gcttcggaag agttccggca aaattatcca
aaaatctagt 1140tcgtgcgaaa cttcagcgct cccgaacacg aagagtacac
ctaatcaaca aaaccctgct 1200accagtaacc gtcctccaca ggtgtattgc
tggcggtgta agggtcacca tgcccctgcc 1260tcttgtccgc aatggaaagc
tgataagcac cgtgcgcaac cttcgcggag ttctgggcca 1320caaactctga
ctaatctcca agctcaagac atc 1353381188DNAPhyseter catodon
38ggggaattgg atcaacgtgc ggcagggggc ttgcgcgcgt acccggcgcc gcgtggtggt
60ccagttgcca aaccgagcgt aattcttcag attggtaagt gccgcgctga gatgctggaa
120cacgtccgcc gcacgcatcg ccatcttctg acggaggtaa gtaaacaagt
ggagcgcgaa 180ctcaaggggt tacatcggtc tgtcggtaag ttggagggca
atttagacgg ctatgtgcct 240accggtgatt cccaacgctg gaaaaaaagt
atcaaggcgt gtctctgccg gtgtcaggaa 300acaattgcaa atctcgagcg
ttgggtgaaa cgtgagatgc atgtttggcg tgaggtattc 360tatcgtttgg
aacggtgggc agaccgtttg gagtctatgg ggggcaagta tccggtgggc
420actaacccgt cgcggcacac agtaagtgtc ggggtagggg gcccggaagg
ctattctcat 480gaagcggata cttatgacta cacggtgtct ccgtatgcta
tcacgccacc gcctgccgcg 540ggtgagttgc ctggtcaaga ggctgtcgag
gcacaacagt accctccatg gggtctgggg 600gaggacgggc aaccaggtcc
gggcgtggac acgcagattt ttgaggaccc tcgcgaattt 660ttgagccact
tagaggagta cctgcggcaa gtagggggga gtgaagagta ctggttatcg
720caaattcaaa atcatatgaa tggccctgcg aagaaatggt gggagttcaa
acaggggtca 780gtcaagaatt gggtcgagtt taagaaagaa tttttgcaat
acagtgaggg tacgttgagt 840cgcgaggcca tccaacgtga actggacctc
cctcagaagc agggggagcc gttagatcaa 900tttttatggc ggaaacgtga
cttataccaa accctctacg ttgacgctga ggaagaagaa 960attattcaat
atgttgtcgg tacgctgcag ccaaagctga agcggttcct ccgtcctcca
1020ctccctaaaa ccttagaaca attaatccaa aaaggcatgg aagttcagga
cgggttagaa 1080caagcggccg aaccggcctc tccgcgtctg ccgccggaag
aggagagtga ggctcttacg 1140cctgcgctca cgagcgaatc agtagcctcc
gatcggacac agccagag 1188391212DNAMeleagris gallopavo 39gggcagcttg
acaatgtgac gaacgcgggg attcacagct ttcaagggca ccgcggcgtc 60gccaacaaac
cgaatgtcat tctgcaaatc ggtaaatgtc gtgctgaaat gcttgagcac
120gttcgtcgta cccatcgtca cttgctttct gaagtatcaa aacaagtgga
gcgggaactc 180aaaggcctgc aaaagtcagt gggtaaattg gagaataacc
tcgaagacca tgtacctaca 240gacaaccagc ggtggaaaaa atctatcaag
gcatgcctcg ctcgttgcca ggagactatt 300gcccatcttg agcggtgggt
gaaacgtgaa atgaacgtat ggaaggaagt attttttcgc 360ttagagaagt
gggctgatcg tcttgaatcg atgggcggca agtactgtcc tggggaacac
420ggcaaacaaa ctgtatctgt cggcgtgggg ggcccggaga tccggccatc
ggaaggggaa 480atttatgatt atgctctcga catgtcccaa atgtatgctc
tcacaccagg gccaggggaa 540gtaccgtcaa ttccgcaagc acacgacagc
taccaatggg tatctgtgag cgaggacgcg 600cctgcctctc cggttgagac
gcaaatcttt gaggacccac atgaattttt gtctcatctt 660gaagaatatc
tcaaacaggt tggcggcaca gaagaatact ggttatctca gatccagaat
720cacatgaacg gcccggctaa aaagtggtgg gagtataagc aagattccgt
aaagaactgg 780gtcgaattca agaaagagtt tcttcaatac tctgagggta
ctctgacgcg cgatgcaatt 840aagcgggagt tagaccttcc acaaaaagag
ggggagcctc ttgaccagtt cctgtggcgt 900aagcgcgacc tctatcagac
actttacgtc gacgctgatg aagaagagat tattcaatat 960gttgtgggta
ccctgcagcc aaagcttaag cgtttcctta gctacccact tccgaaaact
1020ctggagcagc tcattcaacg cggtaaggaa gtgcagggca acatggacca
ctctgaagag 1080cctagcccgc agcgcactcc tgaaatccaa tcaggtgaca
gtgtggagtc aatgccgccg 1140tcaaccaccg cttctccggt acctagcaac
gggacgcaac cagagcctcc aagcccaccg 1200gctacagtca tc
1212401227DNAPogona vitticeps 40gggcaacttg agaatattaa ccaaggttcc
ctgcacgcgt ttcagggtca tcgcggcgtg 60gtccataaca acaagcctaa cgttattctc
cagatcggga agtgccgcgc cgaaatgctg 120gagcatgtgc ggcgcaccca
tcgccatttg ctcactgaag tatcaaaaca ggtggagcgt 180gagttgaagg
ggttgcagaa aagtgtaggc aaacttgaaa ataatttaga agaccacgta
240ccaagtgcgg ctgagaacca acgctggaag aagtcgatta aagcctgctt
agcgcgttgt 300caggagacca ttgcgaactt ggaacgctgg gttaaacgtg
agatgaatgt ttggaaggag 360gtctttttcc gcttagagcg ctgggcagat
cgcctcgaat ccgggggtgg caagtactgc 420catgcagacc agggtcgcca
aactgtcagc gtaggtgttg gtggtcctga agtgcgtccg 480tctgaaggtg
aaatttacga ttacgcgttg gatatgagcc aaatgtacgc cttgactccg
540ccgcctatgg gtgatgttcc agtaattcct cagccgcatg acagttatca
gtgggtgaca 600gatccggaag aagcgccacc aagtccggtt gagacacaaa
ttttcgagga ccctcgggag 660tttctgaccc atcttgagga ttatttaaaa
caagtcggcg ggacagagga atattggctc 720tcacagatcc aaaatcatat
gaatgggcca gcgaaaaagt ggtgggaata taaacaggat 780agtgtgaaga
actggcttga gttcaaaaaa gaattcttgc agtactcaga aggcacgtta
840acgcgggacg ctattaaaca ggaacttgac cttccacaaa aagaagggga
accgctggat 900caattcctct ggcgcaaacg cgatttgtac caaactctct
acgtcgaggc agaagaagag 960gaggtcatcc aatatgtagt tggcacactg
caaccaaaac tgaagcggtt tctttctcat 1020ccgtacccta aaaccctgga
gcaactcatc cagcgcggga aggaagttga ggggaatttg 1080gacaatagtg
aagaaccgtc tccacagcgg accccagaac atcagctggg ggacagtgtg
1140gaatctttgc cgcctagtac tacggcttcg cctgccggtt cggataaaac
gcaacctgag 1200attagcttac ctccaactac agtcatt 1227411212DNAAlligator
sinensis 41gggcaattag attcggtaac caatgcgggc gtccacacct accagggcca
tcggagcgtc 60gccaataaac ctaacgtcat tcttcaaatc gggaaatgtc ggactgagat
gctggagcat 120gtccgtcgga ctcatcgcca cctgctcaca gaagtgtcaa
agcaagtgga acgtgaactc 180aagggcttac agaagagcgt gggcaaactg
gaaaacaatc ttgaagacca tgtcccaact 240gacaatcagc ggtggaagaa
gtcaatcaag gcatgtctcg cgcgttgcca agagaccatt 300gctcaccttg
agcggtgggt gaaacgtgaa atgaacgtgt ggaaggaggt gttcttccgg
360ttagaacgct gggccgaccg ccttgaatca atgggtggta aatactgccc
gacggactct 420gcacgtcaga cagttagcgt tggggtgggg ggcccggaaa
ttcggcctag tgaaggcgaa 480atctatgact acgcgctcga tatgagccaa
atgtacgctc ttacgccgtc accgggcgaa 540ttgccgtccg tccctcaacc
gcatgattca taccagtggg tcactagtcc ggaagacgct 600ccggcgtcac
cagttgaaac gcaggtattc gaggatcctc gggagttctt gtgtcatttg
660gaagagtacc tgaagcaggt tggcggtaca gaggaatatt ggctgagcca
gattcagaat 720catatgaatg gtcctgcaaa aaagtggtgg gaatataaac
aagacacggt taagaattgg 780gtggaattca agaaggagtt cttacaatac
agtgagggta cacttacccg tgatgcgatt 840aagcgggaat tagacctccc
gcaaaaggac ggtgagcctc tggatcaatt tttatggcgt 900aagcgtgacc
tctatcagac attatacatt gatgccgatg aagaacagat cattcagtac
960gtcgtgggga cattgcaacc taaactcaag cggttcttgt cctatccact
tccaaaaact 1020cttgaacaat taatccagaa agggaaggag gtgcagggtt
cacttgacca cagcgaggag 1080ccgagtcctc aacgtgcgag cgaggctcgg
acgggcgata gtgtggaaac cttgccgcct 1140tctaccacta catcaccaaa
tacgtcatct ggtacacagc cagaggcacc atcgcctcca 1200gcgacggtaa tc
1212421212DNAAlligator mississippiensis 42gggcagttag acagtgtgac
taacgccggg gtgcatacgt accaggggca ccgcggggtc 60gccaataagc caaatgtaat
tctccagatt gggaagtgtc gtacagagat gttggaacat 120gtccgtcgca
ctcatcgcca cttgctcacc gaggtctcca aacaagtaga acgcgaactc
180aaggggctcc agaagagtgt tgggaagttg gagaataacc tcgaagacca
cgttccgaca 240gataaccaac ggtggaaaaa gtctattaaa gcctgtctcg
cccgttgtca agagacaatc 300gcacacttgg aacgctgggt caaacgggag
atgaatgtgt ggaaggaagt cttcttccgt 360ctcgagcggt gggcggatcg
tttagaaagt atgggcggta aatattgccc aactgactcg 420gctcgtcaaa
cggtgtcggt tggcgtaggc ggcccggaaa ttcgccctag cgagggtgag
480atctatgact atgcacttga catgagtcag atgtatgcgt taactccgtc
gccaggggag 540cttccaagta ttccacagcc tcacgatagt tatcaatggg
taacttctcc tgaagacgcc 600ccagcatccc cagttgagac acaagtattc
gaggaccctc gtgagtttct ctgtcacctc 660gaggagtacc ttaaacaggt
aggcgggacc gaagagtact ggttatcgca aatccaaaac 720catatgaatg
gtcctgccaa aaagtggtgg gagtataaac aagatactgt gaagaattgg
780gtagagttca agaaagagtt cttacagtac tctgagggga cgttaactcg
tgatgcgatc 840aagcgcgaat tggatttacc tcagaaggac ggcgagccac
tcgaccagtt cttatggcgc 900aagcgtgact tgtatcaaac cctttatatc
gatgctgacg aggaacaaat tatccagtac 960gtagtcggta cgttgcaacc
aaaacttaaa cgctttctga gctacccatt acctaaaacg 1020ttggagcaac
tgatccagaa aggtaaagag gtgcaaggga gcctggatca tagtgaagaa
1080ccgagccctc agcgggcttc tgaagctcgg accggtgata gcgtcgaatc
tttaccacct 1140agtaccacaa ccagcccgaa tgcgtcatct ggtacccaac
ctgaagcgcc ttccccacct 1200gctacagtca tt 1212431224DNAGekko
japonicus 43gggcagctcg agaatgtcaa ccatgggaac ctccattctt ttcaaggtca
tcgcggcggc 60gtcgccaaca agccaaacgt tatcttgcag atcggtaaat gtcgtgcaga
gatgctggac 120cacgtccggc ggacccaccg gcatttactg acagaggtat
cgaaacaggt tgaacgtgag 180ttgaaggggt tacagaaatc agtagggaaa
ttagaaaata acttagaaga ccatgtccct 240tcagccgttg aaaaccagcg
ttggaaaaaa tcgatcaagg cctgcctttc ccgctgccaa 300gagaccattg
cccaccttga gcgttgggtg aagcgcgaga tgaacgtatg gaaagaggtt
360ttcttccgct tagagcggtg ggcagatcgg ttggaatctg ggggcgggaa
atattgtcac 420ggtgataatc atcgtcaaac agtatcagtc ggtgttggcg
gccctgaggt acgtccatct 480gaaggcgaaa tttacgatta cgctctcgac
atgtcgcaaa tgtacgcttt aacaccgcct 540agcccagggg atgtgcctgt
agttagccag ccgcacgaca gctatcagtg ggttacggtt 600ccggaggata
cccctccatc cccggtggag acgcaaatct tcgaggaccc acgggagttc
660ttgacccact tagaggatta cttaaagcaa gtggggggta cagaggaata
ttggttatct 720cagatccaga atcacatgaa cgggccagcc aagaagtggt
gggagtataa gcaagactca 780gtaaaaaatt ggctcgagtt taagaaggaa
ttccttcagt attccgaggg gacacttacg 840cgcgacgcta tcaaggaaga
acttgacctc ccgcaaaagg acggggaacc tcttgatcag 900ttcctgtggc
gcaagcgcga cttgtaccag accctgtacg tggaggcgga tgaggaggag
960gtgatccagt atgttgtggg gactttacaa cctaaattaa agcgttttct
ctcacaccct 1020tacccgaaaa cgttagagca acttatccaa cggggcaaag
aggtggaagg gaacctcgac 1080aattcagagg aaccaacacc tcagcgtact
ccagaacacc aactgtgtgg ttctgtagaa 1140tcgctgcctc cttcctctac
cgtcagtcca gtggctagcg atggtactca acctgagact 1200tcgccattgc
cagcgactgt tatt 1224441365DNAHomo sapiens 44gggccattga cgttgttaca
agactggtgt cgtggtgaac atttaaacac ccgccggtgc 60atgttgatcc tcggtatccc
agaagattgc ggcgaggatg agttcgaaga gacacttcag 120gaggcgtgtc
gccatttagg gcggtaccgc gtgatcggcc gcatgttccg tcgtgaggaa
180aatgcccaag cgatcctctt ggaattggcg caggatattg actatgcctt
actccctcgg 240gaaatccctg ggaaaggcgg gccttgggag gtaattgtga
agccgcgtaa ttccgacggc 300gaattcttaa atcggcttaa tcgctttctt
gaagaggagc gccgtacggt ctccgatatg 360aaccgtgttt tgggctcgga
tactaactgt tcagctcctc gtgtcaccat tagtcctgaa 420ttctggactt
gggcacagac gctgggcgca gctgtccaac cattgctcga acagatgctc
480taccgggagt tacgggtctt cagtggcaat acgatttcca tcccaggtgc
tctcgctttt 540gacgcgtggc tggagcatac cacggaaatg cttcaaatgt
ggcaggtgcc tgaaggggag 600aaacggcggc gcttgatgga gtgtttgcgg
gggccagccc tgcaagtcgt tagtgggtta 660cgtgcatcga atgccagtat
cactgtcgaa gagtgtcttg ctgcactgca gcaggtattc 720ggtccagtgg
aaagtcataa gattgcccaa gtaaagttat gcaaagctta ccaggaggct
780ggggaaaaag taagcagctt cgttttgcgt ttggagccac tgcttcagcg
tgctgtagaa 840aacaacgtgg tcagtcgccg caatgtcaac caaacacgtc
ttaagcgtgt tctgtcgggc 900gccacccttc ctgacaagct gcgtgataaa
ttgaagttaa tgaaacagcg ccgtaaaccg 960ccgggtttct tggcgttggt
taaactgtta cgtgaagagg aggagtggga ggccacctta 1020gggccagacc
gcgagtcatt ggaggggtta gaagtggcac cgcgcccgcc agcacggatt
1080acgggtgttg gcgcagtacc tcttccggca tccgggaatt catttgatgc
ccgtccttcg 1140caagggtacc ggcgccgtcg gggtcgtggt cagcaccgtc
ggggcggcgt tgctcgtgca 1200ggctctcgtg gctctcgtaa gcggaaacgg
cacaccttct gctattcctg tggtgaggat 1260ggccatattc gtgtccaatg
cattaaccct agcaatctcc tgttggctaa ggagaccaaa 1320gagattttgg
aagggggaga acgtgaagcg caaacgaatt cacgt 1365451344DNAHomo sapiens
45ggggctctta cgctcttaga agactggtgt aagggtatgg acatggaccc gcggaaggct
60ctcctgattg taggtattcc gatggaatgc agtgaggtgg aaatccagga tacagttaaa
120gctggtcttc aacctctgtg cgcttatcgt gtactcggcc gtatgttccg
gcgggaggat 180aatgcgaagg ctgttttcat tgagctggca gacaccgtga
attacaccac gttaccgtct 240cacattccgg gtaaaggggg ttcctgggaa
gtcgttgtta aacctcggaa ccctgacgac 300gagttccttt ctcggcttaa
ctacttcttg aaagatgagg gccgctcgat gacggatgtc 360gcccgggcac
tggggtgctg tagcttacct gcggaatcac tggacgcgga agtaatgcca
420caggtccgct ccccaccatt agaacctcca aaagagagta tgtggtaccg
taagttaaaa 480gtgtttagtg gtaccgcgtc gccttcgccg ggggaggaga
catttgagga ctggttagag 540caagtcaccg agatcatgcc tatctggcaa
gtatctgaag ttgaaaagcg ccgtcggtta 600ctggagtcac tccggggccc
ggcactctca attatgcgcg tgttacaagc caataacgat 660agcattaccg
ttgaacagtg tttggatgca ttaaagcaga tctttggcga caaggaagac
720ttccgtgcct ctcaatttcg ttttcttcaa acgtccccta aaattgggga
gaaggtgagt 780acgttcctgc tgcgtttaga gccactcttg caaaaggccg
ttcacaagag cccactttcg 840gtacgtagta ctgatatgat tcggttaaag
cacctgttgg cacgcgtagc catgaccccg 900gcactgcgtg gtaaactcga
attactcgac caacgcgggt gcccacctaa ttttcttgag 960ctgatgaagc
tgatccggga tgaggaagag tgggagaata ctgaagctgt gatgaaaaat
1020aaagagaaac cttcaggtcg tggccgcggt gcatcaggcc gtcaagctcg
cgccgaggcc 1080agtgtaagtg ctccgcaagc aacagtccaa gcacgtagct
tctctgattc tagcccgcag 1140acgattcagg ggggcttacc acctcttgtc
aagcgtcggc gccttttggg ttcggagagc 1200acacgtgggg aagaccacgg
gcaagctact tatccgaaag cagagaatca gactccaggg 1260cgtgagggcc
cgcaggcggc tggggaggaa cttggtaatg aggccggggc cggcgcgatg
1320tcccacccga aaccgtggga aacc 1344461197DNAHomo sapiens
46ggggctgtga caatgctcca ggactggtgc cgttggatgg gcgtgaacgc tcggcggggg
60ctgttaatct taggtatccc tgaagactgt gacgatgcag agttccaaga gtcgttagaa
120gctgcactcc gtcctatggg tcactttact gtactcggta aggccttccg
cgaggaagac 180aacgctaccg ctgcgctggt ggaattagat cgcgaggtta
attacgcact tgttccacgc 240gaaattccgg gcaccggcgg gccttggaac
gtcgtgttcg ttcctcggtg ctccggcgag 300gaattcctgg ggttaggccg
cgtgttccac tttcctgaac aggagggcca aatggtagaa 360tcggttgcgg
gggcactggg ggtaggtctg cgccgcgtgt gttggttacg ctcgatcggg
420caagctgtac aaccatgggt agaagctgtt cgctgccaaa gcttaggggt
atttagtggt 480cgtgatcaac ctgcacctgg tgaagaaagc ttcgaggtct
ggttggatca tacgaccgag 540atgttgcatg tgtggcaagg cgtgtcggaa
cgggaacggc gccgtcgtct gctggaaggg 600ctgcgtggca cagccttaca
acttgtacat gccttactgg cagaaaatcc ggcacggaca 660gcacaagatt
gcttggctgc attagcccaa gtttttggtg ataacgaaag ccaggcaacg
720attcgtgtta aatgtttgac agcccaacag cagagtggcg aacgcctctc
tgcgttcgtt 780ctccgcttag aagtacttct gcaaaaggct atggagaagg
aagcattggc gcgcgcgtca 840gcggatcggg tgcgtcttcg tcagatgctg
acacgcgcac atctcacaga gccgttggat 900gaagccttac ggaaattgcg
tatggcaggg cgttctccgt cttttttgga aatgctcggc 960ttagtacgcg
agtcagaggc ctgggaggca agtctggctc ggtccgtccg ggcgcaaacc
1020caggagggtg caggggcccg ggcgggggcc caagcagttg cgcgtgccag
cactaaggtt 1080gaagctgtac ctggtggccc tggccgggag ccagaaggtc
tcctccaagc cgggggccaa 1140gaagcggaag aacttctcca agagggctta
aagccggttt tagaggaatg tgacaat 1197471197DNAHomo sapiens
47ggggcggtca ccatgttgca agactggtgt cggtggatgg gcgtgaatgc tcggcggggt
60ttattgatct tgggtatccc agaagactgt gacgacgccg agtttcagga gtcgctcgag
120gccgcccttc gtccaatggg gcattttacg gttctgggca aggtgttccg
tgaagaggat 180aacgctacag cagctcttgt ggagcttgac cgtgaggtga
attatgcgtt agtacctcgc 240gagattccag gtaccggtgg gccatggaac
gtagtcttcg tcccacgttg ctcgggggag 300gaatttctgg ggcttgggcg
cgtattccac tttccagaac aggaagggca gatggtcgaa 360agcgtagcag
gcgctcttgg cgttggtctc cggcgcgtgt gctggttacg ctccatcggc
420caagcagtcc aaccatgggt tgaagccgta cgctatcaat ctttaggtgt
cttctcaggc 480cgtgaccagc cggcgcctgg tgaggaatcc ttcgaagtct
ggctcgatca tacaactgag 540atgctgcatg tatggcaagg tgtctcagag
cgggaacggc ggcggcggtt attagagggg 600ctccgtggga ctgcgctcca
attagtacat gcgcttttgg ccgaaaatcc agcccgtact 660gcccaagatt
gtctggcagc actcgcccaa gtattcggcg acaacgaatc gcaggcaaca
720atccgcgtaa agtgtcttac agcacagcag cagtcagggg aacgtcttag
tgcgttcgtt 780ctgcggctgg aagtgttact ccagaaagcc atggaaaagg
aggcattggc tcgcgcgagc 840gctgaccgtg tacgtctgcg gcaaatgctt
actcgcgcac atctcaccga gcctctcgat 900gaagcactgc ggaaactgcg
catggcaggc cgcagcccgt ctttcctgga aatgttaggc 960ttagtccggg
agtccgaagc ctgggaggcc agtctggcac ggtcagtgcg ggcacaaacg
1020caagagggtg caggggcacg ggcgggtgca caagcagttg cacgtgcctc
cactaaagtt 1080gaggcagtgc cgggtgggcc aggccgtgaa ccggagggtt
tgcgccaagc cggcgggcag 1140gaagccgaag aattactcca agaaggttta
aaaccggttt tggaggaatg cgataac 1197481425DNAHomo sapiens
48ggggtggaag atttggcggc atcttacatc gtattaaagc ttgagaacga aatccggcag
60gcgcaggtcc aatggttaat ggaggaaaac gccgccctgc aggcccagat ccctgaactt
120caaaagtcgc aagccgcgaa ggagtatgat cttctgcgta aatcttcgga
ggcgaaggag 180ccgcaaaaac tgccagaaca tatgaatcca ccggccgctt
gggaagcaca aaagactcca 240gagtttaagg aaccacagaa acctcctgaa
ccacaggatt tgcttccttg ggagccgcct 300gctgcctggg agttgcaaga
agcaccggct gcccctgagt cactggctcc gcctgcaacc 360cgtgagtctc
agaaaccacc tatggcgcat gaaatcccta ctgtattgga ggggcaaggg
420cctgccaaca cacaagacgc tacgattgct caagaaccaa agaatagcga
gccgcaagac 480cctccaaata tcgagaaacc tcaggaagct ccggaatatc
aagaaacagc ggcacagttg 540gagtttttag aacttcctcc acctcaggag
ccactcgaac cgagcaatgc gcaagaattt 600ctcgagttgt cggctgccca
ggagtcctta gaaggcctca ttgtagttga aacgtccgcg 660gcttcggagt
tcccacaggc tcctatcggg cttgaagcca ccgactttcc gctgcagtac
720acgcttacct tctctggcga cagccagaag ttgccagaat ttttggtcca
actctacagt 780tatatgcggg tacgtgggca cttataccct accgaggcgg
cgttagtgtc gtttgtaggc 840aattgtttct cagggcgcgc gggctggtgg
tttcagttgc ttttggatat ccagtcgcct 900ctgttagaac agtgtgaaag
ttttatcccg gttctccaag acacatttga caatccggaa 960aacatgaagg
acgcaaacca atgcatccac cagctttgtc agggcgaggg tcatgtggcc
1020acacacttcc acctcattgc acaagagctt aattgggatg aaagcacgct
gtggatccag 1080ttccaggaag gcctggcctc atccatccag gatgaacttt
cccatacatc gcctgctacc 1140aacctgagtg atctgattac tcaatgcatc
tcattagagg aaaagcctga cccaaacccg 1200ttagggaagt cctcctcggc
ggagggggat ggcccggaaa gtccgccagc agaaaaccaa 1260cctatgcaag
ctgcgatcaa ttgtcctcac atttccgaag cagagtgggt tcgttggcac
1320aaaggccggc tttgtctcta ttgcggctat ccgggtcact tcgcacgtga
ttgcccagtg 1380aagccacacc aggcgttaca ggcagggaac attcaggctt gccaa
142549717DNAHomo sapiens 49ggggtgcagc cgcagactag caaagctgaa
tcgccggctc tcgctgcctc accgaacgca 60caaatggatg acgttattga tacattaacc
tccctgcgtc tgacgaattc ggctctgcgg 120cgggaggcta gcactcttcg
ggccgagaaa gcaaatttaa ctaatatgct cgagtcagtg 180atggccgagt
taacgctgtt acggacccgt gcgcggattc cgggggccct gcagattacg
240ccaccaattt cgtctattac tagcaacggt actcgcccga tgacgactcc
tccaactagt 300ttacctgaac cgttttctgg cgatcctggc cggttagctg
gtttccttat gcagatggac 360cgttttatga tctttcaagc tagccggttt
ccaggggagg cagagcgtgt tgcgttcctg 420gtgtcgcgct taactggcga
agcagaaaaa tgggccattc ctcacatgca accagactct 480cctttgcgta
acaactatca aggcttctta gcagagttac ggcggaccta taagagcccg
540ttgcgtcacg cccggcgggc gcaaatccgg aagacatcgg cctcgaaccg
ggcagtccgt 600gaacgccaaa tgctttgccg gcaacttgca tcagcaggta
caggcccatg cccggtacac 660cctgctagta acgggacttc cccggcaccg
gcattaccag cacgggcgcg taactta 71750339DNAHomo sapiens 50ggggacggtc
gggtacagtt gatgaaggct ttattggctg gccctttacg tccggcggca 60cgccgttggc
ggaatcctat tccatttcca gagacttttg atggggatac tgatcgcctc
120ccggagttta tcgtccaaac ttcgtcctac atgttcgttg acgaaaatac
tttctctaac 180gacgctctga aagtgacatt tctcattacc cggctgacag
gtccagcctt gcaatgggtc 240attccgtaca ttcgtaaaga aagcccgctt
cttaacgact atcggggttt cctggccgag 300atgaagcggg tttttgggtg
ggaagaggac gaggacttt 33951339DNAHomo sapiens 51ggggaaggtc
gggtgcaact tatgaaagcg ttgcttgccc gcccgcttcg tccagcagca 60cgtcgctggc
ggaatccaat tcctttcccg gagacttttg acggggacac cgatcggctc
120ccagagttca ttgtgcagac gtcaagctat atgttcgtgg atgagaacac
gttctctaac 180gacgcgttga aagtgacttt cttaattacg cgtttgactg
gcccggcttt acaatgggtg 240attccataca ttaagaaaga gtcaccgctt
ctcagtgatt atcgcggttt tttagccgag 300atgaagcggg tcttcgggtg
ggaagaagac gaagacttt 339521092DNAHomo sapiens 52gggccgcgtg
ggcgttgccg tcaacaaggt cctcggattc cgatttgggc agcggccaac 60tatgccaacg
cccacccgtg gcaacaaatg gataaggctt cgccaggcgt tgcttacaca
120cctttggttg atccttggat tgagcggcct tgttgcggtg acacggtttg
tgtgcgcacc 180acaatggaac agaagagcac agcgtcaggc acttgtggtg
gtaagcctgc tgagcgtggt 240cctctcgcgg ggcatatgcc gagctcacgc
ccacatcggg ttgatttctg ttgggttcct 300ggtagcgacc caggcacatt
cgacggcagt ccatggctct tagatcgctt tttggcgcaa 360cttggtgatt
acatgagttt tcactttgaa cactaccagg acaatatcag ccgtgtctgc
420gagattcttc gtcggttaac gggccgcgct caggcatggg ctgctcctta
cctggacggg 480gaccttccac tgccagacga ctacgaattg ttttgtcaag
accttaagga ggtagtacag 540gaccctaaca gtttcgccga gtatcacgcc
gtggtgactt gtccactccc tcttgcttcg 600tcccaacttc ctgtagctcc
tcagcttccg gtggtacgcc aataccttgc gcgcttcttg 660gagggccttg
ctttggatat gggtacggcg cctcggtcac tcccggccgc tatggccaca
720ccggcagtct ccggctcgaa ctccgtttct cgttctgcct tatttgaaca
acaactcaca 780aaggaatcca ctccaggccc gaaagagcca cctgttctcc
ctagctcgac ttgctctagc 840aaaccgggtc ctgtcgaacc agccagttca
caacctgaag aggctgctcc taccccggtg 900ccgcgtttgt cagagtcggc
taacccaccg gctcagcgtc cagaccctgc tcaccctggt 960ggtcctaaac
cacaaaaaac cgaagaggaa gttttagaaa ctgaggggga ccaggaagtt
1020agcctgggga cgccgcagga ggtcgtagaa gcgccggaaa caccaggtga
accaccgctc 1080agccctgggt tc 109253438DNAHomo sapiens 53ggggttgatg
aattggtgct cttgttgcac gcgctgttaa tgcgccatcg ggcgctttcc 60attgaaaatt
ctcagttgat ggagcaactt cgcttgttgg tctgcgaacg ggcgagcctt
120cttcgtcagg tacgtccgcc gagctgtcca gtgccatttc ctgagacttt
taacggggag 180tcatcacggt tacctgagtt catcgtccaa accgcaagct
atatgttagt taatgaaaat 240cgcttttgca atgacgcaat gaaagtcgct
tttttgatta gccttcttac tggtgaagca 300gaagaatggg tcgtcccata
cattgagatg gattcaccaa ttcttgggga ctaccgtgcg 360ttcttggatg
agatgaagca gtgttttggg tgggacgatg atgaagatga cgacgatgag
420gaagaggagg atgactat 438541647DNAHomo sapiens 54gggcctgtgg
atttaggtca ggctttgggg ttgttgccat ccctcgctaa ggccgaagat 60tcccaattta
gcgaaagcga tgcagcttta caggaggaat tgtcttctcc ggaaaccgca
120cggcaacttt ttcgtcaatt tcgctatcaa gtcatgtcgg ggcctcatga
aacactgaaa 180cagttacgga agttatgttt tcagtggctg caacctgaag
tccatacaaa ggaacaaatc 240ctcgaaattc tgatgctgga acagttcttg
accattctgc ctggtgaaat tcagatgtgg 300gtccgcaagc agtgccctgg
tagtggggag gaggcggtta cgttagtaga atccctgaaa 360ggtgatccac
aacggctctg gcaatggatc tccatccaag tcctgggtca ggatatcctg
420tctgagaaaa tggagtcacc ttcttgccag gtgggcgaag tggagccaca
cctggaagtt 480gtacctcagg aactggggtt agagaattca tcttcagggc
cgggggaact tctttcgcac 540atcgtgaaag aggagtctga cactgaagca
gagttggcgt tagcggcatc ccagccagct 600cgtttggaag aacggctgat
tcgggatcag gaccttgggg cgtccctcct cccggcagca 660ccgcaggagc
aatggcgtca attagacagc actcaaaaag aacaatattg ggacctgatg
720ctggagacct acggcaaaat ggtatccggc gcgggtatct cacacccgaa
gtccgattta 780acgaactcaa ttgagttcgg tgaagagttg gcaggtattt
atttacatgt aaacgaaaag 840attccgcggc ctacctgcat tggtgaccgc
caagaaaacg acaaagaaaa ccttaatttg 900gaaaaccatc gtgaccagga
attattacat gccagctgcc aggcctcggg cgaagtgcca 960tcccaggcat
cgttacgtgg cttctttacc gaggacgaac ctggttgctt cggcgaaggg
1020gagaaccttc ctgaggcact tcagaatatc caggatgagg ggactggcga
acagctgagc 1080ccgcaagaac gcattagtga aaaacagttg ggtcaacatt
tgccaaatcc gcactcgggg 1140gagatgtcga cgatgtggct tgaagaaaaa
cgggagacca gccagaaagg ccaaccacgt 1200gcaccaatgg cgcagaaatt
gccaacgtgc cgcgaatgtg gcaaaacgtt ttatcgcaat 1260agtcaactta
tctttcacca acgcacacac accggtgaga catattttca atgcaccatc
1320tgcaaaaagg cgtttctccg gtcatctgat ttcgtgaaac atcagcggac
tcatactggc 1380gaaaaacctt gtaaatgtga ctattgtggc aagggcttta
gtgattttag cgggcttcgg 1440catcacgaga agatccatac cggcgagaag
ccatacaagt gtccaatctg tgagaaatct 1500ttcatccagc gcagtaattt
taaccgccac caacgggttc acaccggtga aaagccttat 1560aaatgctcgc
attgtggcaa gagcttcagc tggagctcct cgctcgataa gcatcaacgt
1620tcacatctgg ggaagaagcc gttccaa 1647551053DNAHomo sapiens
55gggactctcc gcttacttga ggattggtgt cgggggatgg acatgaaccc acgtaaggcc
60cttcttatcg ccgggatttc ccagtcatgt tcagtcgccg agattgaaga ggcgctccaa
120gccgggcttg ctcctttagg cgagtatcgt ctccttgggc ggatgtttcg
ccgcgatgaa 180aatcgcaaag tagcgttggt tggtctcaca gctgaaacta
gccatgcgct tgtacctaaa 240gaaattcctg gtaaaggcgg gatctggcgg
gttattttta aaccaccgga cccggacaat 300acgtttcttt ctcgtttgaa
tgagttcctc gcgggcgagg ggatgacggt gggggaactt 360agtcgtgctc
ttggtcacga aaatgggtca ttagaccctg aacagggtat gattccggaa
420atgtgggcgc cgatgctggc acaggctctg gaggctctcc aaccggcttt
acagtgcctt 480aagtacaaga agctgcgcgt tttttcaggg cgcgagtctc
cagagccggg tgaggaggaa 540ttcggccgtt ggatgttcca taccacccag
atgatcaaag cgtggcaggt gccggatgtc 600gagaaacgcc gccggctgtt
ggaatcactc cgcgggccgg cacttgacgt tattcgggtt 660ctgaaaatta
acaacccgtt aattacggta gatgaatgtt tgcaagcact tgaagaggtc
720tttggggtga ctgacaatcc tcgggaattg caagtaaaat acttaacgac
ctaccataag 780gacgaggaga aattatcagc ctacgtactg cggctggaac
cgctgctgca gaagctcgtc 840cagcgggggg ctattgaacg ggacgctgtt
aatcaggctc gcctggatca ggtaatcgct 900ggggcggtac ataaaactat
ccgccgtgag ctgaacctgc ctgaagacgg gccggcgcca 960ggctttcttc
aactcctcgt tttgattaag gattacgagg cagctgaaga ggaggaagca
1020ttacttcagg ccattcttga agggaacttt act 1053562124DNAHomo sapiens
56gggacagaac ggcgtcgcga cgaattaagt gaagaaatta ataatcttcg tgaaaaggtt
60atgaaacaga gtgaggaaaa caacaatctt caatcccaag tccagaaact cactgaggag
120aatactacac tccgtgagca agttgaacct acacctgaag atgaagatga
cgacattgag 180ttgcggggcg cagcagccgc agccgcgcct ccgccgccga
tcgaggagga atgcccggag 240gatttaccgg aaaaatttga tggtaatccg
gacatgttag cgccattcat ggcccagtgc 300caaattttta tggaaaagtc
tacgcgcgat tttagtgtag atcgcgtacg tgtatgtttt 360gtgacgagca
tgatgactgg tcgcgcagcc cgttgggcgt cagcgaaatt ggagcggtcg
420cactacctga tgcataatta cccggcgttc atgatggaga tgaaacacgt
gtttgaagac 480ccgcagcggc gggaggtggc caaacgcaag atccggcggt
tgcggcaggg catgggcagc 540gtaattgatt atagtaatgc gtttcaaatg
attgcgcagg atctggattg gaatgaacct 600gctctcattg atcaatatca
tgaagggctt agtgaccata ttcaagagga actctctcac 660ctggaagtgg
ctaaatctct ctccgccctt attggccaat gcattcatat tgagcgccgt
720cttgcacgtg ctgctgccgc tcggaaaccg cgtagtccac cacgggcttt
agtgctccca 780catatcgcgt cacaccatca agtagatcct actgagccag
tggggggtgc acgcatgcgc 840ttaacccaag aagaaaagga acgtcgtcgt
aagctgaatt tatgcctgta ctgcggcact 900ggtggccatt atgccgataa
ctgtcctgcc aaagccagta agtcaagccc ggctgggaaa 960cttccaggtc
ctgccgtcga gggcccttct gctaccggcc cagagattat ccgctccccg
1020caagacgatg cgtcgtcgcc tcatctccag gtaatgctcc aaatccacct
ccctggccgg 1080cacacactct ttgtccgggc gatgattgac tctggggcgt
ctggtaattt tattgatcac 1140gagtatgttg ctcaaaatgg tatccctctc
cggatcaaag actggcctat tctggttgaa 1200gccatcgatg gccgtccgat
cgcgagcggt cctgtggttc atgaaacgca tgacctcatc 1260gttgatctgg
gtgaccaccg tgaagtatta tcctttgatg tgactcagtc accgtttttt
1320ccagttgttt tgggcgtccg ttggctttcg actcacgatc ctaacatcac
gtggtcgaca 1380cggtcgattg tcttcgattc ggaatattgt cgttatcatt
gccgcatgta ttcaccaatt 1440ccgccgtctc tcccgccgcc tgcgccgcaa
cctcctctgt attacccggt ggacggttac 1500cgtgtttacc agccagttcg
ctactactac gtacaaaacg tgtacacgcc tgttgatgaa 1560cacgtgtacc
cagatcaccg cctggtcgac cctcatattg agatgatccc gggtgcgcac
1620tcgatcccat cgggccatgt ttattccttg tctgagccag aaatggccgc
cttacgggat 1680tttgtggccc ggaatgtcaa agacggcctg attaccccga
caattgcacc aaacggtgct 1740caggtgttgc aggtgaagcg gggctggaag
ttgcaagtca gctatgattg tcgtgcgcca 1800aacaacttca ctattcagaa
ccaatatcca cgtctcagca tccctaatct cgaggaccag 1860gcacatcttg
caacatatac tgaatttgta cctcagattc ctggctatca gacttatcct
1920acgtatgctg cctacccaac atacccggta ggtttcgcat ggtacccagt
aggccgggac 1980gggcagggcc gctctttata tgttcctgtc atgattacat
ggaacccgca ttggtaccgc 2040cagcctccgg tcccacagta cccacctcct
caacctccac cacctccgcc gcctcctcca 2100ccgccacctt cttactcgac atta
21245760DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 57atgcatcacc atcaccatca cggctcaggg
tctggtagcg aaaatctgta cttccagggg 605820PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 58Met
His His His His His His Gly Ser Gly Ser Gly Ser Glu Asn Leu1 5 10
15Tyr Phe Gln Gly 20596PRTArtificial SequenceDescription of
Artificial Sequence Synthetic 6xHis tag 59His His His His His His1
5606PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 60Gly Ser Gly Ser Gly Ser1 5617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 61Glu
Asn Leu Tyr Phe Gln Gly1 56226DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 62aagctcattt cctggtatga caacga
266325DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 63agggtctctc tcttcctctt gtgct 256425DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
64gctcaacctg ggaactgcat ctgat 256525DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
65taatcctgtt tgctccccac gcttt 256622DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
66ggcccctcag ctccagtgat tc 226725DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 67cctgttgtca ctctcctggc
tctga 256824DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 68gccaagacat aagaaacctc gcct
246924DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 69gtgaatcaac atcctccctc cgtc 247025PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
PeptideMISC_FEATURE(1)..(25)This sequence may encompass 1-5 "Glu
Ala Ala Ala Lys" repeating units 70Glu Ala Ala Ala Lys Glu Ala Ala
Ala Lys Glu Ala Ala Ala Lys Glu1 5 10 15Ala Ala Ala Lys Glu Ala Ala
Ala Lys 20 257125PRTArtificial SequenceDescription of Artificial
Sequence Synthetic PeptideMISC_FEATURE(1)..(25)This sequence may
encompass 1-5 "Glu Ala Ala Ala Arg" repeating units 71Glu Ala Ala
Ala Arg Glu Ala Ala Ala Arg Glu Ala Ala Ala Arg Glu1 5 10 15Ala Ala
Ala Arg Glu Ala Ala Ala Arg 20 257250PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
PolypeptideMISC_FEATURE(1)..(50)This sequence may encompass 1-10
"Gly Gly Gly Gly Ser" repeating units 72Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45Gly Ser
507340PRTArtificial SequenceDescription of Artificial Sequence
Synthetic PolypeptideMISC_FEATURE(1)..(40)This sequence may
encompass 1-10 "Gly Gly Gly Ser" repeating units 73Gly Gly Gly Ser
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser1 5 10 15Gly Gly Gly
Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 20 25 30Gly Gly
Gly Ser Gly Gly Gly Ser 35 407418PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Peptide 74Lys Glu Ser Gly Ser Val
Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser1 5 10 15Leu
Asp7514PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 75Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys
Ser Thr1 5 107610PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 76Gly Gly Ala Ala Asn Leu Val Arg Gly
Gly1 5 107710PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 77Ser Gly Arg Ile Gly Phe Leu Arg Thr
Ala1 5 10785PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 78Ser Gly Arg Ser Ala1 5794PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 79Gly
Phe Leu Gly1804PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 80Ala Leu Ala Leu1815PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
PeptideMOD_RES(3)..(3)S-ethylcysteine 81Pro Ile Cys Phe Phe1
5825PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(3)..(3)Ser or ThrMOD_RES(4)..(4)Leu or
IleMOD_RES(5)..(5)Ser or Thr 82Pro Arg Xaa Xaa Xaa1
5834PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 83Asp Glu Val Asp1846PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 84Gly
Trp Glu His Asp Gly1 5858PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 85Arg Pro Leu Ala Leu Trp Arg
Ser1 5
* * * * *