U.S. patent application number 16/593302 was filed with the patent office on 2020-08-27 for method of enhancing delivery of therapeutic compounds to the eye.
The applicant listed for this patent is Wayne State University. Invention is credited to Elena IVANOVA, Zhuo-Hua PAN.
Application Number | 20200268647 16/593302 |
Document ID | / |
Family ID | 1000004816199 |
Filed Date | 2020-08-27 |
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United States Patent
Application |
20200268647 |
Kind Code |
A1 |
PAN; Zhuo-Hua ; et
al. |
August 27, 2020 |
METHOD OF ENHANCING DELIVERY OF THERAPEUTIC COMPOUNDS TO THE
EYE
Abstract
The invention provides methods for enhancing the delivery of
therapeutic compounds to the eye of a subject by administering
plasmin or derivatives thereof and the therapeutic compounds to the
eye.
Inventors: |
PAN; Zhuo-Hua; (Troy,
MI) ; IVANOVA; Elena; (White Plains, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wayne State University |
Detroit |
MI |
US |
|
|
Family ID: |
1000004816199 |
Appl. No.: |
16/593302 |
Filed: |
October 4, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15676268 |
Aug 14, 2017 |
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16593302 |
|
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14777420 |
Sep 15, 2015 |
9730888 |
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PCT/US2014/026224 |
Mar 13, 2014 |
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15676268 |
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61785015 |
Mar 14, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/7088 20130101;
A61K 36/05 20130101; A61K 38/484 20130101; A61K 9/0048 20130101;
A61K 48/0075 20130101; C12Y 304/21007 20130101; A61K 38/36
20130101; A61K 38/16 20130101; A61K 31/713 20130101; A61K 48/0008
20130101 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61K 31/713 20060101 A61K031/713; A61K 38/36 20060101
A61K038/36; A61K 31/7088 20060101 A61K031/7088; A61K 36/05 20060101
A61K036/05; A61K 38/16 20060101 A61K038/16; A61K 38/48 20060101
A61K038/48; A61K 48/00 20060101 A61K048/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under
EY017130 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method of enhancing the delivery of a therapeutic agent to an
eye of a subject comprising administering a plasmin or derivative
thereof and the therapeutic agent to the eye.
2. The method of claim 1, wherein the plasmin or derivative thereof
is a miniplasmin or a microplasmin (ocriplasmin).
3. The method of claim 1, wherein the therapeutic agent is selected
from a nucleic acid, a small molecule, an antibody, or a
peptide.
4. The method of claim 3, wherein the nucleic acid is a nucleic
acid expression vector, a plasmid, or an siRNA.
5. The method of claim 4, wherein nucleic acid expression vector is
a viral vector comprising a transgene.
6. The method of claim 5, wherein the transgene is an opsin.
7. The method of claim 6, wherein the opsin is selected from the
group consisting of channelrhodopsin, halorhodopsin, melanopsin,
pineal opsin, bacteriorhodopisin, and proteorhodopsin, or a
functional variant thereof.
8. The method of claim 7, wherein said transgene is operably linked
to a cell-specific promoter.
9. The method of claim 8, wherein the therapeutic agent is
encapsulated in a nanoparticle, a polymer, or a liposome.
10. The method of claim 9, wherein the therapeutic agent is
selected from the group consisting of ranibizumab antibody FAB
(Lucentis), VEGF Trap fusion molecule (VEGF Trap-Eye), macugen
pegylated polypeptide (Pegaptanib), and bevacimzumab (Avastin).
11. The method of claim 1, wherein the subject is suffering from an
ocular disease or disorder.
12. The method of claim 1, wherein the plasmin or derivative
thereof and the therapeutic agent are delivered concurrently or
sequentially.
13. The method of claim 1, wherein the therapeutic agent is
delivered to a retinal cell.
14. The method of claim 13, wherein the retinal cell is a retinal
ganglion cell, a retinal bipolar cell, a retinal horizontal cell,
an amacrine cell, a photoreceptor cell, Muller glial cell, or a
retinal pigment epithelial cell.
15. The method of claim 1, wherein the administration is to the
vitreous of the eye.
16. A method of increasing light sensitivity or improving or
restoring vision in a subject comprising administering a plasmin or
derivative thereof and a viral vector that encodes an opsin to the
vitreous of the eye.
17. The method of claim 16, wherein said opsin is selected from the
group consisting of channelrhodopsin, halorhodopsin, melanopsin,
pineal opsin, bacteriorhodopisin, and proteorhodopsin, or a
functional variant thereof.
18. The method of claim 16, wherein the subject has an ocular
disease or disorder.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 14/777,420 filed on Sep. 15, 2015, which is a
371 of International Application No. PCT/US2014/026224, filed Mar.
13, 2014, which claims priority to, and the benefit of, U.S.
Provisional Application No. 61/785,015, filed on Mar. 14, 2013; the
contents of each of which are hereby incorporated by reference in
its entirety.
INCORPORATION OF SEQUENCE LISTING
[0003] The contents of the text file named
"RTRO-704/C01US_SeqList.txt," which was created on Aug. 14, 2017
and is 25.7 KB in size, are hereby incorporated by reference in
their entirety.
FIELD OF THE INVENTION
[0004] This invention relates generally to methods of enhancing the
delivery of therapeutic compounds to the eye.
BACKGROUND OF THE INVENTION
[0005] The eye is a complex optical system that detects light,
converts the light to a set of electrical signals, and transmits
the signals to the brain, ultimately generating a representation of
our world. Ocular diseases and disorders can cause diminished
visual acuity, diminished light sensitivity, and blindness.
[0006] Delivery of therapeutic compounds to specific ocular tissues
affected by an ocular disease or disorder, such as the retina, is a
challenge. Current methods, such as intravitreal injection or
implanted drug delivery devices, are still limited in the efficacy
of delivery. Specifically, the therapeutic agents are often
localized only to the immediate areas surrounding the delivery
site, and fail to permeate or diffuse beyond intervening ocular
structures or throughout the targeted ocular tissue, thereby
severely limiting the efficacy of such therapeutics. Thus, there
exists a long-felt need for methods to enhance the delivery of
therapeutic compounds to the eye.
SUMMARY OF THE INVENTION
[0007] The invention provides a solution for the long-felt need for
methods to enhance or improve the delivery of therapeutic compounds
to the eye.
[0008] The present invention features a method of enhancing the
delivery of a therapeutic agent to an eye of a subject by
administering a plasmin or derivative thereof and the therapeutic
agent to the eye. The present invention also features the use of a
composition comprising a plasmin or derivative thereof for delivery
to the eye of a subject for enhancing the delivery of a therapeutic
agent.
[0009] In one aspect, the plasmin or derivative thereof is a
miniplasmin or a microplasmin (Ocriplasmin). The plasmin or
derivative thereof encompassed in the present invention includes
amino acid sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ
ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or
functional variants or fragments thereof.
[0010] In one aspect, the therapeutic agent is selected from a
small molecule, a nucleic acid, an antibody, or a peptide. The
nucleic acid is a nucleic acid expression vector (i.e., a viral
vector), a plasmid, or an siRNA. For example, the viral vector is a
AAV viral vector (i.e., recombinant AAV or rAAV) that encodes a
transgene. Preferably, the transgene encodes a gene product that
increases or restores light sensitivity, increases light detection,
increases photosensitivity, increases visual evoked potential, or
restores vision to the blind. More preferably, the transgene is an
opsin gene. Examples of opsin genes include, but are not limited
to, channelrhodopsins (i.e., channelrhodopsin-1,
channelrhodopsin-2, Volvox carteri channelrhodopsins 1 or 2),
melanopsin, pineal opsin, photopsins, halorhodopsin,
bacteriorhodopisin, proteorhodopsin, or any functional variants or
fragments thereof.
[0011] Other examples of therapeutic agents include, but are not
limited to ranibizumab antibody FAB (Lucentis), VEGF Trap fusion
molecule (VEGF Trap-Eye), macugen pegylated polypeptide
(Pegaptanib), and bevacimzumab (Avastin). Any of the therapeutic
agents used in the present invention may be encapsulated in a
nanoparticle, a polymer, or a liposome.
[0012] In one aspect, the plasmin or derivative thereof and the
therapeutic agent are delivered concurrently or sequentially.
[0013] The present invention provides a method in which the
therapeutic agent is delivered to a retinal cell. The retinal cell
is a retinal ganglion cell, a retinal horizontal cell, a retinal
bipolar cell, an amacrine cell, a photoreceptor cell, a Muller
glial cell, or a retinal pigment epithelial cell.
[0014] In one aspect, the plasmin or derivative thereof and the
therapeutic agent is administered to the vitreous of the eye.
[0015] The present invention further provides a method of
increasing or restoring light sensitivity in a subject comprising
administering a plasmin or derivative thereof and a viral vector
that encodes an opsin to the vitreous of the eye. The present
invention also provides a method of improving or restoring vision
in a subject comprising administering a plasmin or derivative
thereof and a viral vector that encodes an opsin to the vitreous of
the eye.
[0016] Uses of a composition comprising a plasmin or derivative
thereof for treating an ocular disease or disorder in a subject are
also provided herein.
[0017] The subject is suffering from an ocular disease or disorder.
Preferably, the ocular disease or disorder is associated with
photoreceptor degeneration.
[0018] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are expressly incorporated by reference in their
entirety. In cases of conflict, the present specification,
including definitions, will control. In addition, the materials,
methods, and examples described herein are illustrative only and
are not intended to be limiting.
[0019] Other features and advantages of the invention will be
apparent from and are encompassed by the following detailed
description and claims.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 is a series of representative GFP fluorescence images
in retinal vertical sections after intravitreal injection of AAV2
vectors (6.times.10.sup.12 vg/ml), AAV2/2-ChR2-GFP-WPRE-hGHpA, in
control (A, B) or co-injection with plasmin (0.025 IU/eye) (C, D).
The vectors were co-injected along with plasmin into the vitreous
space of adult C56BL/6J mice at age of approximately one month.
Transduction efficiency was evaluated one month after virus
injection by immunostaining and cell counting.
[0021] FIG. 2 is a series of representative GFP fluorescence and
DAPI staining images demonstrating the effects of plasmin on
AAV-mediated transduction efficiency and the potential
neurotoxicity in retinal ganglion cells. AAV2 vectors
(2.times.10.sup.12 vg/ml), AAV2/2-ChR2-GFP-WPRE-hGHpA, was injected
in control (A, E), or was co-injected at (B, F) low (L: 0.005 IU),
(C, G) middle (M: 0.025 IU), and (D, H) high (H: 0.100 IU
plasmin/eye) concentrations. Retinal ganglion cells were stained by
DAPI (A-D).
[0022] FIG. 3 is two graphs showing the quantitative assessment of
the effects of plasmin on (A) AAV-mediated transduction efficiency
and (B) potential neurotoxicity of plasmin in retinal ganglion
cells. Co-injection of plasmin at low (L: 0.005 IU), middle (M:
0.025 IU), and high (H: 0.100 IU plasmin/eye) concentrations
significantly increased the AAV-mediated transduction efficiency in
retinal ganglion cells (A). Co-injection of plasmin did not show
any significant neurotoxicity to retinal ganglion cells. The
ganglion cell counts were assessed from multiple unit areas of 223
.mu.m.times.167 .mu.m. * p<0.05; ** p<0.005.
[0023] FIG. 4 is a series of representative fluorescence images of
mCherry-expressing retinal bipolar cells in retinal whole-mounts.
AAV2 vectors (2.times.10.sup.12 vg/ml) with Y444F capsid mutation
carrying mCherry under control of a mGluR6 promoter were
co-injected along with plasmin of three doses (L: 0.005 IU, M:
0.025 IU, and H: 0.100 IU/eye) into the vitreous space of adult
C56BL/6J mice at age of approximately one month. Transduction
efficiency was evaluated one month after virus injection by
immunostaining and cell counting.
[0024] FIG. 5 is three graphs showing quantitative data for the
effects of plasmin on AAV-mediated transduction efficiency in
retinal bipolar cells. A) Center; B) Mid-region; and C) Periphery.
The counts of mCherry expressing retinal bipolar cell were assessed
from multiple unit areas of 223 .mu.m.times.167 .mu.m. * p<0.05;
** p<0.01; ***p<0.001.
DETAILED DESCRIPTION
[0025] The present invention provides methods for enhanced delivery
of therapeutic compounds or agents to the eye of a subject by
administering a plasmin or derivative thereof and the therapeutic
agent to the eye. In some embodiments, the plasmin or derivative
thereof and the therapeutic agent may be delivered to the vitreous
for enhanced delivery to the retina and retinal cells. The retinal
cells include, for example, photoreceptor cells (e.g., rods, cones,
and photosensitive retinal ganglion cells), horizontal cells,
retinal bipolar cells, amacrine cells, retinal ganglion cells,
Muller glial cells, and retinal pigment epithelial cells. In other
embodiments, the plasmin or derivative thereof and the therapeutic
agent may be delivered to, for example, the posterior segment, the
anterior segment, the sclera, the choroid, the conjunctiva, the
iris, the lens, or the cornea.
[0026] The retina is a complex tissue in the back of the eye that
contains specialized photoreceptor cells called rods and cones. The
photoreceptors connect to a network of nerve cells for the local
processing of visual information. This information is sent to the
brain for decoding into a visual image. The retina is susceptible
to a variety of diseases, including age-related macular
degeneration (AMD), diabetic retinopathy (DR), retinitis pigmentosa
(RP), glaucoma, and other inherited retinal degenerations, uveitis,
retinal detachment, and eye cancers (ocular melanoma and
retinoblastoma). Each of these can lead to visual loss or complete
blindness.
[0027] Delivery of therapeutic compounds to the retina is a
challenge, due to the complex structure of the eye. Intravitreal
injection and vitreal delivery devices are frequently used to
deliver therapeutic compounds to the retina, however the efficiency
of delivery is impaired by the inner limiting membrane (ILM) and
the multiple layers of cells of the retina.
Plasmin
[0028] Plasmin is a serine protease that is present in the blood
that degrades fibrin blood clots and other blood plasma proteins.
Specifically, plasmin cleaves fibrin, fibronectin, thrombospondin,
laminin, proaccelerin, and Von Willebrand Factor (VWF) into soluble
products. Plasmin exhibits preferential cleavage at the carboxyl
side of Lysine and Arginine residues with higher selectivity than
trypsin.
[0029] Specifically, plasmin originates from a zymogen, or inactive
precursor protein, called plasminogen (PLG). The amino acid
sequence of plasminogen is known in the art, for example, Genbank
Accession Number NP_000292, and listed below:
TABLE-US-00001 (SEQ ID NO: 1)
MEHKEVVLLLLLFLKSGQGEPLDDYVNTQGASLFSVTKKQLGAGSIEECAA
KCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSE
CKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCR
NPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGL
ECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWE
LCDIPRCTTPPPSSGPTYQCLKGTGENYGNVAVTVSGHTCQHWSAQTPHTH
ANRTPENFPCKNLDENYCRNPDGKRPWCHTTNSQVRWEYCKIPSCDSSPVS
TEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRH
QKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEAS
VVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPH
RHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAA
PSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLIS
PEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRK
DIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGL
LKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVC
FEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN
[0030] The nucleic acid of plasminogen is known in the art, for
example, Genbank Accession Number NM_000301, and as listed
below:
TABLE-US-00002 (SEQ ID NO: 2)
GAATCATTAACTTAATTTGACTATCTGGTTTGTGGATGCGTTTACTCTCAT
GTAAGTCAACAACATCCTGGGATTGGGACCCACTTTCTGGGCACTGCTGGC
CAGTCCCAAAATGGAACATAAGGAAGTGGTTCTTCTACTTCTTTTATTTCT
GAAATCAGGTCAAGGAGAGCCTCTGGATGACTATGTGAATACCCAGGGGGC
TTCACTGTTCAGTGTCACTAAGAAGCAGCTGGGAGCAGGAAGTATAGAAGA
ATGTGCAGCAAAATGTGAGGAGGACGAAGAATTCACCTGCAGGGCATTCCA
ATATCACAGTAAAGAGCAACAATGTGTGATAATGGCTGAAAACAGGAAGTC
CTCCATAATCATTAGGATGAGAGATGTAGTTTTATTTGAAAAGAAAGTGTA
TCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTC
CAAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCA
CAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAA
CTACTGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTAC
TGATCCAGAAAAGAGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGA
ATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAGACCAT
GTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCACACGCTCATGG
ATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGTCG
TAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAACAA
GCGCTGGGAACTTTGTGACATCCCCCGCTGCACAACACCTCCACCATCTTC
TGGTCCCACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAA
TGTGGCTGTTACCGTGTCCGGGCACACCTGTCAGCACTGGAGTGCACAGAC
CCCTCACACACATAACAGGACACCAGAAAACTTCCCCTGCAAAAATTTGGA
TGAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGGTGCCATAC
AACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTC
CTCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTAAC
CCCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGCAC
ATCCTCCACCACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGAC
ACCACACCGGCACCAGAAGACCCCAGAAAACTACCCAAATGCTGGCCTGAC
AATGAACTACTGCAGGAATCCAGATGCCGATAAAGGCCCCTGGTGTTTTAC
CACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGCTCAGG
AACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGT
AGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCG
AGGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGC
CCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACAAATCCACGGGC
GGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGTCC
CTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCC
TCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAA
GAAATGTCCTGGAAGGGTTGTAGGGGGGTGTGTGGCCCACCCACATTCCTG
GCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGG
CACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAA
AGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACCCAAGAAGT
GAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGA
GCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCAT
CACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGC
CTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTATTT
TGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGT
GTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTG
TGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGG
TCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTC
TTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGT
TTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTAATTGGA
CGGGAGACAGAGTGACGCACTGACTCACCTAGAGGCTGGAACGTGGGTAGG
GATTTAGCATGCTGGAAATAACTGGCAGTAATCAAACGAAGACACTGTCCC
CAGCTACCAGCTACGCCAAACCTCGGCATTTTTTGTGTTATTTTCTGACTG
CTGGATTCTGTAGTAAGGTGACATAGCTATGACATTTGTTAAAAATAAACT
CTGTACTTAACTTTGATTTGAGTAAATTTTGGTTTTGGTCTTCAACATTTT
CATGCTCTTTGTTCACCCCACCAATTTTTAAATGGGCAGATGGGGGGATTT
AGCTGCTTTTGATAAGGAACAGCTGCACAAAGGACTGAGCAGGCTGCAAGG
TCACAGAGGGGAGAGCCAAGAAGTTGTCCACGCATTTACCTCATCAGCTAA
GCGAGGGCTTGACATGCATTTTTACTGTCTTTATTCCTGACACTGAGATGA
ATTTTTCAAAGCTGCAACATGTATGGGGAGTCATGCAAACCGATTCTGTTA
TTGGGAATGAAATCTGTCACCGACTGCTTGACTTGAGCCCAGGGGACACGG
AAGCAGAGAGCTGTATATGATGGAGTGAACCGGTCCATGGTGTGTAACACA
AGACCAACTGAGAGTCTGAATGTTATTCTGGGGCACACGTGAGTCTAGGAT
TGGTGCCAAGAGCATGTAAATGAACAACAAGCAAATATTGAAGGTGGACCA
CTTATTTCCCATTGCTAATTGCCTGCCCGGTTTTGAAACAGTCTGCAGTAC
AACACGGTCACAGGAGAATGACCTGTGGGAGAGATACATGTTTAGAGGAAG
AGAAAGGACAAAGGCACACGTTTTACCATTTAAAATATTGTTACCAAACAA
AAATATCCATTCAAAATACAATTTAACAATGCAACAGTCATCTTACAGCAG
CAGAAATGCAGAGAAAAGCAAAACTGCAAGTGATGTGAATAAAGGGTGAAT
GTAGTCTCAAATCCTCAAA
[0031] The signal peptide sequence of plasminogen is 19 amino acids
long. Thus, the plasminogen sequence without the signal peptide
encompasses amino acids from positions 20-810 of the plasminogen
sequence. The signal peptide sequence is as follows:
TABLE-US-00003 (SEQ ID NO: 3) MEHKEVVLLLLLFLKSGQG
[0032] The plasmin heavy chain A is 561 amino acids, comprising the
amino acid sequence provided below:
TABLE-US-00004 (SEQ ID NO: 4)
EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKE
QQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNG
ITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKR
YDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSK
FPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQ
CLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCR
NPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQD
CYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCR
NPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSE
EDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKN
YCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGR
[0033] The short form of the plasmin heavy chain A is 483 amino
acids. The amino acid sequence of the short form of the plasmin
heavy chain A is as follows:
TABLE-US-00005 (SEQ ID NO: 5)
VYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLE
ENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISK
TMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDP
NKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSA
QTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSC
DSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSS
MTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKC
SGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDW
AAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCD
VPQCAAPSFDCGKPQVEPKKCPGR
[0034] The amino acid sequence of the activation peptide comprises
the following amino acid sequence:
TABLE-US-00006 (SEQ ID NO: 6)
EPLDDYVNIQGASLFSVIKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKE
QQCVIMAENRKSSIIIRMRDVVLFEKK
[0035] The plasmin light chain B is 230 amino acids. The amino acid
sequence of the plasmin light chain B is as follows:
TABLE-US-00007 (SEQ ID NO: 7)
VVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPS
SYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVI
PACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYE
FLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGC
ARPNKPGVYVRVSRFVTWIEGVMRNN
[0036] In some preferred embodiments, the plasmin can be a
miniplasmin or a microplasmin or a derivative thereof. Miniplasmin
and microplasmin are produced upon the activation of
miniplasminogen and microplasminogen by plasminogen activators such
as, but not limited to, streptokinase, staphylokinase, tissue-type
plasminogen activator or urokinase. Miniplasminogen and
microplasminogen are derived from plasminogen, which is a single
chain glycoprotein that is an important component of mammalian
blood. Human plasminogen is a multi-domain protein of 791 residues
(SEQ ID NO:1), composed of an N-terminal pre-activation domain,
five homologous kringle domains each of about 80 amino acids, a
serine protease catalytic domain and inter-domain connecting
sequences. Plasmin or plasminogen activators cleave the peptide
bonds between Arg-68 and Met-69, or Lys-77 and Lys-78 or Lys-78 and
Val-79 at the N-terminal of human plasminogen, resulting in shorter
proenzymes called Lys-plasminogens (for example, proteins
consisting of amino acids 69-791 or 78-791 or 79-791). Additional
cleavage by the enzyme elastase removes the first four kringle
domains producing the proenzyme, miniplasminogen (typically amino
acids 442-791). Further cleavage of the fifth kringle yields the
proenzyme, microplasminogen (typically amino acids 543-791). The
kringles of plasminogen contain lysine-binding sites that mediate
specific binding of plasminogen to substrates such as fibrin. The
proenzyme forms of plasminogen are activated to their enzymatically
active form by the cleavage of the peptide bond between Arg-561 and
Val-562 to yield a disulfide bonded double chain form of the
corresponding protein. The product of activation of a plasminogen
protein is called a plasmin Thus, the product of Lys-plasminogen
activation is called Lys-plasmin, while the products of activation
of miniplasminogen and microplasminogen, are referred to as
miniplasmin and microplasmin, respectively. Like plasmin,
miniplasmin and microplasmin possess catalytic activity. An
advantage of miniplasmin and microplasmin over plasmin is their
smaller size compared to plasmin Thus, both microplasmin and
miniplasmin are expected to have faster diffusion rates in the
vitreous than plasmin.
[0037] The plasmin of the present invention may comprise any one of
the plasminogen-related sequences described herein, for example,
any one of SEQ ID NOs: 1 and 3-7, or a functional fragment or
variant thereof.
[0038] The plasmin may also be ocriplasmin (JETREA.RTM.) or
variants or derivatives thereof. Ocriplasmin is a recombinant
truncated form of human plasmin produced by recombinant DNA
technology in a Pichia pastoris expression system. Ocriplasm is a
protein made up of 249 amino acids with a molecular weight of 27.2
kDa, and has two peptide chains. The amino acid sequence for the
truncated heavy chain is as follows:
TABLE-US-00008 (SEQ ID NO: 8) APSFDCGKPQVEPKKCPGR
The amino acid sequence for the light chain is as follows:
TABLE-US-00009 (SEQ ID NO: 9)
VVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPS
SYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVI
PACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYE
FLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGC
ARPNKPGVYVRVSRFVTWIEGVMRNN
The present invention further encompasses variants and derivatives
of ocriplasmin.
[0039] The plasmin may be isolated from blood using known isolation
techniques. Alternatively, plasminogen may be isolated from the
blood using known isolation techniques, and then incubated with
proteases that cleave plasminogen into active plasmin to produce
purified or isolated plasmin suitable for use in the methods
described herein.
[0040] The plasmin may be synthesized chemically using commercially
available peptide synthesizers to produce purified or isolated
plasmin suitable for use in the methods described herein. Chemical
synthesis of peptides and proteins can be used for the
incorporation of modified or unnatural amino acids, including
D-amino acids and other small organic molecules. Replacement of one
or more L-amino acids in a peptide or protein with the
corresponding D-amino acid isoforms can be used to increase
resistance to enzymatic hydrolysis, and to enhance one or more
properties of biological activity, i.e., functional potency or
duration of action. Other modifications to the plasmin can be
introduced, for example, cross-linking to change the conformation
to alter the potency, selectivity or stability of the plasmin.
[0041] For example, the plasmin may be purchased from a commercial
vendor, such as Sigma Aldrich, Catalog Number P1867. The present
invention also encompasses variants or derivatives of the plasmin
supplied by Sigma Aldrich (Cat. No. P1867).
[0042] The plasmin may be a recombinant plasmin obtained by methods
well known in the art for recombinant protein expression and
purification. A DNA molecule encoding a plasmin or a variant or
analog thereof can be generated from known DNA sequences or by
deducing the nucleic acid sequences from the amino acid sequence
based on known codon usage. The DNA molecule encoding a plasmin can
be cloned into a suitable vector, such as a cloning or expression
vector, by any of the methodologies known in the art. The cloning
or expression vectors contain all the components required for
additional cloning of the plasmin DNA, such as restriction enzyme
sites, or for the expression of the plasmin, such as a
host-specific promoter, and optionally, enhancer sequences. The
expression vector can be introduced and expressed in a host cell. A
host cell can be any prokaryotic or eukaryotic cell. For example,
the plasmin can be expressed in bacterial cells (i.e., E. coli),
yeast, insect cells (i.e., Sf9), or mammalian cells. Other suitable
host cells are known to those skilled in the art. The host cell can
be used to produce or overexpress the plasmin, variant or
derivative thereof in culture. Then the biologically expressed
plasmin, variant or derivative thereof may be purified using known
purification techniques, such as affinity chromatography, to
produce purified or isolated plasmin suitable for use in the
methods described herein.
[0043] In some embodiments, a variant, derivative or analog of a
plasmin may be preferred. Variants, derivatives and analogs of
plasmin can be identified or generated by one ordinarily skilled in
the art. The plasmin variants, derivatives and analogs can be
generated, for example, by using the recombinant methods or methods
of synthesis described herein. Plasmin derivatives known in the art
include miniplasmin and microplasmin are also suitable for use in
the methods disclosed herein.
[0044] As used herein, the term "derivative" and "variant" may be
used interchangeably and refers to a plasmin that differs from
naturally occurring plasmin, but retains the essential properties
thereof. For example, the plasmin derivative may be a biologically
active fragment of plasmin, for example, a truncated plasmin. The
biologically active fragment contains the catalytic domain of
plasmin and possesses serine protease catalytic activity.
Alternatively, the plasmin derivative may be a mutated plasmin,
wherein at least 1 amino acid, at least 2, at least 3, at least 4,
at least 5, at least 6, at least 7, at least 8, at least 9, at
least 10, at least 20, at least 30, at least 40, or at least 50
amino acids are mutated from the wild-type plasmin. Mutated plasmin
may have altered sequences by substitutions, additions, or
deletions, that still result in functionally equivalent molecules.
In one embodiment, the plasmin derivative is about 99%, about 98%,
about 97%, about 96%, about 95%, about 90%, about 85%, about 80%,
about 75%, about 70%, about 65%, about 60%, about 55%, or about 50%
identity to wild-type plasmin. In some instances, the mutation may
increase the potency (protease activity), stability, or number of
targets of the plasmin. In another aspect, the mutation may
decrease the potency, stability, or number of targets of the
plasmin. The mutation may be a conservative amino acid
substitution. Alternatively, the mutation may be a non-conservative
amino acid substitution. In another embodiment, the plasmin
derivative may be chemically modified, for example, by the addition
of a chemical moiety that alters activity or stability.
[0045] The term "% identity," in the context of two or more nucleic
acid or polypeptide sequences, refer to two or more sequences or
subsequences that are the same or have a specified percentage of
amino acid residues or nucleotides that are the same, when compared
and aligned for maximum correspondence, as measured using one of
the following sequence comparison algorithms or by visual
inspection. For example, % identity is relative to the entire
length of the coding regions of the sequences being compared.
[0046] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequence coordinates are designated,
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
Percent identity is determined using search algorithms such as
BLAST and PSI-BLAST (Altschul et al., 1990, J Mol Biol 215:3,
403-410; Altschul et al., 1997, Nucleic Acids Res 25:17,
3389-402).
[0047] The term "analog" as used herein refers to compounds or
peptides that retain the essential protease activity of the
plasmin. For example, the analog may bear some amino acid sequence
similarity to or no sequence similarity to naturally occurring
plasmin. However all plasmin analogs retain the functional
capability of protease cleavage of any one of the targets of
plasmin (i.e., laminin, fibrin), and/or the preferential cleavage
at the carboxyl side of Lysine and Arginine residues.
[0048] Plasmin derivatives and analogs can be identified by one
ordinarily skilled in the art by screening combinatorial libraries
of mutants of plasmin or general therapeutic or pharmaceutical
compounds.
Methods of Use
[0049] The present invention provides methods for enhancing the
delivery of a therapeutic agent to the eye. The methods of the
present invention include administering a plasmin and a therapeutic
agent to the eye. The therapeutic agent may be delivered to the eye
by any method known in the art. Routes of administration include,
but are not limited to, intravitreal, intracameral,
subconjunctival, subtenon, retrobulbar, posterior juxtascleral, or
topical. Delivery methods include, for example, injection by a
syringe and a drug delivery device, such as an implanted vitreal
delivery device (i.e., VITRASERT.RTM.).
[0050] Preferably, the therapeutic agent is administered to the
vitreous by intravitreal injection for delivery of therapeutic
agents to the retina. In some embodiments, the methods of the
present invention provide enhanced delivery to cells of the retina.
Exemplary retinal cells, include, but are not limited to,
photoreceptor cells (e.g., rods, cones, and photosensitive retinal
ganglion cells), horizontal cells, bipolar cells, amacrine cells,
retinal ganglion cells, Muller glial cell, and retinal pigment
epithelial cells.
[0051] In one embodiment, the plasmin or derivative thereof is
administered concurrently or sequentially with the therapeutic
agent. For concurrent administration, the plasmin or derivative
thereof can be formulated with the therapeutic agent in a single
composition suitable for delivery, for example, injection, by
methods known in the art. Alternatively, the plasmin or derivative
thereof can be injected in separate compositions, simultaneously or
sequentially. In a preferred embodiment, the plasmin may be
administered prior to administration of the therapeutic agent.
[0052] Such formulations comprise a pharmaceutically and/or
physiologically acceptable vehicle, diluent, carrier or excipient,
such as buffered saline or other buffers, e.g., HEPES, to maintain
physiologic pH. For a discussion of such components and their
formulation, see, generally, Gennaro, A E., Remington: The Science
and Practice of Pharmacy, Lippincott Williams & Wilkins
Publishers; 2003 or latest edition). See also, WO00/15822. If the
preparation is to be stored for long periods, it may be frozen, for
example, in the presence of glycerol.
[0053] The dosage of plasmin or derivative thereof to be
administered can be optimized by one of ordinary skill in the art.
Delivery to certain target ocular tissues may require lower doses
of plasmin or higher doses of plasmin, depending on the location of
the target tissue, intervening ocular structures, and ability of
the agent to penetrate the target tissue. Preferably, the dose of
plasmin administered is about 0.001 UI (enzyme units) per eye,
0.025 UI per eye, about 0.05 UI per eye, about 0.075 UI per eye,
about 0.100 UI per eye, about 0.150 UI per eye, or about 0.200 UI
per eye.
[0054] In some embodiments, the methods for enhanced delivery
disclosed herein may provide increased efficacy of a therapeutic
agent. Increased efficacy of the therapeutic agent can be
determined by measuring the therapeutic effect of the therapeutic
agent. Treatment is efficacious if the treatment leads to clinical
benefit such as, alleviation of a symptom in the subject. For
example, in a degenerative retinal disease, such as retinitis
pigmentosa, treatment is efficacious when light sensitivity or
another aspect of vision is improved or restored. When treatment is
applied prophylactically, "efficacious" means that the treatment
retards or prevents an ocular disease or disorder or prevents or
alleviates a symptom of clinical symptom of an ocular disease or
disorder. Efficaciousness is determined in association with any
known method for diagnosing or treating the particular ocular
disease or disorder.
[0055] In some embodiments, the therapeutic agent is a nucleic acid
or a nucleic acid expression vector (i.e. a viral vector) encoding
a therapeutic transgene or an siRNA species (i.e., short hairpin or
microRNA). With regard to such therapeutic agents, the enhanced
delivery of a therapeutic agent provided by the methods disclosed
herein may result in increased transduction efficiency. Increased
transduction efficiency can be determined by measuring the level of
expression of the transgene introduced by the viral vector.
Therapeutic Agents
[0056] The therapeutic agents to be delivered to the eye by the
methods described herein are any therapeutic agents known in the
art for treating, alleviating, reducing, or preventing a symptom of
an ocular disease, an ocular disorder, or an ocular condition. The
therapeutic agent may be a small molecule, a nucleic acid, an
antibody, or a peptide.
[0057] Examples of small molecules suitable for use in the methods
described herein include, but are not limited to, tyrosine kinase
inhibitors, antibiotics, anti-inflammatory agents, glucocorticoids,
opioid antagonists, and other enzyme inhibitors.
[0058] Examples of nucleic acids suitable for use in the methods
described herein include, but are not limited to, viral vectors
encoding therapeutic transgenes (i.e., channelopsins, or
halorhodopsin), RNA interference molecules (i.e., short hairpins,
siRNA, or microRNAs). In a particularly preferred embodiment, the
therapeutic agents are viral vectors encoding transgenes for gene
therapy. Particularly preferred viral vectors are rAAV vectors that
encode channelopsins or halorhodopsins for expression in the retina
to restore light sensitivity.
[0059] Examples of antibodies suitable for use in the methods
described herein include, but are not limited to, ranibizumab
(Lucentis.RTM.), VEGF antibodies (Eylea.RTM.), bevacizumab
(Avastin.RTM.), infliximab, etanercept, and adalimumab.
[0060] Examples of proteins or peptides suitable for use in the
methods described herein include, but are no limited to,
microplasmin (Ocriplasmin, Jetrea.RTM.), macugen pagylated
polypeptide (Pegaptanib), and integrin peptides. In some aspects,
the peptide therapeutic is a collection of peptides, containing two
or more peptides.
[0061] Any of the therapeutic agents described herein may be
optionally encapsulated in a carrier, such as a nanoparticle, a
polymer, or a liposome. These carrier agents may serve to further
enhance the delivery of the therapeutic agent to the eye. In some
aspects, the carrier agents may alter the properties of the
therapeutic agents, such as increasing the stability (half-life) or
providing sustained-release properties to the therapeutic agents.
Alternatively, the carrier may protect the therapeutic agent from
the proteolytic activities of plasmin if formulated in the same
composition for delivery.
Gene Therapy
[0062] As a large number of ocular diseases and disorders result
from aberrant gene expression in various ocular tissues, gene
therapy possesses increasing potential as an effective therapy.
However, the efficacy of gene therapy in the eye has been limited
due to the challenges of effective delivery and transduction of the
therapeutic viral vectors throughout any ocular tissue.
[0063] Thus, the present invention provides methods for increased
efficiency of delivery of transgenes to the eye for treating an
ocular disease or disorder, or for restoring or improving vision.
Transgenes of particular interest for restoration of
photosensitivity or vision include photosensitive proteins, such as
opsin genes or rhodopsin genes. As used herein, "transgene" refers
to a polynucleotide encoding a polypeptide of interest, wherein the
polynucleotide is present in a nucleic acid expression vector
suitable for gene therapy (e.g., a viral vector such as AAV).
[0064] Previous studies have shown that injection of a recombinant
adeno-associated viral vector encoding a transgene, such as
channelopsin-2, results in poor delivery of the vector and low
expression of Chop2 in the inner retinal cells, especially bipolar
cells. In non-human primates, AAV-mediated gene transfection was
found to be more efficient in peripheral retina, fovea, and along
blood vessels, suggesting that inner limiting membrane (ILM), which
is the boundary between the retina and the vitreous space, is a
major barrier (Ivanova et al., 2010).
[0065] The present invention provides a solution to this problem by
using plasmin or derivatives thereof to dissolve the components the
ILM, such as laminin and fibronectin. Accordingly, therapeutic
agents will have greater accessibility to the retina, specifically
the cells of the inner retina such as the retinal bipolar cells,
retinal ganglion cells, Muller glial cells, and retinal pigment
epithelial cells. The methods described herein provide enhanced
delivery of therapeutic compounds, such as therapeutic viral
vectors. The enhanced delivery of viral vectors is demonstrated by
increased transduction efficiency, increased expression of the
therapeutic transgene (i.e., Chop2), and increased efficacy of the
therapeutic compound (i.e., increased light sensitivity or
restoration of vision).
[0066] Nucleic acid expression vectors suitable for use in gene
therapy are known in the art. For example, the nucleic acid
expression vector is a viral vector. The viral vectors can be
retroviral vectors, adenoviral vectors, adeno-associated vectors
(AAV), or lentiviral vectors, or any engineered or recombinant
viral vector known in the art. Particularly preferred viral vectors
are adeno-associated vectors, for example, AAV-1, AAV-2, AAV-3,
AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12 or
any engineered or recombinant AAV known in the art. In a
particularly preferred embodiment, the vector is recombinant AAV-2
(rAAV2).
[0067] In some embodiments, a recombinant adeno-associated viral
(rAAV) vector comprises a capsid protein with a mutated tyrosine
residue which enables to the vector to have improved transduction
efficiency of a target cell, e.g., a retinal bipolar cell (e.g. ON
or OFF retinal bipolar cells; rod and cone bipolar cells). In some
cases, the rAAV further comprises a promoter (e.g., mGluR6, or
fragment thereof) capable of driving the expression of a protein of
interest in the target cell.
[0068] In one embodiment, a mutation may be made in any one or more
of tyrosine: residues of the capsid protein of AAV 1-12 or hybrid.
AAVs. In specific embodiments these are surface exposed tyrosine
residues. In a related embodiment the tyrosine residues are part of
the VP1, VP2, or VP3 capsid protein. In exemplary embodiments, the
mutation may be made at one or more of the following amino acid
residues of an AAV-VP3 capsid protein: Tyr252, Tyr272, Tyr444,
Tyr500, Tyr700, Tyr704, Tyr730; Tyr275, Tyr281, Tyr508, Tyr576,
Tyr612, Tyr673 or Tyr720. Exemplary mutations are
tyrosine-to-phenylalanine mutations including, but not limited to,
Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F,
Y508F, Y576F, Y612G, Y673F and Y720F. in a specific embodiment
these mutations are made in the AAV2 serotype. In some cases, an
AAV2 serotype comprises a Y444F mutation and/or an AAV8 serotype
comprises a Y733F mutation, wherein 444 and 733 indicate the
location of a point tyrosine mutation of the viral capsid. In
further embodiments, such mutated AAV2 and AAV8 serotypes encode a
light-sensitive protein and also comprise a modified mGluR6
promoter to drive expression of such light-sensitive protein. Such
AAV vectors are described in, for example, Petrs-Silva et al., Mol
Ther., 2011 19:293-301).
[0069] In some embodiments, the expression of the therapeutic
transgene is driven by a constitutive promoter, i.e., CAG promoter,
CMV promoter, LTR. In other embodiments, the promoter is an
inducible or a cell-specific promoter. Cell type-specific promoters
that enable transgene expression in specific subpopulations of
cells, i.e., retinal neuron cells or degenerating cells, may be
preferred. These cells may include, but are not limited to, a
retinal ganglion cell, a photoreceptor cell, a bipolar cell, a rod
bipolar cell, an ON-type cone bipolar cell, a retinal ganglion
cell, a photosensitive retinal ganglion cell, a horizontal cell, an
amacrine cell, an AII amacrine cell, or a retinal pigment
epithelial cell. Cell type-specific promoters are well known in the
art. Particularly preferred cell type-specific promoters include,
but are not limited to mGluR6, NK-3, and Pcp2(L7). Cell
type-specific promoters modified using recombinant DNA techniques
known in the art to increase efficiency of expression and selective
targeting are also encompassed in the present invention. For
example, a modified mGluR6 promoter contains a combination of
regulatory elements from the mGluR6 gene, as described in U.S.
Provisional Application No. 61/951,360, hereby incorporated by
reference in its entirety.
[0070] In one embodiment of the present invention, the therapeutic
transgene can be any light-sensitive opsin. The opsin family of
genes includes vertebrate (animal) and invertebrate opsins. Animal
opsins are G-protein coupled receptors (GPCRs) with 7-transmembrane
helices which regulate the activity of ion channels. Invetertebrate
rhodopsins are usually not GPCRs but are light-sensitive or
light-activated ion pumps or ion channels.
[0071] As referred to herein, an opsin gene or light-sensitive
protein includes, but is not limited to, channelrhodopsins, or
channelopsins, (i.e., ChR1, ChR2, vChR1 from Volvox carteri, vChR2,
and other variants identified from any vertebrate, invertebrate, or
microbe), halorhodopsins (NpHR), melanopsins, pineal opsins,
photopsins, bacteriorhodopisins, proteorhodopsins and functional
variants or chimeras thereof. A light-sensitive protein of this
invention can occur naturally in plant, animal, archaebacterial,
algal, or bacterial cells, or can alternatively be created through
laboratory techniques. Examples of opsin genes are discussed in
further detail below.
[0072] Examples of channelrhodopsins, or channelopsins, as
transgenes in the present invention include channelrhodopsins Chop1
(also known as ChR1) (GenBank accession number AB058890/AF385748)
and Chop2 (also known as ChR2) (GenBank accession number
AB058891/AF461397) are two rhodopsins from the green alga
Chlamydomonas reinhardtii (Nagel, 2002; Nagel, 2003) Channelopsins
are a seven transmembrane domain proteins that become
photo-switchable (light sensitive) when bound to the chromophore
all-trans-retinal. Channelopsins, when linked to a retinal molecule
via Schiff base linkage forms a light-gated, nonspecific, inwardly
rectifying, cation channel, called a channelrhodopsin. These
light-sensitive channels that, when expressed and activated in
neural tissue, allow for a cell to be depolarized when stimulated
with light (Boyden, 2005). A Chop2 fragment (315 amino acids) has
been shown to efficiently increase photosensitivity and vision in
mouse models of photoreceptor degeneration (Bi et al., Neuron,
2006, and U.S. Pat. No. 8,470,790; both of which are hereby
incorporated by reference). Chop2 mutants and variants as described
in PCT Publication WO 2013/134295 (hereby incorporated by
reference) may also be expressed using the promoters described
herein. The present invention also provides for use of Volvox
carteri channelrhodopsins (i.e., vChR1 and vChR2).
[0073] NpHR (Halorhodopsin) (GenBank accession number EF474018) is
from the haloalkaliphilic archaeon Natronomonas pharaonic. In
certain embodiments variants of NpHR can be created. In specific
embodiments single or multiple point mutations to the NpHR protein
can result in NpHR variants. In specific embodiments a mammalian
codon optimized version of NpHR can be utilized. In one embodiment
NpHR variants are utilized. In one specific embodiment eNpHR
(enhanced NpHR) is utilized. Addition of the amino acids FCYENEV to
the NpHR C-terminus along with the signal peptide from the (3
subunit of the nicotinic acetylcholine receptor to the NpHR
N-terminus results in the construction of eNpHR.
[0074] Melanopsin (GenBank accession number 6693702) is a
photopigment found in specialized photosensitive ganglion cells of
the retina that are involved in the regulation of circadian
rhythms, pupillary light reflex, and other non-visual responses to
light. In structure, melanopsin is an opsin, a retinylidene protein
variety of G-protein-coupled receptor. Melanopsin resembles
invertebrate opsins in many respects, including its amino acid
sequence and downstream signaling cascade. Like invertebrate
opsins, melanopsin appears to be a bistable photopigment, with
intrinsic photoisomerase activity. In certain embodiments variants
of melanopsin can be created. In specific embodiments single or
multiple point mutations to the melanopsin protein can result in
melanopsin variants.
[0075] Light-sensitive proteins may also include proteins that are
at least about 10%, at least about 20%, at least about 25%, at
least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 70%, at least about 80%, at least about 90%, at
least about 95%, or at least about 99% identical to any of the
light-sensitive proteins described herein (i.e., ChR1, ChR2, vChR1,
vChR2, NpHR and melanopsin). The light-sensitive proteins of the
present invention may also include proteins that have at least one
mutation. The mutation may be a point mutation.
[0076] In some embodiments, light-sensitive proteins can modulate
signaling within neural circuits and bidirectionally control
behavior of ionic conductance at the level of a single neuron. In
some embodiments the neuron is a retinal neuron, a retinal bipolar
cell (e.g. ON or OFF retinal bipolar cells; rod and cone bipolar
cells), a retinal ganglion cell, a photoreceptor cell, or a retinal
amacrine cell.
[0077] In some embodiments, a polyA tail can be inserted downstream
of the transgene in an expression cassette or nucleic acid
expression vector of the present invention. Suitable polyA tails
are known in the art, and include, for example, human growth
hormone poly A tail (hGHpA), bovine growth hormone polyA tail
(bGHpA), bovine polyA, SV40 polyA, and AV40pA.
[0078] Upon illumination by the preferred dose of light radiation,
rhodopsin proteins opens the pore of the channel, through which
H.sup.+, Na.sup.+, K.sup.+, and/or Ca.sup.2+ ions flow into the
cell from the extracellular space. Activation of the rhodopsin
channel typically causes a depolarization of the cell expressing
the channel Depolarized cells produce graded potentials and or
action potentials to carry information from the
rhodopsin-expressing cell to other cells of the retina or brain, to
increase light sensitivity or restore vision. Methods of improving
vision or light sensitivity by administration of a vector encoding
a channelopsin (or variant thereof) are described in
PCT/US2007/068263, the contents of which are herein incorporated in
its entirety.
[0079] Accordingly, a dual rhodopsin system can be used to
recapitulate the ON and OFF pathways integral to visual processing
and acuity. Briefly, a Chop2 protein of the present invention can
be specifically targeted to ON type retinal neurons (i.e., ON type
ganglion cells and/or ON type bipolar cells), while a
hypopolarizing light sensor (i.e., halorhodopsin or other chloride
pump known in the art) can be targeted to OFF type retinal neurons
(i.e. OFF type ganglion cells and/or OFF type bipolar cells) to
create ON and OFF pathways. The specific targeting to preferred
cell subpopulations can be achieved through the use of different
cell type-specific promoters. For example, Chop2 expression may be
driven by the mGluR6 promoter for targeted expression in ON-type
retinal neurons (i.e., ON type ganglion cells and/or ON type
bipolar cells) while a hypopolarizing channel, such as
halorhodopsin, expression is driven by the NK-3 promoter for
targeted expression in OFF-type retinal neurons (i.e., OFF type
ganglion cells and/or OFF type bipolar cells).
[0080] An alternative approach to restore ON and OFF pathways in
the retina is achieved by, expressing a depolarizing light sensor,
such as ChR2, to rod bipolar cells or AII amacrine. In this
approach, the depolarization of rod bipolar cells or AII amacrine
cells can lead to the ON and OFF responses at the levels of cone
bipolar cells and the downstream retinal ganglion cells. Thus, the
ON and OFF pathways that are inherent in the retina are
maintained.
[0081] An effective amount of rAAV virions carrying a nucleic acid
sequence encoding the rhodopsin DNA under the control of the
promoter of choice, preferably a constitutive CMV promoter or a
cell-specific promoter such as mGluR6, is preferably in the range
of between about 10.sup.10 to about 10.sup.13 rAAV infectious units
in a volume of between about 25 and about 800 .mu.l per injection.
The rAAV infectious units can be measured according to McLaughlin,
S K et al., 1988, J Virol 62:1963. More preferably, the effective
amount is between about 10.sup.10 and about 10.sup.12 rAAV
infectious units and the injection volume is preferably between
about 50 and about 150 .mu.l. Other dosages and volumes, preferably
within these ranges but possibly outside them, may be selected by
the treating professional, taking into account the physical state
of the subject (preferably a human), who is being treated,
including, age, weight, general health, and the nature and severity
of the particular ocular disorder.
[0082] It may also be desirable to administer additional doses
("boosters") of the present nucleic acid(s) or rAAV compositions.
For example, depending upon the duration of the transgene
expression within the ocular target cell, a second treatment may be
administered after 6 months or yearly, and may be similarly
repeated. Neutralizing antibodies to AAV are not expected to be
generated in view of the routes and doses used, thereby permitting
repeat treatment rounds.
[0083] The need for such additional doses can be monitored by the
treating professional using, for example, well-known
electrophysiological and other retinal and visual function tests
and visual behavior tests. The treating professional will be able
to select the appropriate tests applying routine skill in the art.
It may be desirable to inject larger volumes of the composition in
either single or multiple doses to further improve the relevant
outcome parameters.
Ocular Disorders
[0084] The ocular disorders for which the methods of the present
invention are intended and may be used to improve one or more
parameters of vision include, but are not limited to, developmental
abnormalities that affect both anterior and posterior segments of
the eye. Anterior segment disorders include glaucoma, cataracts,
corneal dystrophy, keratoconus. Posterior segment disorders include
blinding disorders caused by photoreceptor malfunction and/or death
caused by retinal dystrophies and degenerations. Retinal disorders
include congenital stationary night blindness, age-related macular
degeneration, congenital cone dystrophies, and a large group of
retinitis-pigmentosa (RP)-related disorders. These disorders
include genetically pre-disposed death of photoreceptor cells, rods
and cones in the retina, occurring at various ages. Among those are
severe retinopathies, such as subtypes of RP itself that progresses
with age and causes blindness in childhood and early adulthood and
RP-associated diseases, such as genetic subtypes of LCA, which
frequently results in loss of vision during childhood, as early as
the first year of life. The latter disorders are generally
characterized by severe reduction, and often complete loss of
photoreceptor cells, rods and cones. Other ocular diseases that may
benefit from the methods described herein include, but are not
limited to, retinoblastoma, ocular melanoma, diabetic retinopathy,
hypertensive retinopathy, any inflammation of the ocular tissues
(i.e., chorioretinal inflammation, scleritis, keratitis, uveitis,
etc.), or infection (i.e., bacterial or viral).
[0085] In particular, the viral-mediated delivery of rhodopsins
using the methods of the present invention useful for the treatment
and/or restoration of at least partial vision to subjects that have
lost vision due to ocular disorders, such as RPE-associated
retinopathies, which are characterized by a long-term preservation
of ocular tissue structure despite loss of function and by the
association between function loss and the defect or absence of a
normal gene in the ocular cells of the subject. A variety of such
ocular disorders are known, such as childhood onset blinding
diseases, retinitis pigmentosa, macular degeneration, and diabetic
retinopathy, as well as ocular blinding diseases known in the art.
It is anticipated that these other disorders, as well as blinding
disorders of presently unknown causation which later are
characterized by the same description as above, may also be
successfully treated by the methods described herein. Thus, the
particular ocular disorder treated by the present invention may
include the above-mentioned disorders and a number of diseases
which have yet to be so characterized.
Restoration of Light Sensitivity
[0086] These methods described herein may be used in subjects of
normal and/or impaired vision. The enhanced delivery of a
therapeutic compound, as described herein, may preserve, improve,
or restore vision. The term "vision" as used herein is defined as
the ability of an organism to usefully detect light as a stimulus
for differentiation or action. Vision is intended to encompass the
following: [0087] 1. Light detection or perception--the ability to
discern whether or not light is present; [0088] 2. Light
projection--the ability to discern the direction from which a light
stimulus is coming; [0089] 3. Resolution--the ability to detect
differing brightness levels (i.e., contrast) in a grating or letter
target; and [0090] 4. Recognition--the ability to recognize the
shape of a visual target by reference to the differing contrast
levels within the target. Thus, "vision" includes the ability to
simply detect the presence of light. The methods of the present
invention can be used to improve or restore vision, wherein the
improvement or restoration in vision includes, for example,
increases in light detection or perception, increase in light
sensitivity or photosensitivity in response to a light stimulus,
increase in the ability to discern the direction from which a light
stimulus is coming, increase in the ability to detect differing
brightness levels, increase in the ability to recognize the shape
of a visual target, and increases in visual evoked potential or
transmission from the retina to the cortex. As such, improvement or
restoration of vision may or may not include full restoration of
sight, i.e., wherein the vision of the patient treated with the
present invention is restored to the degree to the vision of a
non-affected individual. The visual recovery described in the
animal studies described below may, in human terms, place the
person on the low end of vision function by increasing one aspect
of vision (i.e., light sensitivity, or visual evoked potential)
without restoring full sight. Nevertheless, placement at such a
level would be a significant benefit because these individuals
could be trained in mobility and potentially in low order
resolution tasks which would provide them with a greatly improved
level of visual independence compared to total blindness. Even
basic light perception can be used by visually impaired
individuals, whose vision is improved using the present
compositions and methods, to accomplish specific daily tasks and
improve general mobility, capability, and quality of life.
[0091] The degree of restoration of vision can be determined
through the measurement of vision before, and preferably after,
administering a vector comprising, for example, DNA encoding a
therapeutic transfene such as Chop2 or halorhodopsin or both.
Vision can be measured using any of a number of methods well-known
in the art or methods not yet established. Vision, as improved or
restored by the present invention, can be measured by any of the
following visual responses: [0092] 1. a light detection response by
the subject after exposure to a light stimulus--in which evidence
is sought for a reliable response of an indication or movement in
the general direction of the light by the subject individual when
the light it is turned on; [0093] 2. a light projection response by
the subject after exposure to a light stimulus in which evidence is
sought for a reliable response of indication or movement in the
specific direction of the light by the individual when the light is
turned on; [0094] 3. light resolution by the subject of a light vs.
dark patterned visual stimulus, which measures the subject's
capability of resolving light vs dark patterned visual stimuli as
evidenced by: [0095] a. the presence of demonstrable reliable
optokinetically produced nystagmoid eye movements and/or related
head or body movements that demonstrate tracking of the target (see
above) and/or [0096] b. the presence of a reliable ability to
discriminate a pattern visual stimulus and to indicate such
discrimination by verbal or non-verbal means, including, for
example pointing, or pressing a bar or a button; or [0097] 4.
electrical recording of a visual cortex response to a light flash
stimulus or a pattern visual stimulus, which is an endpoint of
electrical transmission from a restored retina to the visual
cortex, also referred to as the visual evoked potential (VEP).
Measurement may be by electrical recording on the scalp surface at
the region of the visual cortex, on the cortical surface, and/or
recording within cells of the visual cortex.
[0098] Thus, improvement or restoration of vision, according to the
present invention, can include, but is not limited to: increases in
amplitude or kinetics of photocurents or electrical response in
response to light stimulus in the retinal cells, increases in light
sensitivity (i.e., lowering the threshold light intensity required
for initiating a photocurrent or electrical response in response to
light stimulus, thereby requiring less or lower light to evoke a
photocurrent) of the retinal cells, increases in number or
amplitude of light-evoked spiking or spike firings, increases in
light responses to the visual cortex, which includes increasing in
visual evoked potential transmitted from the retina or retinal
cells to the visual cortex or the brain.
[0099] Both in vitro and in vivo studies to assess the various
parameters of the present invention may be used, including
recognized animal models of blinding human ocular disorders. Large
animal models of human retinopathy, e.g., childhood blindness, are
useful. The examples provided herein allow one of skill in the art
to readily anticipate that this method may be similarly used in
treating a range of retinal diseases.
[0100] While earlier studies by others have demonstrated that
retinal degeneration can be retarded by gene therapy techniques,
the present invention demonstrates a definite physiological
recovery of function, which is expected to generate or improve
various parameters of vision, including behavioral parameters.
[0101] Behavioral measures can be obtained using known animal
models and tests, for example performance in a water maze, wherein
a subject in whom vision has been preserved or restored to varying
extents will swim toward light (Hayes, J M et al., 1993, Behav
Genet 23:395-403).
[0102] In models in which blindness is induced during adult life or
congenital blindness develops slowly enough that the individual
experiences vision before losing it, training of the subject in
various tests may be done. In this way, when these tests are
re-administered after visual loss to test the efficacy of the
present compositions and methods for their vision-restorative
effects, animals do not have to learn the tasks de novo while in a
blind state. Other behavioral tests do not require learning and
rely on the instinctiveness of certain behaviors. An example is the
optokinetic nystagmus test (Balkema G W et al., 1984, Invest
Ophthalmol Vis Sci. 25:795-800; Mitchiner J C et al., 1976, Vision
Res. 16:1169-71).
[0103] The present invention may also be used in combination with
other forms of vision therapy known in the art to improve or
restore vision. For example, the use of visual prostheses, which
include retinal implants, cortical implants, lateral geniculate
nucleus implants, or optic nerve implants. Thus, in addition to
genetic modification of surviving retinal neurons using the present
methods, the subject being treated may be provided with a visual
prosthesis before, at the same time as, or after the molecular
method is employed. The effectiveness of visual prosthetics can be
improved with training of the individual, thus enhancing the
potential impact of the Chop2 transformation of patient cells as
contemplated herein. Training methods, such as habituation training
characterized by training the subject to recognize recognize (i)
varying levels of light and/or pattern stimulation, and/or (ii)
environmental stimulation from a common light source or object as
would be understood by one skilled in the art; and orientation and
mobility training characterized by training the subject to detect
visually local objects and move among said objects more effectively
than without the training. In fact, any visual stimulation
techniques that are typically used in the field of low vision
rehabilitation are applicable here.
[0104] As used herein, by a "subject" is meant an individual. Thus,
the "subject" can include domesticated animals (e.g., cats, dogs,
etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.),
laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.),
and birds. "Subject" can also include a mammal, such as a primate
or a human. Preferably, the subject is a human. A "subject in need
thereof" is a subject suffering from or at risk of developing or
suffering from an ocular disease or disorder. A subject at risk of
developing or suffering from an ocular disease or disorder can be
diagnosed by a physician or ocular specialist using routine methods
in the art.
EXAMPLES
Example 1: Plasmin Increases Delivery of AAV2-Vector Encoding Chop2
to the Retina
[0105] Delivery of a therapeutic viral construct encoding a
functional GFP-channelopsin-2 protein to the retina was examined
Injection of 6.times.10.sup.12 vg/ml of AAV2 vector
AAV2/2-ChR2-GFP-WPRE-hGHpA in control (FIG. 1A) or co-injection
with plasmin (FIG. 1B) was performed into the vitreous space of the
eye of one month old C56BL/6J. The concentration of plasmin
injected with vector was 0.025 IU/eye. After one month, the mice
retinas were isolated, and retinal vertical sections were prepared.
The sections were immunostained and cells were counted
Immunofluorescence analysis of the sections showed that
co-injection with plasmin increased the transduction efficiency of
the therapeutic AAV2-ChR2-GFP vector, as evidenced by increased
fluorescence in comparison to control (FIG. 1).
Example 2: Plasmin Increases Transduction Efficiency in Retinal
Ganglion Cells
[0106] Using the same experimental set-up as in Example 1, a vector
(2.times.10.sup.12 vg/ml) encoding GFP was co-injected with either
control or varying concentrations of plasmin: low (L=0.005 IU/eye),
middle (M=0.025 IU/eye), and high (H=0.100 IU/eye). After 1 month,
retinal whole mounts were prepared and immunostained.
Representative images for each plasmin concentration and control
are shown in FIG. 2. As shown, treatment with plasmin increases GFP
expression.
[0107] To further quantify these results, GFP-expressing retinal
ganglion cells were counted from multiple unit areas of 223
.mu.m.times.167 .mu.m. The results are presented in FIG. 3A. As
shown, treatment with low, middle and high doses of plasmin
resulted in statistically significantly increased levels of GFP
expression in retinal ganglion cells.
[0108] Neurotoxicity as a result of plasmin injection was also
examined. The retinal whole mounts were stained with DAPI for
cell-counting. The number of cells over multiple unit areas of 223
.mu.m.times.167 .mu.m were counted and compared between control and
low, middle and high doses of plasmin. As shown in FIG. 3B, the
cell counts were not found to differ significantly between control
and plasmin-treated retinas (p=0.74). As such, the tested
concentrations of plasmin were not shown to have any neurotoxic
effect to the retinal ganglion cells, thereby indicating that
plasmin is safe for use in the eye, even at high doses.
Example 3: Plasmin Increases Transduction Efficiency in Retinal
Bipolar Cells
[0109] Comparison of the transduction efficiency of a viral vector
encoding mCherry fluorescent protein when co-injected with
different concentrations of plasmin was assessed in vivo.
Specifically, overall levels and the localization of mCherry
expression throughout the retina were examined. An AAV2 vector with
an Y444F capsid mutation carrying mCherry under control of an
mGluR6 promoter were injected at a concentration of
2.times.10.sup.12 vg/ml. The mGluR6 promoter directs expression of
mCherry specifically to the retinal bipolar cells.
[0110] The AAV2 mCherry vector was co-injected with three doses of
plasmin, high (H=0.100 IU/eye), middle (M=0.025 IU/eye), and low
(L=0.005 IU/eye). After 1 month, the retinas were isolated and
retinal whole-mounts were prepared. Transduction efficiency was
evaluated by immunostaining of mCherry for immunofluorescence
analysis and cell counting. Cells were counted from multiple unit
areas of 223 .mu.m.times.167 .mu.m.
[0111] Injection of the vectors without plasmin did not result in
uniform mCherry expression in retinal bipolar cells across the
entire retina (FIG. 4, control, top panels). Transduction
efficiency was low in the center (A) and middle (B) retina, but
high in the periphery. Co-injection of the AAV2 mCherry vector with
increasing dosages of plasmin (low, middle and high, bottom panels)
resulted in increased transduction efficiency at each retinal
region in a dose-dependent manner.
[0112] The qualitative results from immunofluorescence images were
verified by cell counting. Quantification of mCherry-expressing
cells when co-injected with or without plasmin showed that plasmin
significantly increased the density of mCherry-expressing retinal
cells. The increase in transduction efficiency with plasmin
compared to control was statistically significant with all three
doses of plasmin at the center of the retina. Middle and high doses
of plasmin resulted in a statistically significant increase in
mCherry expression at the mid-region and periphery of the retina.
These results show that plasmin enhances transduction efficiency
throughout the retina, including the peripheral, middle, and center
regions of the retina.
Other Embodiments
[0113] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
[0114] The patent and scientific literature referred to herein
establishes the knowledge that is available to those with skill in
the art. All United States patents and published or unpublished
United States patent applications cited herein are incorporated by
reference. All published foreign patents and patent applications
cited herein are hereby incorporated by reference. All other
published references, documents, manuscripts and scientific
literature cited herein are hereby incorporated by reference.
[0115] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
91809PRTHomo sapiens 1Met Glu His Lys Glu Val Val Leu Leu Leu Leu
Leu Phe Leu Lys Ser1 5 10 15Gly Gln Gly Glu Pro Leu Asp Asp Tyr Val
Asn Thr Gln Gly Ala Ser 20 25 30Leu Phe Ser Val Thr Lys Lys Gln Leu
Gly Ala Gly Ser Ile Glu Glu 35 40 45Cys Ala Ala Lys Cys Glu Glu Asp
Glu Glu Phe Thr Cys Arg Ala Phe 50 55 60Gln Tyr His Ser Lys Glu Gln
Gln Cys Val Ile Met Ala Glu Asn Arg65 70 75 80Lys Ser Ser Ile Ile
Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys 85 90 95Lys Val Tyr Leu
Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg 100 105 110Gly Thr
Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser 115 120
125Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser
130 135 140Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp
Pro Gln145 150 155 160Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys
Arg Tyr Asp Tyr Cys 165 170 175Asp Ile Leu Glu Cys Glu Glu Glu Cys
Met His Cys Ser Gly Glu Asn 180 185 190Tyr Asp Gly Lys Ile Ser Lys
Thr Met Ser Gly Leu Glu Cys Gln Ala 195 200 205Trp Asp Ser Gln Ser
Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe 210 215 220Pro Asn Lys
Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu225 230 235
240Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu
245 250 255Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly
Pro Thr 260 265 270Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Gly
Asn Val Ala Val 275 280 285Thr Val Ser Gly His Thr Cys Gln His Trp
Ser Ala Gln Thr Pro His 290 295 300Thr His Asn Arg Thr Pro Glu Asn
Phe Pro Cys Lys Asn Leu Asp Glu305 310 315 320Asn Tyr Cys Arg Asn
Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr 325 330 335Thr Asn Ser
Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp 340 345 350Ser
Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu 355 360
365Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr
370 375 380Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln
Ser Trp385 390 395 400Ser Ser Met Thr Pro His Arg His Gln Lys Thr
Pro Glu Asn Tyr Pro 405 410 415Asn Ala Gly Leu Thr Met Asn Tyr Cys
Arg Asn Pro Asp Ala Asp Lys 420 425 430Gly Pro Trp Cys Phe Thr Thr
Asp Pro Ser Val Arg Trp Glu Tyr Cys 435 440 445Asn Leu Lys Lys Cys
Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro 450 455 460Pro Val Val
Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys465 470 475
480Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val
485 490 495Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His
Arg His 500 505 510Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly
Leu Glu Lys Asn 515 520 525Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly
Gly Pro Trp Cys Tyr Thr 530 535 540Thr Asn Pro Arg Lys Leu Tyr Asp
Tyr Cys Asp Val Pro Gln Cys Ala545 550 555 560Ala Pro Ser Phe Asp
Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys 565 570 575Pro Gly Arg
Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro 580 585 590Trp
Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly 595 600
605Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu
610 615 620Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala
His Gln625 630 635 640Glu Val Asn Leu Glu Pro His Val Gln Glu Ile
Glu Val Ser Arg Leu 645 650 655Phe Leu Glu Pro Thr Arg Lys Asp Ile
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Val Ile Pro Ala Cys Leu Pro Ser Pro 675 680 685Asn Tyr Val Val Ala
Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly 690 695 700Glu Thr Gln
Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu705 710 715
720Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly
725 730 735Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly
Gly Thr 740 745 750Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val
Cys Phe Glu Lys 755 760 765Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser
Trp Gly Leu Gly Cys Ala 770 775 780Arg Pro Asn Lys Pro Gly Val Tyr
Val Arg Val Ser Arg Phe Val Thr785 790 795 800Trp Ile Glu Gly Val
Met Arg Asn Asn 80523538DNAHomo sapiens 2gaatcattaa cttaatttga
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3538319PRTHomo sapiens 3Met Glu His Lys Glu Val Val Leu Leu Leu Leu
Leu Phe Leu Lys Ser1 5 10 15Gly Gln Gly4561PRTHomo sapiens 4Glu Pro
Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser1 5 10 15Val
Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala 20 25
30Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His
35 40 45Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser
Ser 50 55 60Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys
Val Tyr65 70 75 80Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr
Arg Gly Thr Met 85 90 95Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys
Trp Ser Ser Thr Ser 100 105 110Pro His Arg Pro Arg Phe Ser Pro Ala
Thr His Pro Ser Glu Gly Leu 115 120 125Glu Glu Asn Tyr Cys Arg Asn
Pro Asp Asn Asp Pro Gln Gly Pro Trp 130 135 140Cys Tyr Thr Thr Asp
Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu145 150 155 160Glu Cys
Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly 165 170
175Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser
180 185 190Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro
Asn Lys 195 200 205Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg
Glu Leu Arg Pro 210 215 220Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg
Trp Glu Leu Cys Asp Ile225 230 235 240Pro Arg Cys Thr Thr Pro Pro
Pro Ser Ser Gly Pro Thr Tyr Gln Cys 245 250 255Leu Lys Gly Thr Gly
Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val 260 265 270Ser Gly His
Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His 275 280 285Asn
Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr 290 295
300Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr
Asn305 310 315 320Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser
Cys Asp Ser Ser 325 330 335Pro Val Ser Thr Glu Gln Leu Ala Pro Thr
Ala Pro Pro Glu Leu Thr 340 345 350Pro Val Val Gln Asp Cys Tyr His
Gly Asp Gly Gln Ser Tyr Arg Gly 355 360 365Thr Ser Ser Thr Thr Thr
Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser 370 375 380Met Thr Pro His
Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala385 390 395 400Gly
Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro 405 410
415Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu
420 425 430Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro
Pro Val 435 440 445Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu
Asp Cys Met Phe 450 455 460Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg
Ala Thr Thr Val Thr Gly465 470 475 480Thr Pro Cys Gln Asp Trp Ala
Ala Gln Glu Pro His Arg His Ser Ile 485 490 495Phe Thr Pro Glu Thr
Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys 500 505 510Arg Asn Pro
Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn 515 520 525Pro
Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro 530 535
540Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro
Gly545 550 555 560Arg5483PRTHomo sapiens 5Val Tyr Leu Ser Glu Cys
Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly1 5 10 15Thr Met Ser Lys Thr
Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser 20 25 30Thr Ser Pro His
Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu 35 40 45Gly Leu Glu
Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly 50 55 60Pro Trp
Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp65 70 75
80Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr
85 90 95Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala
Trp 100 105 110Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser
Lys Phe Pro 115 120 125Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn
Pro Asp Arg Glu Leu 130 135 140Arg Pro Trp Cys Phe Thr Thr Asp Pro
Asn Lys Arg Trp Glu Leu Cys145 150 155 160Asp Ile Pro Arg Cys Thr
Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr 165 170 175Gln Cys Leu Lys
Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val 180 185 190Thr Val
Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His 195 200
205Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu
210 215 220Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys
His Thr225 230 235 240Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys
Ile Pro Ser Cys Asp 245 250 255Ser Ser Pro Val Ser Thr Glu Gln Leu
Ala Pro Thr Ala Pro Pro Glu 260 265 270Leu Thr Pro Val Val Gln Asp
Cys Tyr His Gly Asp Gly Gln Ser Tyr 275 280 285Arg Gly Thr Ser Ser
Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp 290 295 300Ser Ser Met
Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro305 310 315
320Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys
325 330 335Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu
Tyr Cys 340 345 350Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val
Val Ala Pro Pro 355 360 365Pro Val Val Leu Leu Pro Asp Val Glu Thr
Pro Ser Glu Glu Asp Cys 370 375 380Met Phe Gly Asn Gly Lys Gly Tyr
Arg Gly Lys Arg Ala Thr Thr Val385 390 395 400Thr Gly Thr Pro Cys
Gln Asp Trp Ala Ala Gln Glu Pro His Arg His 405 410 415Ser Ile Phe
Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn 420 425 430Tyr
Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr 435 440
445Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala
450 455 460Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys
Lys Cys465 470 475 480Pro Gly Arg678PRTHomo sapiens 6Glu Pro Leu
Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser1 5
10 15Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala
Ala 20 25 30Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln
Tyr His 35 40 45Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg
Lys Ser Ser 50 55 60Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu
Lys Lys65 70 757230PRTHomo sapiens 7Val Val Gly Gly Cys Val Ala His
Pro His Ser Trp Pro Trp Gln Val1 5 10 15Ser Leu Arg Thr Arg Phe Gly
Met His Phe Cys Gly Gly Thr Leu Ile 20 25 30Ser Pro Glu Trp Val Leu
Thr Ala Ala His Cys Leu Glu Lys Ser Pro 35 40 45Arg Pro Ser Ser Tyr
Lys Val Ile Leu Gly Ala His Gln Glu Val Asn 50 55 60Leu Glu Pro His
Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu65 70 75 80Pro Thr
Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val 85 90 95Ile
Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val 100 105
110Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln
115 120 125Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro
Val Ile 130 135 140Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn
Gly Arg Val Gln145 150 155 160Ser Thr Glu Leu Cys Ala Gly His Leu
Ala Gly Gly Thr Asp Ser Cys 165 170 175Gln Gly Asp Ser Gly Gly Pro
Leu Val Cys Phe Glu Lys Asp Lys Tyr 180 185 190Ile Leu Gln Gly Val
Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn 195 200 205Lys Pro Gly
Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu 210 215 220Gly
Val Met Arg Asn Asn225 230819PRTArtificial SequenceRecombinant
truncated form of human plasmin 8Ala Pro Ser Phe Asp Cys Gly Lys
Pro Gln Val Glu Pro Lys Lys Cys1 5 10 15Pro Gly
Arg9230PRTArtificial SequenceRecombinant truncated form of human
plasmin 9Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp
Gln Val1 5 10 15Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly
Thr Leu Ile 20 25 30Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu
Glu Lys Ser Pro 35 40 45Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala
His Gln Glu Val Asn 50 55 60Leu Glu Pro His Val Gln Glu Ile Glu Val
Ser Arg Leu Phe Leu Glu65 70 75 80Pro Thr Arg Lys Asp Ile Ala Leu
Leu Lys Leu Ser Ser Pro Ala Val 85 90 95Ile Thr Asp Lys Val Ile Pro
Ala Cys Leu Pro Ser Pro Asn Tyr Val 100 105 110Val Ala Asp Arg Thr
Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln 115 120 125Gly Thr Phe
Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile 130 135 140Glu
Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln145 150
155 160Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser
Cys 165 170 175Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys
Asp Lys Tyr 180 185 190Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly
Cys Ala Arg Pro Asn 195 200 205Lys Pro Gly Val Tyr Val Arg Val Ser
Arg Phe Val Thr Trp Ile Glu 210 215 220Gly Val Met Arg Asn Asn225
230
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