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Patent Analysis of

Treating glaucoma, cardiovascular diseases, and renal diseases

Updated Time 12 June 2019

Patent Registration Data

Publication Number

US10058597

Application Number

US15/187471

Application Date

20 June 2016

Publication Date

28 August 2018

Current Assignee

MAYO FOUNDATION FOR MEDICAL EDUCATION AND RESEARCH

Original Assignee (Applicant)

MAYO FOUNDATION FOR MEDICAL EDUCATION AND RESEARCH

International Classification

C12N15/00,A61K48/00,C12N9/02,A61K38/44,A61K38/00

Cooperative Classification

A61K38/44,A61K48/005,C12Y114/99001,C12N9/0083,A61K48/00

Inventor

POESCHLA, ERIC M.,BARRAZA, ROMAN A.

Patent Images

This patent contains figures and images illustrating the invention and its embodiment.

US10058597 Treating glaucoma, cardiovascular diseases, 1 US10058597 Treating glaucoma, cardiovascular diseases, 2 US10058597 Treating glaucoma, cardiovascular diseases, 3
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Abstract

This document provides methods and materials related to treating glaucoma, ocular hypertension, cardiovascular diseases, and renal diseases. For example, this document provides isolated nucleic acid molecules and viral vectors (e.g., lentiviral vectors) containing isolated nucleic acid molecules. Methods for reducing intraocular pressure as well as symptoms and progression of cardiovascular and renal diseases also are provided.

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Claims

1. A viral vector comprising a nucleic acid encoding a polypeptide having cyclooxygenase-2 activity, wherein said nucleic acid comprises the sequence set forth in SEQ ID NO:3.

2. The viral vector of claim 1, wherein said viral vector is a lentiviral vector.

3. The viral vector of claim 1, wherein said nucleic acid is a template for an mRNA molecule encoding said polypeptide, wherein said mRNA has an increased stability in cells as compared to the stability of an mRNA molecule transcribed from the sequence set forth in SEQ ID NO:2.

4. The viral vector of claim 1, wherein said viral vector comprises a nucleic acid sequence encoding a polypeptide having prostaglandin F2α receptor activity, wherein (i) said nucleic acid sequence encoding said polypeptide having prostaglandin F2α receptor activity comprises a nucleic acid sequence that encodes the same amino acid sequence as set forth in SEQ ID NO:4 and comprises a codon sequence different than the codons set forth in SEQ ID NO:5, (ii) said nucleic acid sequence encoding said polypeptide having prostaglandin F2α receptor activity comprises five or more different codon sequences compared to the codon sequences set forth in SEQ ID NO:5, (iii) said nucleic acid sequence encoding said polypeptide having prostaglandin F2α receptor activity comprises the sequence set forth in SEQ ID NO:6, or (iv) said polypeptide having prostaglandin F2α receptor activity comprises the sequence set forth in SEQ ID NO:4.

5. The viral vector of claim 4, wherein said nucleic acid sequence encoding said polypeptide having prostaglandin F2α receptor activity is a template for an mRNA molecule encoding said polypeptide having prostaglandin F2α receptor activity, wherein said mRNA has an increased stability in cells as compared to the stability of an mRNA molecule transcribed from the sequence set forth in SEQ ID NO:5.

6. The viral vector of claim 4, wherein said nucleic acid sequence encoding said polypeptide having prostaglandin F2α receptor activity comprises a nucleic acid sequence that encodes the same amino acid sequence as set forth in SEQ ID NO:4 and comprises a codon sequence different than the codons set forth in SEQ ID NO:5.

7. The viral vector of claim 4, wherein said nucleic acid sequence encoding said polypeptide having prostaglandin F2α receptor activity comprises five or more different codon sequences compared to the codon sequences set forth in SEQ ID NO:5.

8. The viral vector of claim 4, wherein said nucleic acid sequence encoding said polypeptide having prostaglandin F2α receptor activity comprises the sequence set forth in SEQ ID NO:6.

9. The viral vector of claim 4, wherein said polypeptide having prostaglandin F2α receptor activity comprises the sequence set forth in SEQ ID NO:4.

10. The viral vector of claim 1, wherein said viral vector comprises a nucleic acid sequence encoding a polypeptide having prostaglandin synthase activity, wherein (i) said nucleic acid sequence encoding said polypeptide having prostaglandin synthase activity comprises the sequence set forth in SEQ ID NO:8 or (ii) said polypeptide having prostaglandin synthase activity comprises the sequence set forth in SEQ ID NO:7.

11. The viral vector of claim 10, wherein said nucleic acid sequence encoding said polypeptide having prostaglandin synthase activity comprises the sequence set forth in SEQ ID NO:8.

12. The viral vector of claim 10, wherein said polypeptide having prostaglandin synthase activity comprises the sequence set forth in SEQ ID NO:7.

13. The viral vector of claim 10, wherein said viral vector comprises nucleic acid encoding a polypeptide having prostaglandin F2α receptor activity and a polypeptide having prostaglandin synthase activity, wherein (i) said nucleic acid sequence encoding said polypeptide having prostaglandin F2α receptor activity comprises a nucleic acid sequence that encodes the same amino acid sequence as set forth in SEQ ID NO:4 and comprises a codon sequence different than the codons set forth in SEQ ID NO:5, (ii) said nucleic acid sequence encoding said polypeptide having prostaglandin F2α receptor activity comprises five or more different codon sequences compared to the codon sequences set forth in SEQ ID NO:5, (iii) said nucleic acid sequence encoding said polypeptide having prostaglandin F2α receptor activity comprises the sequence set forth in SEQ ID NO:6, or (iv) said polypeptide having prostaglandin F2α receptor activity comprises the sequence set forth in SEQ ID NO:4.

14. A viral vector comprising a nucleic acid encoding a polypeptide having cyclooxygenase-2 activity and a nucleic acid sequence encoding a polypeptide having prostaglandin F2α receptor activity, wherein said polypeptide having cyclooxygenase-2 activity comprises the sequence set forth in SEQ ID NO:1, and wherein (i) said nucleic acid sequence encoding said polypeptide having prostaglandin F2α receptor activity comprises a nucleic acid sequence that encodes the same amino acid sequence as set forth in SEQ ID NO:4 and comprises a codon sequence different than the codons set forth in SEQ ID NO:5, (ii) said nucleic acid sequence encoding said polypeptide having prostaglandin F2α receptor activity comprises five or more different codon sequences compared to the codon sequences set forth in SEQ ID NO:5, (iii) said nucleic acid sequence encoding said polypeptide having prostaglandin F2α receptor activity comprises the sequence set forth in SEQ ID NO:6, or (iv) said polypeptide having prostaglandin F2α receptor activity comprises the sequence set forth in SEQ ID NO:4.

15. The viral vector of claim 14, wherein said viral vector is a lentiviral vector.

16. The viral vector of claim 14, wherein said nucleic acid encoding said polypeptide having cyclooxygenase-2 activity is a template for an mRNA molecule encoding said polypeptide having cyclooxygenase-2 activity, wherein said mRNA has an increased stability in cells as compared to the stability of an mRNA molecule transcribed from the sequence set forth in SEQ ID NO:2.

17. The viral vector of claim 14, wherein said nucleic acid encoding said polypeptide having cyclooxygenase-2 activity comprises a nucleic acid sequence that encodes the same amino acid sequence as set forth in SEQ ID NO:1 and comprises a codon sequence different than the codons set forth in SEQ ID NO:2.

18. The viral vector of claim 14, wherein said nucleic acid encoding said polypeptide having cyclooxygenase-2 activity comprises five or more different codon sequences compared to the codon sequences set forth in SEQ ID NO:2.

19. The viral vector of claim 14, wherein said nucleic acid encoding said polypeptide having cyclooxygenase-2 activity comprises the sequence set forth in SEQ ID NO:3.

20. The viral vector of claim 14, wherein said nucleic acid sequence encoding said polypeptide having prostaglandin F2α receptor activity is a template for an mRNA molecule encoding said polypeptide having prostaglandin F2α receptor activity, wherein said mRNA has an increased stability in cells as compared to the stability of an mRNA molecule transcribed from the sequence set forth in SEQ ID NO:5.

21. The viral vector of claim 14, wherein said nucleic acid sequence encoding said polypeptide having prostaglandin F2α receptor activity comprises a nucleic acid sequence that encodes the same amino acid sequence as set forth in SEQ ID NO:4 and comprises a codon sequence different than the codons set forth in SEQ ID NO:5.

22. The viral vector of claim 14, wherein said nucleic acid sequence encoding said polypeptide having prostaglandin F2α receptor activity comprises five or more different codon sequences compared to the codon sequences set forth in SEQ ID NO:5.

23. The viral vector of claim 14, wherein said nucleic acid sequence encoding said polypeptide having prostaglandin F2α receptor activity comprises the sequence set forth in SEQ ID NO:6.

24. The viral vector of claim 14, wherein said polypeptide having prostaglandin F2α receptor activity comprises the sequence set forth in SEQ ID NO:4.

25. The viral vector of claim 14, wherein said viral vector comprises a nucleic acid sequence encoding a polypeptide having prostaglandin synthase activity, wherein (i) said nucleic acid sequence encoding said polypeptide having prostaglandin synthase activity comprises the sequence set forth in SEQ ID NO:8 or (ii) said polypeptide having prostaglandin synthase activity comprises the sequence set forth in SEQ ID NO:7.

26. The viral vector of claim 25, wherein said nucleic acid sequence encoding said polypeptide having prostaglandin synthase activity comprises the sequence set forth in SEQ ID NO:8.

27. The viral vector of claim 25, wherein said polypeptide having prostaglandin synthase activity comprises the sequence set forth in SEQ ID NO:7.

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Claim Tree

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Description

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in treating glaucoma, cardiovascular diseases, and renal diseases. For example, this document relates to methods and materials that can be used to reduce intraocular pressure.

2. Background Information

Glaucoma is characterized by a loss of visual function due to damage to the optic nerve. The several morphologically or functionally distinct types of glaucoma are typically characterized by elevated intraocular pressure, which is considered to be causally related to the pathological course of the disease. Ocular hypertension is a condition where intraocular pressure is elevated but no apparent loss of visual function has occurred. Such patients are considered to be at high risk for the eventual development of the visual loss associated with glaucoma. If glaucoma or ocular hypertension is detected early and treated promptly with medications that effectively reduce elevated intraocular pressure, loss of visual function or its progressive deterioration can generally be ameliorated. Drug therapies that have proven to be effective for the reduction of intraocular pressure include both agents that decrease aqueous humor production and agents that increase the outflow facility.

SUMMARY

This document provides methods and materials related to treating glaucoma, ocular hypertension, cardiovascular diseases, and renal diseases. For example, this document provides isolated nucleic acid molecules encoding a polypeptide having COX-2 activity as well as isolated nucleic acid molecules encoding a polypeptide having prostaglandin F receptor activity. In addition, this document provides viral vectors (e.g., lentiviral vectors) containing nucleic acid encoding a polypeptide having COX-2 activity, a polypeptide having prostaglandin F receptor activity, a polypeptide having prostacyclin IP receptor activity, a polypeptide having prostaglandin synthase activity, a polypeptide having prostacyclin synthase activity, or combinations thereof. Such isolated nucleic acid molecules and viral vectors can be used to reduce intraocular pressure and to treat cardiovascular and renal diseases. For example, viral vectors provided herein can be administered to the eye or eyes of a human patient having elevated intraocular pressure, thereby reducing the patient's risk of developing glaucoma. Viral vectors provided herein also can be administered to the heart of a human patient having cardiovascular disease, thereby reducing the patient's risk of having a myocardial infarction. In some cases, viral vectors provided herein can be administered to the kidneys of a human patient having renal disease, thereby reducing the patient's risk of having renal failure.

This document also provides methods and materials for reducing intraocular pressure. For example, the methods provided herein can include administering a viral vector such as a lentiviral vector to one or both eyes. Such methods can be used to treat existing glaucoma or can be used to slow or prevent the onset of glaucoma. In some cases, the methods and materials provided herein can be used to reduce a human patient's risk of developing glaucoma. In some cases, the methods and materials provided herein can be used to increase a mammal's ability to respond to an intraocular pressure reducing treatment such as Latanoprost (xalatan) eye drops.

This document also provides methods and materials for treating cardiovascular and renal diseases. For example, the methods provided herein can include administering a viral vector systemically, administering a viral vector to the heart (e.g., via a catheter), or administering a viral vector to one or both kidneys (e.g., via a urethral catheter or during dialysis). Such methods can be used to reduce the severity of a symptom of a cardiovascular or renal disease (e.g., hypertension or renal fibrosis) and can be used to reduce the progression of a cardiovascular or renal disease (e.g., to reduce progressive loss of function of the heart or kidneys).

In general, one aspect of this document features a method for treating a mammal having glaucoma or elevated intraocular pressure. The method comprises, or consists essentially of, administering a nucleic acid to an eye of the mammal under conditions effective to reduce intraocular pressure of the eye, wherein the nucleic acid comprises a nucleic acid sequence encoding a polypeptide having cyclooxygenase-2 activity and a nucleic acid sequence encoding a polypeptide having prostaglandin F receptor activity. The nucleic acid can be administered to said eye using a viral vector (e.g., a lentiviral vector).

In another aspect of this document features a method for treating a mammal having glaucoma or elevated intraocular pressure. The method comprises, or consists essentially of, administering a viral vector to an eye of the mammal under conditions effective to reduce intraocular pressure of the eye, where the viral vector comprises a nucleic acid encoding a polypeptide having cyclooxygenase-2 activity. The mammal can be a human. The viral vector can be a lentiviral vector. The administering step can comprise contacting the eye with a solution containing the viral vector. The solution can be a saline solution or a physiologically acceptable buffered solution. The solution can comprise between 103 and 1012 lentivirus particles per mL (e.g., between 104 and 1011 lentivirus particles per mL; between 105 and 1010 lentivirus particles per mL; between 106 and 1010 lentivirus particles per mL; or between 106 and 109 lentivirus particles per mL). The nucleic acid can be a template for an mRNA molecule encoding the polypeptide, where the mRNA has an increased stability in cells as compared to the stability of an mRNA molecule transcribed from the sequence set forth in SEQ ID NO:2. The nucleic acid can comprise a nucleic acid sequence that encodes the same amino acid sequence as set forth in SEQ ID NO:1 and can comprise a codon sequence different than the codons set forth in SEQ ID NO:2. The nucleic acid sequence can comprise five or more different codon sequences compared to the codon sequences set forth in SEQ ID NO:2. The nucleic acid can comprise the sequence set forth in SEQ ID NO:3. The polypeptide can comprise the sequence set forth in SEQ ID NO:1. The viral vector can comprise a nucleic acid sequence encoding a polypeptide having prostaglandin F receptor activity. The nucleic acid sequence encoding the polypeptide having prostaglandin F receptor activity can be a template for an mRNA molecule encoding the polypeptide having prostaglandin F receptor activity, where the mRNA has an increased stability in cells as compared to the stability of an mRNA molecule transcribed from the sequence set forth in SEQ ID NO:5. The nucleic acid sequence encoding the polypeptide having prostaglandin F receptor activity can comprise a nucleic acid sequence that encodes the same amino acid sequence as set forth in SEQ ID NO:4 and can comprise a codon sequence different than the codons set forth in SEQ ID NO:5. The nucleic acid sequence encoding the polypeptide having prostaglandin F receptor activity can comprise five or more different codon sequences compared to the codon sequences set forth in SEQ ID NO:5. The nucleic acid sequence encoding the polypeptide having prostaglandin F receptor activity can comprise the sequence set forth in SEQ ID NO:6. The polypeptide having prostaglandin F receptor activity can comprise the sequence set forth in SEQ ID NO:4. The viral vector can comprise a nucleic acid sequence encoding a polypeptide having prostaglandin synthase activity. The nucleic acid sequence encoding the polypeptide having prostaglandin synthase activity can comprise the sequence set forth in SEQ ID NO:8. The polypeptide having prostaglandin synthase activity can comprise the sequence set forth in SEQ ID NO:7. The viral vector can comprise nucleic acid encoding a polypeptide having prostaglandin F receptor activity and a polypeptide having prostaglandin synthase activity. The method can be effective to reduce the intraocular pressure by at least 10 percent. The method can be effective to reduce the intraocular pressure by at least 20 percent. The method can be effective to reduce the intraocular pressure by at least 30 percent. The viral vector can be a feline immunodeficiency virus vector.

In another aspect, this document features a method for treating a mammal having a cardiovascular or renal disease. The method comprises, or consists essentially of, administering a viral vector to the mammal under conditions effective to reduce the severity of a symptom of the cardiovascular or renal disease, where the viral vector comprises a nucleic acid encoding a polypeptide having cyclooxygenase-2 activity. The mammal can be a human. The viral vector can be a lentiviral vector. The nucleic acid can be a template for an mRNA molecule encoding the polypeptide, where the mRNA has an increased stability in cells as compared to the stability of an mRNA molecule transcribed from the sequence set forth in SEQ ID NO:2. The nucleic acid can comprise a nucleic acid sequence that encodes the same amino acid sequence as set forth in SEQ ID NO:1 and can comprise a codon sequence different than the codons set forth in SEQ ID NO:2. The nucleic acid sequence can comprise five or more different codon sequences compared to the codon sequences set forth in SEQ ID NO:2. The nucleic acid can comprise the sequence set forth in SEQ ID NO:3. The polypeptide can comprise the sequence set forth in SEQ ID NO:1. The viral vector can comprise a nucleic acid sequence encoding a polypeptide having prostacyclin IP receptor activity. The nucleic acid can comprise a nucleic acid sequence that encodes the same amino acid sequence as set forth in SEQ ID NO:9 and can comprise a codon sequence different than the codons set forth in SEQ ID NO:10. The nucleic acid sequence encoding the polypeptide having prostacyclin IP receptor activity can comprise the sequence set forth in SEQ ID NO:10. The polypeptide having prostacyclin IP receptor activity can comprise the sequence set forth in SEQ ID NO:9. The viral vector can comprise a nucleic acid sequence encoding a polypeptide having prostacyclin synthase activity. The nucleic acid sequence encoding the polypeptide having prostacyclin synthase activity can comprise the sequence set forth in SEQ ID NO:12. The polypeptide having prostacyclin synthase activity can comprise the sequence set forth in SEQ ID NO:11. The viral vector can comprise nucleic acid encoding two or more polypeptides selected from the group consisting of a polypeptide having cyclooxygenase-2 activity, a polypeptide having prostacyclin IP receptor activity, and a polypeptide having prostacyclin synthase activity. The symptom can be reduced by 25%. The symptom can be reduced by 50%. The symptom can be reduced by 75%. The symptom can be reduced by 100%.

In another aspect, this document features a viral vector comprising a nucleic acid encoding a polypeptide having cyclooxygenase-2 activity. The viral vector can be a lentiviral vector. The nucleic acid can be a template for an mRNA molecule encoding the polypeptide, where the mRNA has an increased stability in cells as compared to the stability of an mRNA molecule transcribed from the sequence set forth in SEQ ID NO:2. The nucleic acid can comprise a nucleic acid sequence that encodes the same amino acid sequence as set forth in SEQ ID NO:1 and can comprise a codon sequence different than the codons set forth in SEQ ID NO:2. The nucleic acid sequence can comprise five or more different codon sequences compared to the codon sequences set forth in SEQ ID NO:2. The nucleic acid can comprise the sequence set forth in SEQ ID NO:3. The polypeptide can comprise the sequence set forth in SEQ ID NO:1. The viral vector can comprise a nucleic acid sequence encoding a polypeptide having prostaglandin F receptor activity. The nucleic acid sequence encoding the polypeptide having prostaglandin F receptor activity can be a template for an mRNA molecule encoding the polypeptide having prostaglandin F receptor activity, where the mRNA has an increased stability in cells as compared to the stability of an mRNA molecule transcribed from the sequence set forth in SEQ ID NO:5. The nucleic acid sequence encoding the polypeptide having prostaglandin F receptor activity can comprise a nucleic acid sequence that encodes the same amino acid sequence as set forth in SEQ ID NO:4 and can comprise a codon sequence different than the codons set forth in SEQ ID NO:5. The nucleic acid sequence encoding the polypeptide having prostaglandin F receptor activity can comprise five or more different codon sequences compared to the codon sequences set forth in SEQ ID NO:5. The nucleic acid sequence encoding the polypeptide having prostaglandin F receptor activity can comprise the sequence set forth in SEQ ID NO:6. The polypeptide having prostaglandin F receptor activity can comprise the sequence set forth in SEQ ID NO:4. The viral vector can comprise a nucleic acid sequence encoding a polypeptide having prostaglandin synthase activity. The nucleic acid sequence encoding the polypeptide having prostaglandin synthase activity can comprise the sequence set forth in SEQ ID NO:8. The polypeptide having prostaglandin synthase activity can comprise the sequence set forth in SEQ ID NO:7. The viral vector can comprise nucleic acid encoding a polypeptide having prostaglandin F receptor activity and a polypeptide having prostaglandin synthase activity. The viral vector can comprise nucleic acid encoding a polypeptide having prostacyclin IP receptor activity. The nucleic acid can comprise a nucleic acid sequence that encodes the same amino acid sequence as set forth in SEQ ID NO:9 and can comprise a codon sequence different than the codons set forth in SEQ ID NO:10. The nucleic acid sequence encoding the polypeptide having prostacyclin IP receptor activity can comprise the sequence set forth in SEQ ID NO:10. The polypeptide having prostacyclin IP receptor activity can comprise the sequence set forth in SEQ ID NO:9. The viral vector can comprise a nucleic acid sequence encoding a polypeptide having prostacyclin synthase activity. The nucleic acid sequence encoding the polypeptide having prostacyclin synthase activity can comprise the sequence set forth in SEQ ID NO:12. The polypeptide having prostacyclin synthase activity can comprise the sequence set forth in SEQ ID NO:11. The viral vector can comprise nucleic acid encoding two or more polypeptides selected from the group consisting of a polypeptide having cyclooxygenase-2 activity, a polypeptide having prostacyclin IP receptor activity, and a polypeptide having prostacyclin synthase activity.

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 to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 contains graphs plotting the percent base composition versus base position in the human COX-2 mRNA (Upper panel), the coding region of the human COX-2 mRNA (middle panel), and the codon-optimized COX-2 cDNA (lower panel).

FIG. 2 contains photomicrographs of cells transfected with transfer constructs containing the wild-type COX-2 cDNA (COX2igWF) or the codon-optimized COX-2 cDNA (XOGWF) upstream of an IRES operably linked to a GFP coding sequence. Cells transfected with a transfer construct containing a GFP coding sequence operably linked to a CMV promoter (GINWF) served as a positive control, and mock transfected cells served as a negative control.

FIG. 3 is a Northern blot analyzing expression of COX-2 mRNA in cells transfected with a transfer construct containing the codon-optimized (XOGWF) or wild-type (COX2igWF) COX-2 cDNA. Cells transfected with a transfer construct containing a GFP coding sequence operably linked to a CMV promoter (GINWF) served as a positive control, and mock transfected cells served as a negative control.

FIG. 4 is a Western blot analyzing expression of COX-2 polypeptides in 293T cells transfected with a transfer construct containing a codon-optimized or wild-type COX-2 cDNA.

FIG. 5 is a schematic diagram of FIV-based lentiviral transfer constructs containing a codon-optimized COX-2 cDNA (pXOGWF), a PGF synthase cDNA (pPGFSigWF), or a codon-optimized prostaglandin F receptor cDNA (pHAFPRigWF). The prostaglandin F receptor cDNA was HA-tagged to enable detection of prostaglandin F receptor polypeptides on Western blots

FIG. 6 is a Western blot analyzing expression of prostaglandin F receptor (FPR), COX-2, and prostaglandin F synthase (PGFS) polypeptides in cells that were mock transfected or transfected with one or more lentiviral transfer vectors containing a cDNA encoding an FPR, COX-2, or PGFS polypeptide.

FIG. 7 is a graph plotting levels of PGF2alpha in 293T cells transfected with a construct containing a COX-2 cDNA, in 293T cells transfected with a construct containing a PGF synthase (PGFS) cDNA, and in 293T cells co-transfected with a construct containing a COX-2 cDNA and a construct containing a PGFS cDNA. FIG. 7 also contains Western blots analyzing expression of COX-2 and PGFS polypeptides in the transfected 293T cells.

FIG. 8 is a chart indicating the therapeutic regimen applied to each subject in the animal study.

FIG. 9 contains a series of graphs plotting intraocular pressure (mm Hg) versus days post injection for the indicated treatment groups.

FIG. 10 is a graph plotting the mean intraocular pressure (IOP) sustained for more than two months in each of the five experimental groups described in FIG. 8 and in eyes treated with the control vector. The p-values were determined using a paired, two-tailed distribution T-test.

FIG. 11 is a listing of an amino acid sequence (SEQ ID NO:1) of a human COX-2 polypeptide.

FIG. 12 is a listing of a wild-type human nucleic acid sequence (SEQ ID NO:2) encoding the amino acid sequence set forth in SEQ ID NO:1.

FIG. 13 is a listing of a codon optimized nucleic acid sequence (SEQ ID NO:3) encoding the amino acid sequence set forth in SEQ ID NO:1. The bold, underlined nucleotides represent nucleotides that were changed relative to the sequence set forth in SEQ ID NO:2.

FIG. 14 is a listing of an amino acid sequence (SEQ ID NO:4) of a human prostaglandin F receptor polypeptide containing an HA tag. The underlined amino acid sequence represents the HA tag.

FIG. 15 is a listing of a wild-type human nucleic acid sequence (SEQ ID NO:5) encoding the amino acid sequence set forth in SEQ ID NO:4.

FIG. 16 is a listing of a codon optimized nucleic acid sequence (SEQ ID NO:6) encoding the amino acid sequence set forth in SEQ ID NO:4. The bold, underlined nucleotides represent nucleotides that were changed relative to the sequence set forth in SEQ ID NO:5.

FIG. 17 is a listing of an amino acid sequence (SEQ ID NO:7) of a human prostaglandin F synthase polypeptide.

FIG. 18 is a listing of a nucleic acid sequence (SEQ ID NO:8) encoding a human prostaglandin F synthase polypeptide.

DETAILED DESCRIPTION

This document provides methods and materials related to treating glaucoma, intraocular hypertension, cardiovascular disease, and renal disease. For example, this document provides isolated nucleic acid molecules encoding a polypeptide having COX-2 activity as well as isolated nucleic acid molecules encoding a polypeptide having prostaglandin F receptor activity. This document also provides viral vectors (e.g., lentiviral vectors) containing a polypeptide having COX-2 activity, a polypeptide having prostaglandin F receptor activity, a polypeptide having prostacyclin IP receptor activity, a polypeptide having prostaglandin synthase activity, a polypeptide having prostacyclin synthase activity, or combinations thereof.

In addition, this document provides methods and materials for reducing intraocular pressure. For example, the methods provided herein can include administering a viral vector such as a lentiviral vector to one or both eyes. Such methods can be used to treat existing glaucoma or can be used to slow or prevent the onset of glaucoma. In some cases, the methods and materials provided herein can be used to reduce a human patient's risk of developing glaucoma.

This document also provides methods and materials for treating cardiovascular diseases (e.g., pulmonary hypertension) and renal diseases (e.g., diabetic nephropathy). For example, the methods provided herein can include administering a viral vector systemically, administering a viral vector to the heart, or administering a viral vector to one or both kidneys. Such methods can be used to reduce the severity of a symptom of a cardiovascular or renal disease (e.g., hypertension or renal fibrosis) and can be used to reduce the progression of a cardiovascular or renal disease (e.g., to reduce progressive loss of function of the heart or kidneys).

The term “nucleic acid” as used herein encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

The term “isolated” as used herein with reference to nucleic acid refers to a naturally-occurring nucleic acid that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5′ end and one on the 3′ end) in the naturally-occurring genome of the organism from which it is derived. For example, an isolated nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a recombinant DNA that exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.

The term “isolated” as used herein with reference to nucleic acid also includes any non-naturally-occurring nucleic acid since non-naturally-occurring nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome. For example, non-naturally-occurring nucleic acid such as an engineered nucleic acid is considered to be isolated nucleic acid. Engineered nucleic acid can be made using common molecular cloning or chemical nucleic acid synthesis techniques. Isolated non-naturally-occurring nucleic acid can be independent of other sequences, or incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or the genomic DNA of a prokaryote or eukaryote. In addition, a non-naturally-occurring nucleic acid can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid sequence.

It will be apparent to those of skill in the art that a nucleic acid existing among hundreds to millions of other nucleic acid molecules within, for example, cDNA or genomic libraries, or gel slices containing a genomic DNA restriction digest is not to be considered an isolated nucleic acid.

An isolated nucleic acid molecule provided herein can contain a nucleic acid sequence encoding a polypeptide having COX-2 activity, a polypeptide having prostaglandin F receptor activity, a polypeptide having prostacyclin IP receptor activity, a polypeptide having prostaglandin synthase activity, a polypeptide having prostacyclin synthase activity, or combinations thereof. Non-limiting examples of nucleic acid sequences encoding a polypeptide having COX-2 activity, a polypeptide having prostaglandin F2α receptor activity, a polypeptide having prostacyclin IP receptor activity, a polypeptide having prostaglandin synthase activity, and a polypeptide having prostacyclin synthase activity are set forth in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:10 (GenBank® GI Number GI:39995095), SEQ ID NO:8, and SEQ ID NO:12 (GenBank® GI Number GI:75517290), respectively. Non-limiting examples of amino acid sequences of polypeptides having COX-2 activity, prostaglandin F2α receptor activity, prostacyclin IP receptor activity, prostaglandin synthase activity, and prostacyclin synthase activity are set forth in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:9 (GenBank® GI Number GI:4506263), SEQ ID NO:7, and SEQ ID NO:11 (GenBank® GI Number GI:2493373), respectively.

Isolated nucleic acid molecules provided herein can contain a sequence that has one or more codons that are different from those found in a wild-type sequence. For example, an isolated nucleic acid molecule provided herein can contain a nucleic acid sequence that encodes a polypeptide having COX-2 activity that is identical to a human COX-2 polypeptide with the nucleic acid sequence having one or more codons that are different from wild-type human nucleic acid encoding that human COX-2 polypeptide. An example of such a nucleic acid molecule is provided in FIG. 13.

Any method can be used to obtain an isolated nucleic acid molecule provided herein including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, PCR can be used to obtain an isolated nucleic acid molecule containing a nucleic acid sequence set forth in FIG. 12. In some cases, the obtained nucleic acid can be mutated to form a codon-optimized sequence such as the sequence set forth in FIG. 13.

Any of the nucleic acid molecules provided herein can be incorporated into a viral vector. For example, a viral vector can be designed to contain an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide having COX-2 activity, a polypeptide having prostaglandin F2α receptor activity, a polypeptide having prostacyclin IP receptor activity, a polypeptide having prostaglandin synthase activity, a polypeptide having prostacyclin synthase activity, or combinations thereof. Examples of viral vectors that can be used include, without limitation, lentiviral vectors (e.g., feline immunodeficiency viral vectors), retroviral vectors (e.g., murine retroviral vectors), foamy virus vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, and herpes virus vectors. Viral vectors can be replication incompetent and can contain few if any viral genes. In some cases, the isolated nucleic acid molecules provided herein can used as naked DNA or can be incorporated into plasmids, transposons, retroelement-based vectors, or phage integrase containing DNA vectors.

This document provides methods for treating glaucoma or intraocular hypertension. Such methods can include administering an isolated nucleic acid molecule provided herein to a mammal in need of treatment (e.g., a human, dog, cat, horse, cow, pig, or monkey). Any method can be used to administer an isolated nucleic acid molecule provided herein. For example, a viral vector provided herein can be administered to a mammal via oral administration or direct administration to one or both eyes. In some cases, a viral vector provided herein can be contained within a solution that can be directly applied to an eye. Any method can be used to administer such a solution to an eye. For example, a solution containing a viral vector provided herein can be administered in a manner similar to the manner used to self administer eye drops.

As described herein, this document provides methods and materials for treating cardiovascular and renal diseases. Cardiovascular diseases include, without limitation, pulmonary hypertension (e.g., pulmonary arteriolar hypertension), arterial thrombosis, myocardial ischemia, myocardial infarction, atherosclerosis, restenosis, and reperfusion injury. Examples of renal diseases include, without limitation, diabetic nephropathy, progressive renal disease, renal fibrosis, renal hypertrophy, and glomerulosclerosis. As described herein, a mammal having a cardiovascular or renal disease can be treated using an isolated nucleic acid molecule provided herein (e.g., an isolated nucleic acid molecule encoding a polypeptide having cyclooxygenase-2 activity, a polypeptide having prostacyclin IP receptor activity, a polypeptide having prostacyclin synthase activity, or any combination thereof). A viral vector containing an isolated nucleic acid molecule provided herein can be used to administer the nucleic acid to a mammal in need of treatment. Viral vectors can be prepared using standard materials (e.g., packaging cell lines and vectors) and methods known to those of ordinary skill in the art.

Viral vectors containing one or more nucleic acid molecules provided herein (e.g., one or more of a nucleic acid encoding a polypeptide having cyclooxygenase-2 activity, a nucleic acid encoding a polypeptide having prostacyclin IP receptor activity, and a nucleic acid encoding a polypeptide having prostacyclin synthase activity) can be administered to a mammal having a cardiovascular or renal disease via numerous routes. For example, a viral vector can be administered systemically (e.g., via intravenous injection). In some cases, a viral vector can be administered directly to the heart. Direct administration of a viral vector to the heart can be achieved using a catheter or a stent, for example, and can performed during a therapeutic manipulation such as arterial bypass surgery. In some cases, a viral vector can be administered directly to one or both kidneys. Various methods can be used to deliver a viral vector to a kidney. For example, a urethral catheter can be used to deliver a viral vector to a kidney. In some cases, a viral vector can be delivered to a kidney during dialysis or by direct injection into the kidney (e.g., CT-guided direct needle injection into the kidney). In some cases, a viral vector can be targeted to the heart or kidneys using liposomes or by expressing a polypeptide on the surface of the viral particle that interacts with another polypeptide that is expressed predominantly or selectively on the surface of heart or kidney cells. In some cases, one or more nucleic acid molecules provided herein can be administered to a mammal having cardiovascular or renal disease by direct injection of the naked nucleic acid molecules into the heart or kidney, or by direct administration of liposomes containing the nucleic acid molecules. As with viral vectors, liposomes also can be targeted to heart or kidney tissue. Viral vectors, naked nucleic acids, and liposomes can be administered to a mammal in a biologically compatible solution or a pharmaceutically acceptable delivery vehicle. Suitable pharmaceutical formulations depend in part on the use and route of delivery. For example, a suitable formulation for direct injection is isotonic and has a neutral pH.

After identifying a mammal as having glaucoma, intraocular hypertension, a cardiovascular disease, or a renal disease, the mammal can be administered a viral vector containing a nucleic acid disclosed herein. A viral vector can be administered to a mammal in any amount, at any frequency, and for any duration effective to achieve a desired outcome (e.g., to reduce the severity of a symptom of glaucoma, cardiovascular disease, or renal disease). In some cases, a viral vector can be administered to a mammal having glaucoma, intraocular hypertension, a cardiovascular disease, or a renal disease to reduce the severity of a symptom or to reduce the progression rate of the condition by 5, 10, 25, 50, 75, 100, or more percent. For example, the severity of a symptom can be reduced in a mammal such that the symptom is no longer detected by the mammal. In some cases, the progression of a condition can be reduced such that no additional progression is detected. Any method can be used to determine whether or not the severity of a symptom or the progression rate of a condition is reduced. For example, a mammal having glaucoma can be tested for intraocular pressure before and after treatment to determine whether the pressure is reduced. In some cases, a mammal can be observed or tested for the severity of a symptom of cardiovascular disease (e.g., high blood pressure, blood clots in arteries and veins, pain isolated to one leg (usually the calf or medial thigh), swelling in the extremity, or varicose veins) before and after treatment to determine whether or not the severity of a symptom is reduced. In some cases, renal biopsy tissue taken from a mammal before and after treatment can be analyzed (e.g., for fibrosis) to determine whether the severity of a symptom is reduced. To determine whether or not progression of a condition (e.g., glaucoma, intraocular hypertension, cardiovascular disease, or renal disease) is reduced, a physical examination can be performed at different time points to determine the stage or severity of the condition. The stage or severity of the condition observed at different time points can be compared to assess the progression rate. After treatment as described herein, the progression rate can be determined again over another time interval to determine whether or not the progression rate has decreased. For example, renal function can be assessed at various time points to determine whether the function is improving, worsening, or staying the same.

An effective amount of a viral vector can be any amount that reduces the severity of a symptom or the progression of a condition (e.g., glaucoma, intraocular hypertension, cardiovascular disease, or renal disease) without producing significant toxicity to the mammal. If a particular mammal fails to respond to a particular amount, then the amount of the viral vector can be increased by, for example, two fold. After receiving this higher concentration, the mammal can be monitored for both responsiveness to the treatment and toxicity symptoms, and adjustments made accordingly. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal's response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, immunocompetency of the mammal, and severity of the condition may require an increase or decrease in the actual effective amount administered.

The frequency of administration can be any frequency that reduces the severity of a symptom or progression rate of a condition without producing significant toxicity to the mammal. For example, the frequency of administration can be from about once in a lifetime to about once a month. The frequency of administration can remain constant or can be variable during the duration of treatment. A course of treatment with a viral vector can include rest periods. For example, a viral vector can be administered over a six month period followed by a three month rest period, and such a regimen can be repeated multiple times. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, immunocompetency of the mammal, and severity of the condition may require an increase or decrease in administration frequency.

An effective duration for administering a viral vector provided herein can be any duration that reduces the severity of a symptom or the progression rate of glaucoma, intraocular hypertension, cardiovascular disease, or renal disease without producing significant toxicity to the mammal. Thus, the effective duration can vary from several days to several weeks, months, or years. In general, the effective duration for the treatment of glaucoma, intraocular hypertension, cardiovascular disease, or renal disease can range in duration from several months to several years. In some cases, an effective duration can be for as long as an individual mammal is alive. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, immunocompetency of the mammal, and severity of the condition.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1—Modulation of Prostaglandin Pathways Reduces Intraocular Pressure

Experiments were conducted to determine whether expression of polypeptides involved in prostaglandin biosynthetic and response pathways can be manipulated to provide a sustained improvement in intraocular pressure involved in diseases such as glaucoma.

Methods and Materials

Cloning FIV-Based Transfer Construct Plasmids

pCOX2igWF: A plasmid having the normal human COX-2 cDNA (obtained from S. Prescott, Huntsman Cancer Institute) was NotI-XbaI-digested to isolate the COX-2 cDNA and blunted with T4 polymerase. pGiNWF (Loewen et al., Investig. Ophthalmol. Vis. Sci., 43:3686-3690 (2002)) was digested with AgeI and EcoRI, and blunted with T4 polymerase to isolate the backbone sequence that was then ligated with the COX-2 cDNA insert. Next, an IRES (internal ribosome entry site)-GFP cassette was blunt-end ligated into the EcoRI site just downstream of COX-2. This insertion resulted in a bicistronic FIV-based transfer construct with COX-2 expression driven by the CMV promoter and GFP expression being driven by the IRES just downstream of the COX-2 cDNA cassette. This plasmid, COX2igWF, was the basis for the cloning of the following transfer constructs.

pXOGWF: A codon-optimized human COX-2 cDNA was designed and synthesized with the assistance of GenScript Corporation custom services (Scotch Plains, N.J.). Codon usage was optimized for usage in mammalian cells. G-C content was optimized, and other factors such as secondary structure and repetitive codons were taken into consideration to achieve codon optimization. The codon-optimized COX-2 cDNA was designed to include flanking restriction sites to enable downstream recombinant cloning strategies. BamHI sites flank the codon-optimized COX-2 gene, and these sites were used to insert the optimized cDNA into the BamHI-digested backbone of COX2igWF. This essentially replaced the wild-type COX-2 cDNA cassette with the codon-optimized COX-2 cDNA.

pGFSigWF: The PGFS cDNA from hPGFS-cDNA pUC8 (obtained from Kikuko Watanabe, University of East Asia, Japan) was removed by EcoRI and SalI digestion, and blunted with T4 DNA polymerase. This cDNA insert was blunt ligated into the BamHI-digested backbone of COX2igWF.

pHAFPRigWF: An HA-tagged prostaglandin F receptor (HAFPR) cDNA plasmid (obtained from G. FitzGerald, University of Pennsylvania, USA), HA-FP, contains the HAFPR cDNA flanked by an upstream KpnI and a downstream NcoI site. The HAFPR cassette was removed by KpnI-NcoI digestion, blunted with T4 DNA polymerase and ligated with T4 DNA ligase into the BamHI-digested backbone of COX2igWF.

pcoFPRigWF: A codon-optimized human HA-tagged FPR cDNA was designed and synthesized with the assistance of GenScript Corporation custom services (Scotch Plains, N.J.). Codon usage was optimized for usage in mammalian cells. G-C content was optimized, and other factors such as secondary structure and repetitive codons were taken into consideration to achieve codon optimization. The codon-optimized HAFPR was designed to include flanking restriction sites for accessible cloning strategies. BamHI sites flank the codon-optimized HAFPR gene and were used to digest and ligate into the BamHI-digested backbone of COX2igWF.

PGF2Alpha Assay

293T cells in 6 well plates were transfected with 2 μg XOGWF or PGFSigWF using the calcium phosphate transfection method as described elsewhere (Loewen et al., Methods Mol. Biol., 229:251-271 (2003)). Media was changed 12 to 16 hours later and collected 24 hours thereafter. Media was filtered (0.2 μm) to remove cells and used in the Prostaglandin F2 α enzyme immunoassay kit (Cayman Chemical, cat. no. 516011) as recommended by the manufacturer's protocol manual.

Northern Blot

293T cells were transfected with equivalent quantities of COX2igWF, XOWGF, or GINWF. Cells were treated with 1 mL Trizol 36 hours post-transfection and stored at −80° C. until RNA purification. Trizol-treated lysates were treated with chloroform, followed by isopropanol, and spun to isolate nucleic acid. Nucleic acid was treated with DNase, followed by RNA extraction with equal volume of phenol:chloroform:isoamyl alcohol (125:24:1). RNA was precipitated with 1/10 volume 3M sodium acetate and 2.5 volumes 100% ethanol.

Isolated total cellular RNA was separated by gel electrophoresis (1.2% agarose gel, 3.75% formaldehyde, 1×MOPS). After gel electrophoresis, RNA was transferred onto a nylon transfer membrane (Nytran Supercharge Membrane, Schleicher & Schuell, cat. no. 10416284).

A beta-actin anti-sense oligo probe was 5′-end labeled using T4 PNK (Promega) and [γ-32P]ATP incubated at 37° C. for 30 minutes followed by heat inactivation at 70° C. for 10 minutes. Labeled primer probe was purified using a quick spin column and hybridized with RNA on nylon membrane overnight at 42° C. The membrane was washed and then exposed to MR X-ray film (Kodak) at −80° C. for five days and developed.

Since all transfer constructs contain the GFP gene cassette to be included in the message, mRNA expression levels for each message were assessed by probing for GFP sequence common to all messages in this experiment.

25 ng of GFP anti-sense oligo probe was randomly labeled using dNTP stock solution, 50 μCi α-dCTP and Klenow. The random labeling reaction occurred at 37° C. for 1 hour and was heat-inactivated at 65° C. for 10 minutes. Random labeled probe was purified using a quick spin column followed by hybridization with the RNA-containing nylon membrane for 5 hours to 3 days at −80° C. The membrane was exposed to MR X-ray film (Kodak) and developed.

Western Blot

For Western blotting, cells were lysed in Tris-buffered saline containing 1% Triton X-100 and 1% NP-40, plus a protease inhibitor cocktail (Complete-mini; Boehringer). Lysates were centrifuged to remove chromatin. Proteins were resolved in sodium dodecyl sulfate-10% polyacrylamide gels and transferred to Immobilon P membranes (Millipore). Blocked membranes were incubated overnight at 4° C. or for 2 hours at room temperature with mouse anti-COX-2 MAb (Cayman Chemical, cat. no. 160112), rat anti-HA MAb (Roche), or rabbit anti-hPGFS Ab (obtained from Kikuko Watanabe), diluted in Tris-buffered saline-5% nonfat milk plus 0.05% Tween 20. After washing, membranes were incubated with the appropriate horseradish peroxidase-tagged secondary antibody. Bound antibodies were detected by ECL (Amersham Pharmacia Biotech).

Vector Production and Titration

Transfections were performed using the calcium phosphate transient transfection method in ten-chamber cell factories (CF10) as described elsewhere (Loewen et al., Methods Mol. Biol., 229:251-271 (2003)). Medium was changed 12 to 16 hours later, and supernatants were collected 48 hours thereafter, filtered through a 0.2 μm-pore-size filter, and concentrated by two rounds of ultracentrifugation. The first spin was performed in a series of 250 mL polyallomer Oakridge ultracentrifuge bottles (Sorvall, cat. no. 54477) at 19,000 rpm in a SureSpin 630 rotor (Sorvall, cat. no. 79367) in a Sorvall Discovery 100SE ultracentrifuge (67,000 grmax) for 6 hours at 4° C. Supernatant was removed, and vector was resupended in 30 mL PBS and centrifuged over a sucrose cushion in a swinging bucket SW41TI rotor at 24,000 rpm for 2 hours at 4° C., and aliquoted and frozen at −80° C.

CrFK cells were transduced with serial dilutions of each vector preparation. 48 hours after transduction, cells were harvested, and titers of each vector preparation were determined by flow cytometry for GFP expression. All preparations were tested for reverse transcriptase (RT) activity as described elsewhere (Saenz et al., J. Virol., 79(24):15175-88 (2005)).

Vector Administration to Cat Anterior Chamber

Experiments were conducted in pathogen-free domestic cats (Harlan, Indianapolis, Ind.). Prior to vector administration, cats were anesthetized with 10 mg/kg intramuscular tiletamine HCl/zolazepam HCl (Telazol; Fort Dodge Laboratories Inc., Fort Dodge, Iowa) injection. Anterior chambers of feline eyes were transcorneally injected with a bolus of 200 μL PBS containing 107 TU of vectors GINWF, XOGWF, PGFSigWF, or HAFPRigWF. Animals receiving two or more different vectors received a total of 2×107 TU and 3×107 TU vector, respectively.

Intraocular Pressure Measurements, Slit Lamp Examinations, & Gonioscopic Observation

Prior to examinations, cats were anesthetized with 10 mg/kg intramuscular tiletamine HCl/zolazepam HCl (Telazol; Fort Dodge Laboratories Inc., Fort Dodge, Iowa) injection. Weekly examinations consisted of slit lamp (Haag-Streit, Mason, Ohio) observation and determination of intraocular pressure using a handheld pneumatonometer (Model 30 Classic; Medtronic, Fridley, Minn.).

Fluorescence of transduced TM was observed with a standard gonioscope (Posner; Ocular Instruments, Bellevue, Wash.) and a microscope (Eclipse E400; Nikon) equipped with a GFP-optimized filter (EF-4 B-2E/C FITC, cat. no. 96107; Nikon).

Results

Codon Optimization of Prostaglandin Pathway mRNAs

Initial studies revealed that human COX-2 and PGF receptor polypeptides were difficult to express using standard methods. By performing in silico analyses, it was discovered that the coding regions of both the COX-2 and PGF receptor mRNAs were aberrantly AU-rich, with a markedly suboptimal codon bias. The skewed codon use of the human COX-2 coding region is very similar in composition to that of lentiviral structural genes. This composition makes lentiviral mRNAs labile, a problem that the viruses overcome with specialized viral polypeptides that stabilize RNA at particular stages of the life cycle. The composition of the prostaglandin pathway mRNAs presumably fosters rapid endogenous turnover. Human codon-optimized versions of the human COX-2 cDNA (FIG. 1) and the PGF receptor cDNA were synthesized. The codon-optimized COX-2 cDNA contains a GC-rich sequence that encodes an amino acid sequence identical to the wild-type COX-2 sequence.

Transfer constructs were generated that contained the wild-type or the codon-optimized COX-2 cDNA upstream of an IRES operably linked to a GFP coding sequence. Cells were transfected with the constructs, and GFP expression levels were observed. A significantly higher level of GFP expression was observed in cells transfected with the construct containing the codon-optimized COX-2 cDNA (XOGWF) as compared to cells transfected with the construct containing the wild-type COX-2 cDNA (COX2igWF; FIG. 2).

Cells transfected with the transfer constructs containing the wild-type or codon-optimized COX-2 cDNA were also analyzed for mRNA levels by Northern blotting. The blots were analyzed with a GFP probe random-labeled using 32P-dCTP. The blots were also analyzed with a β-actin probe 5′-labeled using 32P-dATP to control for equal loading. The level of mRNA was much higher in cells transfected with the construct containing the codon-optimized COX-2 cDNA than in cells transfected with the construct containing the wild-type COX-2 cDNA (FIG. 3).

Recombinant DNA constructs containing the codon-optimized COX-2 cDNA or the wild-type COX-2 cDNA were also used to transfect 293T cells, and lysates from the transfected cells were analyzed for COX-2 expression by Western blotting. Expression of COX-2 polypeptides was higher in cells transfected with the construct containing the codon-optimized COX-2 cDNA than in cells transfected with the construct containing the wild-type COX-2 cDNA (FIG. 4).

These results indicate that codon optimization of the COX-2 coding region increases the stability of the transcribed RNA, resulting in increased expression at the polypeptide level. The wild-type COX-2 coding region, not just the 3′ untranslated region as was previously recognized, prevents significant polypeptide expression. In contrast, PGF synthase does not have an aberrant RNA base composition and does not require codon optimization.

Effect of the Expression of Prostaglandin Pathway Polypeptides on IOP In Vivo

Lentiviral transfer constructs based on the feline immunodeficiency virus (FIV) vector system (Poeschla et al., Nat. Med., 4(3):354-7 (1998)) were generated which contained a human codon-optimized COX-2 cDNA, a human PGF synthase cDNA, or a human codon-optimized PGF receptor cDNA (FIG. 5). Levels of COX-2, PGF synthase, and PGF receptor polypeptides in cells transfected with one or more of the transfer constructs were analyzed by Western blotting, and expression of each of the polypeptides was detected (FIG. 6).

Production of PGF2alpha was measured in 293T cells transfected with a construct containing a COX-2 or a PGF synthase (PGFS) cDNA, and in 293T cells co-transfected with a construct containing a COX-2 cDNA and a construct containing a PGF synthase cDNA. Production of PGF2alpha was observed in the presence of COX-2 polypeptides and correlated strongly with the expression level of COX-2 polypeptides (FIG. 7). Co-expression of COX-2 and PGFS resulted in an even greater level of PGF2alpha production than expression of COX-2 alone (FIG. 7). Synthesis of PGF2alpha was increased up to 0.9×104-fold in the transfected cells relative to synthesis of PGF2alpha in control cells. Expression of PGFS alone did not increase PGF2alpha levels, indicating that COX-2 is a rate-limiting polypeptide in the prostaglandin synthesis pathway.

The effect of expression of prostaglandin pathway polypeptides on intraocular pressure (IOP) was investigated in a large animal model developed for glaucoma studies and described elsewhere (Loewen et al., Invest. Ophthalmol. Vis. Sci., 43(12):3686-90 (2002)). Fifteen domestic cats were divided into five groups, with three cats in each group. The anterior chamber of the right eye of each cat was injected with one or more lentiviral vectors containing a COX-2, PGFS, or prostaglandin F receptor (FPR) cDNA. The anterior chamber of the left eye of each cat was injected with 107-108 TU of a control eGFP vector (FIG. 8). The animals were monitored serially for intraocular pressure (IOP) and clinical effects.

The lentiviral vectors were well-tolerated in the animals and produced marked, sustained (two months at present, with observation of all animals ongoing), and highly significant IOP decreases (the mean over the entire two months was 4.2 mm Hg, p<0.002) compared to the IOP levels in eyes treated with the control vector. A combination of vectors containing COX-2 and PGF receptor cDNAs produced the largest IOP decrease (mean=5.6 mm Hg, 38% reduction, p<5×10−14; FIGS. 9 and 10).

These results indicate that major prostaglandin biosynthetic and response pathways can be manipulated. Codon optimization of the COX-2 coding region profoundly augments mRNA stability. Sustained, substantial, highly statistically significant decreases in IOP were achieved in a large animal model.

Other Embodiments

It is to be understood that 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.

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tgctgcagcc acccctgcca gaaccggggc gtgtgcatga gcgtgggctt cgaccagtac 120

aagtgcgatt gcacccggac cggcttctac ggcgaaaact gcagcacccc cgagttcctg 180

acccggatca aactgttcct gaaacccacc cccaacaccg tgcactacat cctgacccac 240

ttcaagggct tctggaacgt ggtgaacaac atccccttcc tgcggaacgc tatcatgagc 300

tacgtgctga ccagccggag ccacctgatc gatagccctc ccacctacaa cgctgactac 360

ggctacaaaa gctgggaagc cttcagcaac ctcagctact acacccgggc tctgcctccc 420

gtgcccgatg attgccccac ccctctgggc gtgaaaggca agaagcagct gcccgatagc 480

aacgagatcg tggaaaaact gctcctgcgg cggaagttca tccccgatcc ccagggcagc 540

aacatgatgt tcgctttctt cgcccagcac ttcacccacc agttcttcaa gaccgatcac 600

aaacggggcc ccgccttcac caacggcctg ggccacggcg tggacctgaa ccacatctac 660

ggcgaaaccc tggctcggca gcggaaactg cggctgttca aggatggcaa aatgaaatac 720

cagatcatcg atggcgagat gtaccctccc accgtgaaag atacccaggc tgaaatgatc 780

tacccccccc aggtgcccga acacctgcgg ttcgctgtgg gccaggaagt gttcggcctg 840

gtgcccggcc tgatgatgta cgctaccatc tggctgcggg aacacaaccg ggtgtgcgat 900

gtgctgaaac aggaacaccc cgaatggggc gatgaacagc tgttccagac cagccggctg 960

atcctgatcg gcgagaccat caagatcgtg atcgaagatt acgtgcagca cctgagcggc 1020

taccacttca aactgaaatt cgaccccgaa ctgctcttca acaaacagtt ccagtaccag 1080

aaccggatcg ctgccgagtt caacaccctc taccactggc accccctgct ccccgacacc 1140

ttccagatcc acgaccagaa atacaactac cagcagttca tctacaacaa cagcatcctg 1200

ctcgaacacg gcatcaccca gttcgtggaa agcttcaccc ggcagatcgc tggccgggtg 1260

gctggcggcc ggaacgtgcc tcctgccgtg cagaaagtga gccaggctag catcgaccag 1320

agccggcaga tgaaatacca gagcttcaac gagtaccgga aacggttcat gctgaagccc 1380

tacgaaagct tcgaagagct gaccggcgaa aaggaaatga gcgctgaact ggaagctctg 1440

tacggcgaca tcgatgctgt ggaactgtac cccgccctcc tggtggagaa accccggccc 1500

gatgccatct tcggcgaaac catggtggaa gtgggcgctc ccttcagcct gaaaggcctg 1560

atgggcaacg tgatctgcag ccccgcttac tggaaaccca gcaccttcgg cggcgaagtg 1620

ggcttccaga tcatcaacac cgccagcatc cagagcctca tctgcaacaa cgtgaaaggc 1680

tgccccttca ccagcttcag cgtgcccgat cccgagctca tcaaaaccgt gaccatcaac 1740

gctagcagca gccggagcgg cctggatgac atcaacccca ccgtgctgct caaagaacgg 1800

agcaccgaac tgtga 1815

<210> SEQ ID NO: 4

<211> LENGTH: 368

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<223> OTHER INFORMATION: codon optimized COX-2 nucleic acid sequence

<400> SEQENCE: 4

Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Met Asn Asn Ser Lys

1 5 10 15

Gln Leu Val Ser Pro Ala Ala Ala Leu Leu Ser Asn Thr Thr Cys Gln

20 25 30

Thr Glu Asn Arg Leu Ser Val Phe Phe Ser Val Ile Phe Met Thr Val

35 40 45

Gly Ile Leu Ser Asn Ser Leu Ala Ile Ala Ile Leu Met Lys Ala Tyr

50 55 60

Gln Arg Phe Arg Gln Lys Ser Lys Ala Ser Phe Leu Leu Leu Ala Ser

65 70 75 80

Gly Leu Val Ile Thr Asp Phe Phe Gly His Leu Ile Asn Gly Ala Ile

85 90 95

Ala Val Phe Val Tyr Ala Ser Asp Lys Glu Trp Ile Arg Phe Asp Gln

100 105 110

Ser Asn Val Leu Cys Ser Ile Phe Gly Ile Cys Met Val Phe Ser Gly

115 120 125

Leu Cys Pro Leu Leu Leu Gly Ser Val Met Ala Ile Glu Arg Cys Ile

130 135 140

Gly Val Thr Lys Pro Ile Phe His Ser Thr Lys Ile Thr Ser Lys His

145 150 155 160

Val Lys Met Met Leu Ser Gly Val Cys Leu Phe Ala Val Phe Ile Ala

165 170 175

Leu Leu Pro Ile Leu Gly His Arg Asp Tyr Lys Ile Gln Ala Ser Arg

180 185 190

Thr Trp Cys Phe Tyr Asn Thr Glu Asp Ile Lys Asp Trp Glu Asp Arg

195 200 205

Phe Tyr Leu Leu Leu Phe Ser Phe Leu Gly Leu Leu Ala Leu Gly Val

210 215 220

Ser Leu Leu Cys Asn Ala Ile Thr Gly Ile Thr Leu Leu Arg Val Lys

225 230 235 240

Phe Lys Ser Gln Gln His Arg Gln Gly Arg Ser His His Leu Glu Met

245 250 255

Val Ile Gln Leu Leu Ala Ile Met Cys Val Ser Cys Ile Cys Trp Ser

260 265 270

Pro Phe Leu Val Thr Met Ala Asn Ile Gly Ile Asn Gly Asn His Ser

275 280 285

Leu Glu Thr Cys Glu Thr Thr Leu Phe Ala Leu Arg Met Ala Thr Trp

290 295 300

Asn Gln Ile Leu Asp Pro Trp Val Tyr Ile Leu Leu Arg Lys Ala Val

305 310 315 320

Leu Lys Asn Leu Tyr Lys Leu Ala Ser Gln Cys Cys Gly Val His Val

325 330 335

Ile Ser Leu His Ile Trp Glu Leu Ser Ser Ile Lys Asn Ser Leu Lys

340 345 350

Val Ala Ala Ile Ser Glu Ser Pro Val Ala Glu Lys Ser Ala Ser Thr

355 360 365

<210> SEQ ID NO: 5

<211> LENGTH: 1107

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 5

atgtatccat atgatgtgcc agattatgct tccatgaaca attccaaaca gctagtgtct 60

cctgcagctg cgcttctttc aaacacaacc tgccagacgg aaaaccggct ttccgtattt 120

ttttcagtaa tcttcatgac agtgggaatc ttgtcaaaca gccttgccat cgccattctc 180

atgaaggcat atcagagatt tagacagaag tccaaggcat cgtttctgct tttggccagc 240

ggcctggtaa tcactgattt ctttggccat ctcatcaatg gagccatagc agtatttgta 300

tatgcttctg ataaagaatg gatccgcttt gaccaatcaa atgtcctttg cagtattttt 360

ggtatctgca tggtgttttc tggtctgtgc ccacttcttc taggcagtgt gatggccatt 420

gagcggtgta ttggagtcac aaaaccaata tttcattcta cgaaaattac atccaaacat 480

gtgaaaatga tgttaagtgg tgtgtgcttg tttgctgttt tcatagcttt gctgcccatc 540

cttggacatc gagactataa aattcaggcg tcgaggacct ggtgtttcta caacacagaa 600

gacatcaaag actgggaaga tagattttat cttctacttt tttcttttct ggggctctta 660

gcccttggtg tttcattgtt gtgcaatgca atcacaggaa ttacactttt aagagttaaa 720

tttaaaagtc agcagcacag acaaggcaga tctcatcatt tggaaatggt aatccagctc 780

ctggcgataa tgtgtgtctc ctgtatttgt tggagcccat ttctggttac aatggccaac 840

attggaataa atggaaatca ttctctggaa acctgtgaaa caacactttt tgctctccga 900

atggcaacat ggaatcaaat cttagatcct tgggtatata ttcttctacg aaaggctgtc 960

cttaagaatc tctataagct tgccagtcaa tgctgtggag tgcatgtcat cagcttacat 1020

atttgggagc ttagttccat taaaaattcc ttaaaggttg ctgctatttc tgagtcacca 1080

gttgcagaga aatcagcaag cacctag 1107

<210> SEQ ID NO: 6

<211> LENGTH: 1107

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<223> OTHER INFORMATION: codon optimized human prostaglandin F2a

receptor polypeptide

<400> SEQENCE: 6

atgtacccct atgacgtgcc agattacgcc tccatgaaca attccaaaca gctcgtgtca 60

cccgcagctg cactgctctc taatacaaca tgccagaccg agaataggct cagcgtgttt 120

ttctctgtga tctttatgac tgtgggcatc ctcagcaact cactggctat cgcaattctg 180

atgaaggcct accagcgctt tcgacagaag agtaaggcct ctttcctcct gctggccagc 240

gggctggtca ttaccgactt tttcggacac ctcatcaatg gagccattgc tgtgttcgtc 300

tatgcctccg ataaggagtg gattagattc gatcagtcaa acgtgctctg ttcaatcttt 360

ggtatctgta tggtcttttc aggtctctgc cctctgctgc tgggctccgt gatggccatt 420

gagcgctgta ttggcgtgac caagcctatc tttcattcta caaagatcac ctccaagcac 480

gtgaagatga tgctgagcgg ggtgtgcctc ttcgctgtct tcattgcact gctgccaatt 540

ctcggccacc gggattacaa gatccaggca tcccgaacct ggtgcttcta caataccgaa 600

gacatcaaag attgggagga taggttctac ctgctcctct ttagtttcct gggcctgctg 660

gctctcggag tgtccctgct gtgtaacgcc atcacaggca tcaccctgct gagagtgaag 720

tttaagtctc agcagcatag acagggcaga agccaccacc tcgagatggt catccagctg 780

ctggccatca tgtgcgtgtc ttgcatctgt tggtctccct tcctggtcac aatggccaac 840

attgggatta atggtaatca cagcctggaa acatgcgaaa caacactgtt tgccctgaga 900

atggcaacct ggaatcagat tctggaccca tgggtgtaca tcctgctcag aaaagccgtg 960

ctgaaaaatc tctacaagct agccagccag tgctgcggcg tgcacgtgat cagcctgcac 1020

atctgggagc tgagcagcat caagaacagc ctgaaggtgg ccgccatcag cgagagcccc 1080

gtggccgaga agagcgccag cacctga 1107

<210> SEQ ID NO: 7

<211> LENGTH: 323

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 7

Met Asp Ser Lys Gln Gln Cys Val Lys Leu Asn Asp Gly His Phe Met

1 5 10 15

Pro Val Leu Gly Phe Gly Thr Tyr Ala Pro Pro Glu Val Pro Arg Ser

20 25 30

Lys Ala Leu Glu Val Thr Lys Leu Ala Ile Glu Ala Gly Phe Arg His

35 40 45

Ile Asp Ser Ala His Leu Tyr Asn Asn Glu Glu Gln Val Gly Leu Ala

50 55 60

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

65 70 75 80

Tyr Thr Ser Lys Leu Trp Ser Thr Phe His Arg Pro Glu Leu Val Arg

85 90 95

Pro Ala Leu Glu Asn Ser Leu Lys Lys Ala Gln Leu Asp Tyr Val Asp

100 105 110

Leu Tyr Leu Ile His Ser Pro Met Ser Leu Lys Pro Gly Glu Glu Leu

115 120 125

Ser Pro Thr Asp Glu Asn Gly Lys Val Ile Phe Asp Ile Val Asp Leu

130 135 140

Cys Thr Thr Trp Glu Ala Met Glu Lys Cys Lys Asp Ala Gly Leu Ala

145 150 155 160

Lys Ser Ile Gly Val Ser Asn Phe Asn Arg Arg Gln Leu Glu Met Ile

165 170 175

Leu Asn Lys Pro Gly Leu Lys Tyr Lys Pro Val Cys Asn Gln Val Glu

180 185 190

Cys His Pro Tyr Phe Asn Arg Ser Lys Leu Leu Asp Phe Cys Lys Ser

195 200 205

Lys Asp Ile Val Leu Val Ala Tyr Ser Ala Leu Gly Ser Gln Arg Asp

210 215 220

Lys Arg Trp Val Asp Pro Asn Ser Pro Val Leu Leu Glu Asp Pro Val

225 230 235 240

Leu Cys Ala Leu Ala Lys Lys His Lys Arg Thr Pro Ala Leu Ile Ala

245 250 255

Leu Arg Tyr Gln Leu Gln Arg Gly Val Val Val Leu Ala Lys Ser Tyr

260 265 270

Asn Glu Gln Arg Ile Arg Gln Asn Val Gln Val Phe Glu Phe Gln Leu

275 280 285

Thr Ala Glu Asp Met Lys Ala Ile Asp Gly Leu Asp Arg Asn Leu His

290 295 300

Tyr Phe Asn Ser Asp Ser Phe Ala Ser His Pro Asn Tyr Pro Tyr Ser

305 310 315 320

Asp Glu Tyr

<210> SEQ ID NO: 8

<211> LENGTH: 972

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 8

atggattcca aacagcagtg tgtaaagcta aatgatggcc acttcatgcc tgtattggga 60

tttggcacct atgcacctcc agaggttccg agaagtaaag ctttggaggt cacaaaatta 120

gcaatagaag ctgggttccg ccatatagat tctgctcatt tatacaataa tgaggagcag 180

gttggactgg ccatccgaag caagattgca gatggcagtg tgaagagaga agacatattc 240

tacacttcaa agctttggtc cacttttcat cgaccagagt tggtccgacc agccttggaa 300

aactcactga aaaaagctca attggactat gttgacctct atcttattca ttctccaatg 360

tctctaaagc caggtgagga actttcacca acagatgaaa atggaaaagt aatatttgac 420

atagtggatc tctgtaccac ctgggaggcc atggagaagt gtaaggatgc aggattggcc 480

aagtccattg gggtgtcaaa cttcaaccgc aggcagctgg agatgatcct caacaagcca 540

ggactcaagt acaagcctgt ctgcaaccag gtagaatgtc atccgtattt caaccggagt 600

aaattgctag atttctgcaa gtcgaaagat attgttctgg ttgcctatag tgctctggga 660

tctcaacgag acaaacgatg ggtggacccg aactccccgg tgctcttgga ggacccagtc 720

ctttgtgcct tggcaaaaaa gcacaagcga accccagccc tgattgccct gcgctaccag 780

ctgcagcgtg gggttgtggt cctggccaag agctacaatg agcagcgcat cagacagaac 840

gtgcaggttt ttgagttcca gttgactgca gaggacatga aagccataga tggcctagac 900

agaaatctcc actattttaa cagtgatagt tttgctagcc accctaatta tccatattca 960

gatgaatatt aa 972

<210> SEQ ID NO: 9

<211> LENGTH: 386

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 9

Met Ala Asp Ser Cys Arg Asn Leu Thr Tyr Val Arg Gly Ser Val Gly

1 5 10 15

Pro Ala Thr Ser Thr Leu Met Phe Val Ala Gly Val Val Gly Asn Gly

20 25 30

Leu Ala Leu Gly Ile Leu Ser Ala Arg Arg Pro Ala Arg Pro Ser Ala

35 40 45

Phe Ala Val Leu Val Thr Gly Leu Ala Ala Thr Asp Leu Leu Gly Thr

50 55 60

Ser Phe Leu Ser Pro Ala Val Phe Val Ala Tyr Ala Arg Asn Ser Ser

65 70 75 80

Leu Leu Gly Leu Ala Arg Gly Gly Pro Ala Leu Cys Asp Ala Phe Ala

85 90 95

Phe Ala Met Thr Phe Phe Gly Leu Ala Ser Met Leu Ile Leu Phe Ala

100 105 110

Met Ala Val Glu Arg Cys Leu Ala Leu Ser His Pro Tyr Leu Tyr Ala

115 120 125

Gln Leu Asp Gly Pro Arg Cys Ala Arg Leu Ala Leu Pro Ala Ile Tyr

130 135 140

Ala Phe Cys Val Leu Phe Cys Ala Leu Pro Leu Leu Gly Leu Gly Gln

145 150 155 160

His Gln Gln Tyr Cys Pro Gly Ser Trp Cys Phe Leu Arg Met Arg Trp

165 170 175

Ala Gln Pro Gly Gly Ala Ala Phe Ser Leu Ala Tyr Ala Gly Leu Val

180 185 190

Ala Leu Leu Val Ala Ala Ile Phe Leu Cys Asn Gly Ser Val Thr Leu

195 200 205

Ser Leu Cys Arg Met Tyr Arg Gln Gln Lys Arg His Gln Gly Ser Leu

210 215 220

Gly Pro Arg Pro Arg Thr Gly Glu Asp Glu Val Asp His Leu Ile Leu

225 230 235 240

Leu Ala Leu Met Thr Val Val Met Ala Val Cys Ser Leu Pro Leu Thr

245 250 255

Ile Arg Cys Phe Thr Gln Ala Val Ala Pro Asp Ser Ser Ser Glu Met

260 265 270

Gly Asp Leu Leu Ala Phe Arg Phe Tyr Ala Phe Asn Pro Ile Leu Asp

275 280 285

Pro Trp Val Phe Ile Leu Phe Arg Lys Ala Val Phe Gln Arg Leu Lys

290 295 300

Leu Trp Val Cys Cys Leu Cys Leu Gly Pro Ala His Gly Asp Ser Gln

305 310 315 320

Thr Pro Leu Ser Gln Leu Ala Ser Gly Arg Arg Asp Pro Arg Ala Pro

325 330 335

Ser Ala Pro Val Gly Lys Glu Gly Ser Cys Val Pro Leu Ser Ala Trp

340 345 350

Gly Glu Gly Gln Val Glu Pro Leu Pro Pro Thr Gln Gln Ser Ser Gly

355 360 365

Ser Ala Val Gly Thr Ser Ser Lys Ala Glu Ala Ser Val Ala Cys Ser

370 375 380

Leu Cys

385

<210> SEQ ID NO: 10

<211> LENGTH: 2103

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 10

aagctgacac acagaccgac acaggcagcg agagacacga ggagcaaagc aagtgaaggc 60

acagacgcac gggacaggag agcctgggca agactggaga gcccagacct gggatggcgg 120

attcgtgcag gaacctcacc tacgtgcggg gctcggtggg gccggccacc agcaccctga 180

tgttcgtggc cggtgtggtg ggcaacgggc tggccctggg catcctgagc gcacggcgac 240

cggcgcgccc ctcggccttc gcggtgctgg tgaccggact ggcggccacc gacctgctgg 300

gcaccagctt cctgagcccg gccgtgttcg tggcctatgc gcgcaacagc tccctgctgg 360

gcctggcccg aggcggcccc gccctgtgcg atgccttcgc cttcgccatg accttcttcg 420

gcctggcgtc catgctcatc ctctttgcca tggccgtgga gcgctgcctg gcgctgagcc 480

acccctacct ctacgcgcag ctggacgggc cccgctgcgc ccgcctggcg ctgccagcca 540

tctacgcctt ctgcgtcctc ttctgcgcgc tgcccctgct gggcctgggc caacaccagc 600

agtactgccc cggcagctgg tgcttcctcc gcatgcgctg ggcccagccg ggcggcgccg 660

ccttctcgct ggcctacgcc ggcctggtgg ccctgctggt ggctgccatc ttcctctgca 720

acggctcggt caccctcagc ctctgccgca tgtaccgcca gcagaagcgc caccagggct 780

ctctgggtcc acggccgcgc accggagagg acgaggtgga ccacctgatc ctgctggccc 840

tcatgacagt ggtcatggcc gtgtgctccc tgcctctcac gatccgctgc ttcacccagg 900

ctgtcgcccc tgacagcagc agtgagatgg gggacctcct tgccttccgc ttctacgcct 960

tcaaccccat cctggacccc tgggtcttca tccttttccg caaggctgtc ttccagcgac 1020

tcaagctctg ggtctgctgc ctgtgcctcg ggcctgccca cggagactcg cagacacccc 1080

tttcccagct cgcctcaggg aggagggacc caagggcccc ctctgctcct gtgggaaagg 1140

aggggagctg cgtgcctttg tcggcttggg gcgaggggca ggtggagccc ttgcctccca 1200

cacagcagtc cagcggcagc gccgtgggaa cgtcgtccaa agcagaagcc agcgtcgcct 1260

gctccctctg ctgacatttc aagctgaccc tgtgatctct gccctgtctt cgggcgacag 1320

gagccagaaa atcagggaca tggctgatgg ctgcggatgc tggaaccttg gcccccaaac 1380

tctggggccg atcagctgct gtttctcctg cggcagggca gtcgctgctg gctctgggaa 1440

gagagtgagg gacagaggaa acgtttatcc tggagtgcag aaagaatggt tctctcaaaa 1500

taaccagtgg cctggccgac ctgctctggc cctggattcc ccatccatct cattgtctaa 1560

atatttagaa ggcggagaag ttcccagagg cttctgtaca gtcaggtctg ctctggtctg 1620

ggtgctggct ccaatctgcg tccacttagg aggcccaact gcccacccca agtccccagg 1680

ggatggccct ccccctctac caagccactc caagagccag cccctttctg ctccacaaaa 1740

accacagtta ttggaaaagc tccctgcctt cccttgccgc tggtccccca ccaggcttgg 1800

gagccctggc atcccaaagg ggcaacggga ggaaggggag gctgctgcat tgtgggtgat 1860

gacgtaggac atgtgcttgg tacaaaaagg gcctgagaca ttccacctag cttgactggc 1920

tgcaagatga gaactggggg ggtgcaggtg gtggggagac agatggagaa gctggcagat 1980

gaagggtggg ggctgcggat cccagggact gccccagaac acaaacctaa gtcctgtgcc 2040

tgtccccagg gtcctgaata aataaaagcc tccttgcaga gcctgaaaaa aaaaaaaaaa 2100

aaa 2103

<210> SEQ ID NO: 11

<211> LENGTH: 500

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 11

Met Ala Trp Ala Ala Leu Leu Gly Leu Leu Ala Ala Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Ser Arg Arg Arg Thr Arg Arg Pro Gly Glu Pro Pro Leu

20 25 30

Asp Leu Gly Ser Ile Pro Trp Leu Gly Tyr Ala Leu Asp Phe Gly Lys

35 40 45

Asp Ala Ala Ser Phe Leu Thr Arg Met Lys Glu Lys His Gly Asp Ile

50 55 60

Phe Thr Ile Leu Val Gly Gly Arg Tyr Val Thr Val Leu Leu Asp Pro

65 70 75 80

His Ser Tyr Asp Ala Val Val Trp Glu Pro Arg Thr Arg Leu Asp Phe

85 90 95

His Ala Tyr Ala Ile Phe Leu Met Glu Arg Ile Phe Asp Val Gln Leu

100 105 110

Pro His Tyr Ser Pro Ser Asp Glu Lys Ala Arg Met Lys Leu Thr Leu

115 120 125

Leu His Arg Glu Leu Gln Ala Leu Thr Glu Ala Met Tyr Thr Asn Leu

130 135 140

His Ala Val Leu Leu Gly Asp Ala Thr Glu Ala Gly Ser Gly Trp His

145 150 155 160

Glu Met Gly Leu Leu Asp Phe Ser Tyr Ser Phe Leu Leu Arg Ala Gly

165 170 175

Tyr Leu Thr Leu Tyr Gly Ile Glu Ala Leu Pro Arg Thr His Glu Ser

180 185 190

Gln Ala Gln Asp Arg Val His Ser Ala Asp Val Phe His Thr Phe Arg

195 200 205

Gln Leu Asp Arg Leu Leu Pro Lys Leu Ala Arg Gly Ser Leu Ser Val

210 215 220

Gly Asp Lys Asp His Met Cys Ser Val Lys Ser Arg Leu Trp Lys Leu

225 230 235 240

Leu Ser Pro Ala Arg Leu Ala Arg Arg Ala His Arg Ser Lys Trp Leu

245 250 255

Glu Ser Tyr Leu Leu His Leu Glu Glu Met Gly Val Ser Glu Glu Met

260 265 270

Gln Ala Arg Ala Leu Val Leu Gln Leu Trp Ala Thr Gln Gly Asn Met

275 280 285

Gly Pro Ala Ala Phe Trp Leu Leu Leu Phe Leu Leu Lys Asn Pro Glu

290 295 300

Ala Leu Ala Ala Val Arg Gly Glu Leu Glu Ser Ile Leu Trp Gln Ala

305 310 315 320

Glu Gln Pro Val Ser Gln Thr Thr Thr Leu Pro Gln Lys Val Leu Asp

325 330 335

Ser Thr Pro Val Leu Asp Ser Val Leu Ser Glu Ser Leu Arg Leu Thr

340 345 350

Ala Ala Pro Phe Ile Thr Arg Glu Val Val Val Asp Leu Ala Met Pro

355 360 365

Met Ala Asp Gly Arg Glu Phe Asn Leu Arg Arg Gly Asp Arg Leu Leu

370 375 380

Leu Phe Pro Phe Leu Ser Pro Gln Arg Asp Pro Glu Ile Tyr Thr Asp

385 390 395 400

Pro Glu Val Phe Lys Tyr Asn Arg Phe Leu Asn Pro Asp Gly Ser Glu

405 410 415

Lys Lys Asp Phe Tyr Lys Asp Gly Lys Arg Leu Lys Asn Tyr Asn Met

420 425 430

Pro Trp Gly Ala Gly His Asn His Cys Leu Gly Arg Ser Tyr Ala Val

435 440 445

Asn Ser Ile Lys Gln Phe Val Phe Leu Val Leu Val His Leu Asp Leu

450 455 460

Glu Leu Ile Asn Ala Asp Val Glu Ile Pro Glu Phe Asp Leu Ser Arg

465 470 475 480

Tyr Gly Phe Gly Leu Met Gln Pro Glu His Asp Val Pro Val Arg Tyr

485 490 495

Arg Ile Arg Pro

500

<210> SEQ ID NO: 12

<211> LENGTH: 1614

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 12

gggggcgggg aactttacct gggagtgggc caggccgcca gccccgccag ccccgccagc 60

cccgccagcc ccgcgatggc ttgggccgcg ctcctcggcc tcctggccgc actgttgctg 120

ctgctgctac tgagccgccg ccgcacgcgg cgacctggtg agcctcccct ggacctgggc 180

agcatcccct ggttggggta tgccttggac tttggaaaag atgctgccag cttcctcacg 240

aggatgaagg agaagcacgg tgacatcttt actatactgg ttgggggcag gtatgtcacc 300

gttctcctgg acccacactc ctacgacgcg gtggtgtggg agcctcgcac caggctcgac 360

ttccatgcct atgccatctt cctcatggag aggatttttg atgtgcagct tccacattac 420

agccccagtg atgaaaaggc caggatgaaa ctgactcttc tccacagaga gctccaggca 480

ctcacagaag ccatgtatac caacctccat gcagtgctgt tgggcgatgc tacagaagca 540

ggcagtggct ggcacgagat gggtctcctc gacttctcct acagcttcct gctcagagcc 600

ggctacctga ctctttacgg aattgaggcg ctgccacgca cccatgaaag ccaggcccag 660

gaccgcgtcc actcagctga tgtcttccac acctttcgcc agctcgaccg gctgctcccc 720

aaactggccc gtggctccct gtcagtgggg gacaaggacc acatgtgcag tgtcaaaagt 780

cgcctgtgga agctgctatc cccagccagg ctggccaggc gggcccaccg gagcaaatgg 840

ctggagagtt acctgctgca cctggaggag atgggtgtgt cagaggagat gcaggcacgg 900

gccctggtgc tgcagctgtg ggccacacag gggaatatgg gtcccgctgc cttctggctc 960

ctgctcttcc ttctcaagaa tcctgaagcc ctggctgctg tccgcggaga gctcgagagt 1020

atcctttggc aagcggagca gcctgtctcg cagacgacca ctctcccaca gaaggttcta 1080

gacagcacac ctgtgcttga tagcgtgctg agtgagagcc tcaggcttac agctgccccc 1140

ttcatcaccc gcgaggttgt ggtggacctg gccatgccca tggcagacgg gcgagaattc 1200

aacctgcgac gtggtgaccg cctcctcctc ttccccttcc tgagccccca gagagaccca 1260

gaaatctaca cagacccaga ggtatttaaa tacaaccgat tcctgaaccc tgacggatca 1320

gagaagaaag acttttacaa ggatgggaaa cggctgaaga attacaacat gccctggggg 1380

gcggggcaca atcactgcct ggggaggagt tatgcggtca acagcatcaa acaatttgtg 1440

ttccttgtgc tggtgcactt ggacttggag ctgatcaacg cagatgtgga gatccctgag 1500

tttgacctca gcaggtacgg cttcggtctg atgcagccgg aacacgacgt gcccgtccgc 1560

taccgcatcc gcccatgaca cagggagcag atggatccac gtgctcgcct ctgc 1614

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Citation

Patents Cited in This Cited by
Title Current Assignee Application Date Publication Date
Vectors, compositions and methods for treating a vascular disorder UNIVERSITY OF TEXAS HEALTH SCIENCE CENTER HOUSTON 08 March 2002 14 November 2002
High level expression of human cyclooxygenase-2 MERCK FROSST CANADA & CO. 21 June 2000 26 March 2002
Adenoviral vector containing cyclooxygenase-2 promoter and uses thereof UAB RESEARCH FOUNDATION, THE 07 December 2001 08 August 2002
Ophthalmic formulation of a selective cyclooxygenase-2 inhibitory drug PHARMACIA & UPJOHN COMPANY 12 July 2001 21 March 2002
Human cyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2 activity MERCK FROSST CANADA, INC. 06 May 1993 06 August 1996
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