A method for increasing outflow facility and reducing intraocular pressure from an eye of a subject having glaucoma includes the step of administering to the eye an amount of an ADP ribosyltransferase protein effective to reduce intraocular pressure and increase outflow facility.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a treatment for glaucoma. While the present invention does not depend on an understanding of the mechanism by which successful treatment is accomplished, it is believed that C3 disrupts the system of focal adhesions and actin and myosin II containing stress fibers, in turn causing changes in cell shape that translate into an increase in aqueous humor outflow facility.

It will be understood, that the use of a genetic construct to provide C3 to an eye of a subject, is considered a desired but not an essential aspect of the administration method. Vectors that are particularly well suited for introduction into non-dividing cells (of which trabecular meshwork cells are an example) are known and are considered desirable for in vivo expression of C3 in vivo in human and non-human animal eyes. A suitable vector can include an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus-based vector, a lentivirus vector, and a plasmid vector. The skilled artisan will appreciate the importance of engineering a vector and its components for efficient use in trabecular meshwork cells. The transduction efficiency of the various delivery systems is known to vary and can depend upon the nature of the vector and its components.

In addition to vectors of the types noted above, non-vector approaches, including direct administration of C3 protein, liposomal delivery of C3, and diffusion of C3 protein from implanted cells encapsulated in a sealed semipermeable membrane capsule, are contemplated.

The use of adenovirus expression vectors and other vector systems for therapeutic transfer of a nucleic acid construct into target tissue to treat glaucoma is described generally in, e.g., Borras, T. et al., "Gene Therapy for Glaucoma: Treating a Multifaceted, Chronic Disease," IOVS, 43:2513 (2002) and papers cited therein in references 25 31, each of which is incorporated by reference herein as if set forth in its entirety. Also incorporated herein by reference in its entirety is Hauswirth, W. W. and L. Beaufrere, "Ocular Gene Therapy: Quo Vadis?," IOVS 41:2821 (2000) which reviews the eye as a gene therapy target and concludes that "ocular gene therapy seems well poised to be among the earliest successful applications" of the technology. The cited papers also provide the skilled artisan with the technical requirements for a suitable expression vector.

The skilled person will appreciate that when a C3-encoding genetic construct is delivered, various aspects can affect expression of C3 from the encoding construct. For example, the vector backbone of the genetic contruct should be suited for efficient transfer into the target trabecular meshwork cells, for long-term maintenance of the construct in the cells and for sustained expression of C3 in the cells. Expression is sustained, e.g., by providing on the construct a transcriptional promoter that supports transcription in target trabecular meshwork cells. In particular, certain lentivirus vectors, namely certain feline immunodeficiency virus vectors, are efficiently transduced into human and non-human trabecular meshwork cells and provide efficient and long-term stable expression of a protein encoded by a polynucleotide provided on the vector. Suitable vectors, and methods for their production and use, are described in Loewen, N., et al., "Long-Term, Targeted Genetic Modification of the Aqueous Humor Outflow Tract Coupled with Noninvasive Imaging of Gene Expression In Vivo," IOVS, 45:3091 (2004) and in Loewen, N., et al., "Preservation of Aqueous Outflow Facility after Second-Generation FIV Vector-Mediated Expression of Marker Genes in Anterior Segments of Human Eyes," IOVS, 43:3686 (2002), each of which is incorporated by reference as if set forth herein in its entirety. Further incorporation by reference is made to the papers cited in the foregoing papers in connection with various starting materials and methods for producing vectors suited for efficient transduction into trabecular meshwork cells. Loewen, N., et al. (2004) provides the skilled person with guidance as to the amount of vector advantageously administered in vivo to cats, a species for which effectiveness of a therapeutic method is generally considered to be a reliable predictor of effectiveness of the method in humans. In cats, amounts in the range of between about 10.sup.6 and 10.sup.8 tranducing units (TU) were administered per eye with good results. The skilled person applying only routine skill can adjust these amounts, if appropriate, to deliver IOP-reducing amounts of vectors to anterior portions of the eye of human or other non-human subjects. Production of lentiviral vectors and delivery into non-dividing human eye cells is also described and claimed in U.S. Pat. No. 6,555,107, incorporated herein by reference as if set forth in its entirety.

Using conventional tools of the molecular biologist, the aforementioned vectors, and others, can be modified to provide a polynucleotide that encodes C3 in the vector downstream from a transcriptional promoter functional in trabecular meshwork cells, such that C3 is produced in the TM cells.

In the accompanying working examples, C3 was encoded by and expressed from a vector provided with the C3 coding sequences in trabecular meshwork cells grown in culture or maintained in anterior segments mounted on organ perfusion culture dishes. In the examples, C3 and a marker, green fluorescent protein (GFP), were expressed upon introduction into the cells by an adenovirus expression vector under transcriptional control of a cytomegalovirus promoter-enhancer. Introduction by injection of genetic material is considered a preferred approach by the inventors, although provision of C3 protein to trabecular meshwork cells in a manner known to the art is also suitable.

It is also noted that the protein encoded by the C3 coding sequence includes a short (7 amino acid) leader sequence. This sequence is important to the bacterial source, but is not of interest or use in the present invention and can be removed from the coding sequence without adverse effect on intracellular Rho targeting. It is also noted that other ADP-ribosyltransferase exoenzymes, such as the C. botulinum C2 toxin, having different targets in the actin microfilament network are known and can be employed in place of exoenzyme C3. However, the C2 enzyme can be more toxic than the C3 enzyme and it would be advantageous to introduce substitution mutations into the enzyme (via the C2-encoding polynucleotide) to modulate the toxicity of C2 before use. A polynucleotide that encodes exoenzyme C2 is available at GenBank accession number D88982 and is attached hereto as SEQ ID NO:3. SEQ ID NO:4 and SEQ ID NO:5 are two components encoded by SEQ ID NO:3. SEQ ID NO:4 has ADP-ribosyltransferase activity.

The skilled artisan will appreciate that in due course further improvements to nucleic acid delivery methods, employing virus- or non-virus based approaches may be developed, and that the invention is sufficiently broad to encompass use of any such methods for providing C3 in trabecular meshwork cells, without regard to the specific delivery vector or method. Further, the C3 protein need not be obtained from C. botulinum as in the examples. As the activities of C3 are well understood, the skilled artisan can readily select a C3 protein source having the characteristic properties of C3, namely a ADP ribosyltransferase that inhibits rho-activated cellular contractility, or a nucleic acid sequence encoding same, for administration in the methods of the invention. It will also be understood that the ability of C3 to function in the methods of the invention may be modulated, particularly enhanced, by introducing one or more changes to amino acid residues of the C3 protein. The skilled artisan can introduce such changes at the nucleic acid level and can monitor outflow facility directed by modified proteins such that modified C3 proteins that yield great outflow facility (and nucleic acids encoding same) can be selected for use in the methods. The present invention will be more fully understood upon consideration of the following non-limiting examples. The examples demonstrate proof of principle, but the skilled artisan will appreciate that the C3 can be administered via any medically acceptable route.

In the accompanying working examples, C3 was expressed in trabecular meshwork cells grown in culture. In the examples, C3 and a marker, green fluorescent protein (GFP), were expressed upon introduction into the cells of an adenovirus expression vector under transcriptional control of a cytomegalovirus promoter-enhancer. Introduction by injection of genetic material can increase persistence of the treating agent in the target tissue and is therefore considered a preferred approach by the inventors, although provision of C3 protein to trabecular meshwork cells in a manner known to the art is also suitable. The use of adenovirus expression vectors and other vector systems for therapeutic transfer of a nucleic acid construct into target tissue to treat glaucoma is described generally in, e.g., Borras, T. et al., "Gene Therapy for Glaucoma: Treating a Multifaceted, Chronic Disease," IOVS, 43:2513 (2002) and papers cited therein at references 25 31, each of which is incorporated by reference herein as if set forth in its entirety. Also incorporated herein by reference in its entirety is Hauswirth, W. W. and L. Beaufrere, "Ocular Gene Therapy: Quo Vadis?," IOVS 41:2821 (2000) which reviews the eye as a gene therapy target and concludes that "ocular gene therapy seems well poised to be among the earliest successful applications" of the technology. The cited papers also provide the skilled artisan with the technical requirements for a suitable expression vector. It will be understood, that the use of a particular adenovirus vector, or an adenovirus vector per se, or, more generally, a genetic construct, to provide C3 to an eye of a subject, is considered a preferred but not an essential administration method. The skilled artisan will appreciate that in due course further improvements to nucleic acid delivery methods, employing virus- or non-virus based approaches will be developed, and that the invention is sufficiently broad to encompass use of any such methods.

Claim 1 of 8 Claims

1. A method for increasing outflow facility of aqueous humor from an eye having a trabecular meshwork, the method comprising the steps of: providing to the trabecular meshwork an amount of an ADP ribosyltransferase protein effective to increase outflow facility.

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