WO2003077796A2

WO2003077796A2 - Ophthalmic solutions for delivery of expression vectors

Info

Publication number: WO2003077796A2
Application number: PCT/US2003/007918
Authority: WO
Inventors: Erlinda M. Gordon; Frederick L. Hall
Original assignee: University Of Southern California
Priority date: 2002-03-15
Filing date: 2003-03-14
Publication date: 2003-09-25
Also published as: AU2003218168A1; AU2003218168A8; WO2003077796A3

Abstract

This invention provides improved ophthalmic solutions useful for storing and/or delivering expression vectors. More specifically, this invention provides a stable aqueous ophthalmic composition comprising an expression vector and a vector stabilizing agent such as serum albumin, sucrose, or lactose. The stabilizing agent is in an amount that maintains the potency (i.e., expression efficiency) of the vector. This invention further provides method of ameliorating ocular diseases or conditions in a mammal using the same opthalmic composition, wherein the vector expresses a protein useful in the treatment of the ocular diseases or conditions. The ophtalmic composition can be administrated topically or systematically. Fig. 1 compares the expression efficiencies of a dnG1 vector in an ophthalmic solution of this invention (A), Genteal Lubricant Eye Drops (B), Three Tears (C), Refresh Tears (D), or Visine Tears (E) using viral titers.

Description

OPHTHALMIC SOLUTIONS FOR DELIVERY OF EXPRESSION VECTORS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 60/364,787, filed March 15, 2002, which is specifically incorporated herein by reference. BACKGROUND OF THE INVENTION

1. Field of the invention: This invention relates to a stable ophthalmic composition containing an expression vector and a vector stabilizing agent, and to methods of ameliorating ocular diseases and disorders by administering an ophthalmic composition of this invention.

2. Description of the Prior Art:

Throughout this application, various publications are referenced by author and date. The disclosures of these publications are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of this invention described and claimed herein.

Gene therapy treatments are rapidly becoming a reality, with several dozen gene therapy protocols approved by the National Institutes of Health, many of which being currently underway. Gene therapy has been proposed as an approach to the treatment of ocular diseases by a number of investigators. These diseases fall into two categories, namely, ocular diseases caused by a specific genetic disorder, whether dominant or recessive, and diseases which have no currently known genetic basis but instead could be treated with the introduction of genes expressing proteins useful in the treatment of the condition.

In the first category, there are a number of diseases for which the underlying genetic defect is known. Autosomal retinitis pigmentosa, both dominant and recessive, may be caused by as many as 50 different mutations in the rhodopsin gene (Bok, Invest. Ophthalm. and Visual Sci. 34(3):473(1993)). Autosomal dominant retinitis punctata albescens, butterfly-shaped pigment dystrophy of the fovea, and adult vitelliform macular dystrophy, have been correlated to a mutation in the peripherin/RDS gene (Kajiwara, et al., Nature Genetics 3:208 (1993); Nichols, et al., Nature Genetics 3:202 (1993); Wells et al., Nature Genetics 3:213 (1993)). Nome's disease (Berger, et al., Nature Genetics 1:199 (1992)), blue cone monochromasy (Nathans, et al., Science 245:831 (1989)), and choroideremia (Cremers, et al., Nature 34,1:61 A (1990); Merry, et al., Proc. Natl. Acad. Sci. USA 89:2135 (1992)) have all been shown to be caused by genetic mutations. The gene for gyrate atrophy involves more than 60 different mutations in the mitochondrial enzyme ornithine aminotransferase (Bok, supra).

In addition to the diseases for which specific genetic mutations are known to cause the phenotype, there are a number of diseases for which the specific genetic component is unknown. These diseases may have a genetic basis or may be caused by other factors resulting in changes in protein expression. For example, age-related macular degeneration is a significant ocular disease among older patients. Retinoblastoma, anterior and posterior uveitis, retinovascular diseases, cataracts, inherited corneal defects such as corneal dystrophies, retinal detachment and degeneration and atrophy of the iris fall into this category, as do retinal diseases which are secondary to glaucoma and diabetes, such as diabetic retinopathy.

Finally, there are a number of conditions which are not genetically based but are still significant ocular diseases. For example, viral infections such as Herpes Simplex Virus (HSV) or cytomegalovirus (CMV) infections frequently cause significant symptoms, and may cause blindness. Retinal detachment, diabetic retinal disease, retinal vein thrombosis, retinal artery embolism, allergic conjunctivitis and other ocular allergic responses, dry eye, lysosomal storage diseases, glycogen storage diseases, disorders of collagen, disorders of glycosaminoglycans and proteoglycans, sphinogolipodoses, mucolipidoses, disorders of amino acid metabolism, dysthyroid eye diseases, anterior and posterior corneal dystrophies, retinal photoreceptor disorders, corneal ulceration and other ocular wounds such as those following surgery are also significant conditions which do not have a known genetic component.

In vitro gene transfer using a retroviral vector has been done on cells deficient in beta-glucoronidase, an enzyme deficiency which is inherited in an autosomal recessive manner. After transformation with the gene coding for the enzyme, the beta-glucuronidase deficient cells exhibited normal enzyme activity (Stramm, et al., Exp. Eye Res. 50:521-532

(1990)).

Recently, two in vivo protocols using adenoviral vectors have been reported (Bennett et al., Investigative Ophthalmology and Visual Science, 35(5):2535 (1994); Li, et al., Investigative Ophthalmology and Visual Science, 35(5):2543 (1994)).

Alcohols are accepted preservatives in commercially available ophthalmic solutions, however, they cannot be used with expression vectors, since the alcohol as well as other chemicals in commercially available ophthalmic solutions either kill the vector or inhibit its potency. Therefore there remains a need for an ophthalmically acceptable aqueous medium for stably storing expression vectors (such as gene therapy vectors) and for delivering the vectors to cells such as corneal keratocytes without inhibiting vector potency or causing eye irritation.

SUMMARY OF THE INVENTION

One aspect of this invention is directed to improved ophthalmic solutions for storing and/or delivering expression vectors such as gene therapy vectors. More specifically, this invention provides a stable aqueous ophthalmic composition comprising an expression vector and a vector stabilizing agent. The vector stabilizing agent is present in an amount that maintains the potency (i.e., expression efficiency) of the expression vector. The vector stabilizing agent is preferably serum albumin, sucrose, or lactose. The ophthalmic solutions are applicable to both viral and non-viral vectors, and may optionally comprise an additional therapeutically active material such as one or more antibiotics and/or vitamins.

Another aspect of this invention is directed to a method of stably storing an expression vector, comprising combining the expression vector with a vector stabilizing agent in an amount that maintains the potency of said vector, wherein the stabilizing agent is serum albumin, sucrose, or lactose.

Yet another aspect of this invention is directed to a method of ameliorating an ocular disease or disorder in a mammal, comprising administering to the mammal an ophthalmic composition in an amount that ameliorates one or more symptoms of said disease or disorder, said solution comprising an expression vector and a vector stabilizing agent in an amount that maintains the potency of said vector, wherein said expression vector expresses a protein useful for treating said disease or disorder. The ophthalmic composition may be delivered topically, e.g., in the form of drops applied directly to the eye, or may be delivered systemically.

Additional advantages and features of this invention shall be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following specification or may be learned by the practice of the invention. The features and advantages of the invention may be realized and attained by means of the instrumentalities, combinations, and methods particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE FIGURES The accompanying drawings, which are incorporated in and form a part of the specification, illustrate non-limiting embodiments of the present invention, and together with the description serve to explain the principles of the invention. In the Figures:

Figures 1A-E compare the expression efficiencies of a dnGl vector in an ophthalmic solution of this invention (A), Genteal™ Lubricant Eye Drops (B), Thera™ Tears (C), Refresh™ Tears (D), or Visine™ Tears (E) using viral titers.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of this invention is directed to improved ophthalmic solutions useful for storing and/or delivering expression vectors. More specifically, this invention provides a stable aqueous ophthalmic composition comprising an expression vector and a vector stabilizing agent. This invention is based on the discovery that incorporating a vector stabilizing agent such as serum albumin, sucrose, or lactose into an aqueous medium containing an expression vector stabilizes the expression vector such that the potency of the vector is preserved. In contrast to conventional ophthalmic solutions, the ophthalmic solutions and compositions of this invention are free of alcohols and other compounds which would otherwise kill or decrease the potency of the expression vector. The term "potency" as used herein refers to the expression efficiency of the vector, that is, the ability of the expression vector to express the gene of interest that was inserted into the vector. Accordingly, as used herein, a "stable" ophthalmic composition refers to an ophthalmic solution of this invention wherein the expression vector has the same or similar expression efficiency as it did prior to storage in an ophthalmic solution of this invention.

As used herein, an "ophthalmic solution" refers to an aqueous medium comprising a vector stabilizing agent of this invention, and an "ophthalmic composition" refers to an aqueous medium comprising an expression vector suspended in an ophthalmic solution, i.e., an aqueous medium comprising a vector stabilizing agent and an expression vector. Further, in contrast to ophthalmic solutions in the art, the ophthalmic solutions and compositions of this invention do not cause eye irritation or corneal ulceration.

Furthermore, ocular adnexa (lower and upper eyelids), contralateral eye, nasal mucosa and nasal septa show no evidence of inflammation, ulceration, thrombosis or hemorrhage when treated with an ophthalmic composition of this invention.

It was further discovered that in addition to having vector stabilizing properties (e.g., preserving the potency of the expression vector), the ophthalmically acceptable compositions of this invention preserve cell integrity and promote wound healing. Furthermore, the inventive ophthalmic compositions have anti-oxidant properties, anti- aggregating properties, and pH buffering capacity.

A necessary component of an ophthalmic composition of this invention is a vector stabilizing agent. The stabilizing agent functions to prevent non-specific binding of the expression vector to surfaces and to prevent molecular aggregation of the vector. The stabilizing agent also enhances cryopreservation of the vector and maintains the integrity of the vector during freeze-thaw transitions. As a result of incorporating a vector stabilizing agent into the ophthalmic solutions of this invention, the need to add conventional chemicals such as alcohols to the ophthalmic compositions or composition is eliminated.

The vector stabilizing agent is preferably serum albumin, sucrose, or lactose. Human serum albumin is a preferred stabilizing agent for both systemic and topical delivery of an expression vector to humans or to non-human mammals. In this embodiment, the ophthalmic composition preferably comprises about 0.5 to about 5 gram percent, and more preferably about 1 to about 2 grams percent, of the human serum albumin. Alternatively, other non-human serum albumins including, but not limited to, bovine, ovine, or porcine serum albumin, can also be utilized as vector stabilizing agents. When a non-human albumin is used as a vector stabilizing agent, the ophthalmic composition comprises about 0.5 to about 5 gram percent, more preferably about 1 to about 2 grams percent of the non-human albumin. For topical vector delivery (e.g., corneal application) in humans or non-human mammals, sucrose or lactose can be used as the vector stabilizing agent in an ophthalmic solution, and in this embodiment the ophthalmic composition preferably comprises between about 2% and about 4 % (w/v) sucrose or lactose. The ophthalmic compositions of this invention comprise a vector stabilizing agent in amount that stabilizes the vector in a useful condition suitable for gene delivery purposes for at least two years when stored at -80° C under typical storage conditions, or for about 3 days when stored at 4° C, or for about 8 hours when stored at room temperature.

When the ophthalmic composition of this invention is formulated as a solution or suspension, the composition is in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, and the like, provided that the carrier does not kill the expression vector or inhibit its potency. The ophthalmic solutions and compositions may also contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, including, but not limited to, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, sodium acetate, sodium lactate, sorbitan monolaurate, triethanolamine oleate, etc. In one embodiment, the ophthalmic solution is titrated to about pH 7.1 to about pH 7.4, more preferably about pH 7.2 to about pH 7.3, with a suitable acid such as 1N HC1. •

The ophthalmic solutions and compositions may also comprise one or more amino acids in conventional amounts. The amino acid may be a water-soluble hydrophilic amino acid such as L-arginine, L-cystine, glycine, L-histidine, L-leucine, L-lysine, L-methionine, L-serine, L-valine, L-threonine, L-tryptophan, L-glutamine, and L-tyrosine. The concentration of the amino acid in the solution is typically from 10 to about 300 mg/L, depending on the specific amino acid.

In another embodiment, the ophthalmic solution or composition also comprises a sugar such as glucose.

In some preferred embodiments, one or more antibiotics are optionally added in conventional amounts to the ophthalmic solution or composition of this invention. Examples of antibiotics suitable for purposes of this invention include, but are not limited to, ciprofloxacin, a penicillin, a semisynthetic penicillin, fluoroquinolone, erythromycin, rifampicin, streptomycin, ofloxacin, norfloxacin, cefazolin, tobramycin, gentamycin, an aminoglycoside, amoxicillin, ampicillin, carbenicillin, ticarcillin, mezlocillin, a cephalosporin, vancomycin, chloramphenicol, clindamycin, bacitracin, polymyxin, spectinomycin, a sulfonamide, trimethoprim, or mixtures thereof. Additionally, one or more auxiliary substances such as vitamins, minerals, etc. are optionally added in conventional amounts to the ophthalmic solution or composition.

Examples of vitamins, minerals, etc., suitable for purposes of this invention include, but are not limited to, inositol, folic acid, pantothenic acid, nicotinic acid amide, riboflavin, thiamine, pyridoxine, choline, ferric nitrate, and pyruvic acid.

The ophthalmic compositions of this invention may be sterilized before use by conventional, well known sterilization techniques, or may be sterile filtered. In one embodiment, the composition is sterilized by filtration using a sterile filter with a pore size of 0.45 μm or less. The resulting aqueous composition may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile carrier prior to administration.

The ophthalmic compositions of this invention comprise an expression vector that expresses a protein of interest for use in the treatment of an ocular disease or disorder, wherein the disease can be treated with the introduction of a gene expressing a protein useful in the treatment of the disease or disorder. One example of an expression vector is a gene therapy vector. In order to express a biologically active gene, the nucleotide sequences encoding gene or derivatives thereof may be inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for directing the expression of the inserted coding sequence in the target cell. Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding the gene of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described by Sambrook, J. et al. (1989; Molecular Cloning. A Laboratory Manual, ch. 4, 8, and 16-17, Cold Spring Harbor Press, Plainview, N.Y.); and Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.).

The ophthalmic compositions of the present invention may be used in connection with a wide variety of expression vectors. Examples of non-viral delivery expression vectors include expression plasmids capable of directing the expression of the therapeutic gene of interest in the target cell. Expression plasmids are autonomously replicating, extrachromosomal circular DNA molecules, distinct from the normal genome and nonessential for cell survival under nonselective conditions capable of effecting the expression of a DNA sequence in the target cell. Plasmids autonomously replicate in bacteria to facilitate bacterial production, but it is not necessary that such plasmids replicate in the target cell in order to achieve the therapeutic effect. The transgene may also be under control of a tissue specific promoter region allowing expression of the transgene only in particular cell types. Those of skill in the art will readily appreciate the variety of expression plasmids which may be useful in the practice of the present invention. The expression plasmid may also contain promoter, enhancer or other sequences aiding expression of the therapeutic gene and/or secretion can also be included in the expression vector. In other instances, the DNA sequence is delivered by a viral delivery system wherein the therapeutic gene of interest is incorporated into a viral genome capable of infecting the target cell, and the gene is operably linked to expression and control sequences such that the gene of interest is expressed under appropriate conditions in the target cell. The vectors useful in the practice of the present invention may also be derived from the viral genomes. Vectors which may be employed include recombinantly modified enveloped or non-enveloped DNA and RNA viruses. Examples of suitable vectors include retroviral vectors such as, but not limited to, a Moloney murine leukemia virus (MuLV)- based retroviral vector, a human immunodeficiency virus (HΙV)-based lentiviral vector, an adenoviral vector, an adeno-associated virus vector, a herpes virus vector, such as a herpes simplex virus-derived vector, and a pseudotyped virus.

The concentration of the expression vector in the ophthalmic compositions can vary widely, depending on the intended use. The concentration required in order to have the desired effect, e.g., to ameliorate one or more symptoms of an ocular disease or disorder, can be determined by those skilled in the art without undue experimentation. The ophthalmic solutions of this invention are conveniently useful for the harvesting of vectors from producer cell cultures and for downstream processing (e.g., vector concentration, diafiltration, buffer exchange, and/or other purification steps) prior to and including "final fill" of cryovials, tubes, bags, ampoules;, or bottles, and for post-fill storage. The inventive solutions and compositions of this invention are compatible with both custom-designed and commercially available final fill and closure systems.

One non-limiting example of an ophthalmic solution formulation of this invention is presented in Table 1. The formulation shown in Table 1 can optionally include additional components, if desired, such as described in Example 1. Of course, suitable chemical equivalents of the listed components in Table 1 can also be replaced with suitable alternatives. For example, L-cysteine can replace L-cystine 2HC1; hydrated forms can replace anhydrous forms of the listed components; sodium phosphate dibasic can replace sodium phosphate monobasic; and pharmaceutically acceptable salts can be substituted for free amino acids. Substantially equivalent concentrations beyond the concentration ranges recited in Table 1 are also encompassed by the present invention.

Table 1. Aqueous ophthalmic compositions for delivery of expression vectors

Another aspect of this invention provides a method of ameliorating an ocular disease or disorder in a mammal, comprising administering to said mammal an ophthalmic composition of this invention in an amount that ameliorates one or more symptoms of the disease or disorder, wherein the composition comprises an expression vector and a vector stabilizing agent, wherein the expression vector expresses a protein which ameliorates one or more symptoms of the disease or disorder. The ophthalmic composition comprises the vector stabilizing agent in an amount that is able to maintain the potency of the vector. In one embodiment the ophthalmic compositions according to this invention can be used topically (e.g., administered as an eye drop) for the treatment of diseases or disorders of the eye without causing local irritant effects and produce beneficial effects surpassing those obtainable with expression vector compositions prepared with conventional ophthalmic solutions. In one embodiment the topical administration delivers the vectors to cells such as corneal keratocytes. In another embodiment, the ophthalmic compositions of this invention can be used for applications of in vivo gene delivery that are suitable for delivery routes other than by topically delivery. For example, the inventive compositions can be used to deliver a therapeutic gene via a delivery route such as, but not limited to, intravitreous, intramuscular, subcutaneous, intraperitoneal, intravenous, intra-arterial, intranasal, sublingual, intrarectal, intrabladder, intravaginal, intracervical, transmembranous (i.e., via an epithelial membrane) delivery routes, and oral, inhalation, sublingual, and other oral epithelial membrane delivery routes. However, for ease of discussion, all compositions of this invention, whether administered topically or systemically, will be referred to as "ophthalmic compositions." Ophthalmic disorders or diseases may be treated by administering the ophthalmic composition of the present invention in an amount effective to treat or ameliorate the disorder, i.e., in an amount that ameliorates one or more symptoms of the disease. The amount of the ophthalmic composition that will be required for the treatment will depend upon the nature and scope of the disorder, and such amounts can be readily determined by those skilled in the art without undue experimentation.

In a preferred embodiment, the expression vector comprises a exogenous nucleic acid that encodes a protein useful in the treatment of ocular diseases. By "ocular disease" is meant a disorder or pathological condition of the eye that is not normal to a mammal in a healthy state. In one embodiment, the ocular disease may be caused by a genetic defect.

Examples of such ocular diseases for which a gene has been identified include, but are not limited to, autosomal retinitis pigmentosa, autosomal dominant retinitis punctata albescens, butterfly-shaped pigment dystrophy of the fovea, adult vitelliform macular dystrophy, Nome's disease, blue cone monochromasy, choroideremia and gyrate atrophy. These may also be referred to as genetic ocular diseases.

In other embodiments, the ocular disease may not be caused by a specific known genotype (although they may be shown in the future to have a genetic component). These ocular diseases include, but are not limited to, age-related macular degeneration, retinoblastoma, anterior and posterior uveitis, retinovascular diseases, cataracts, inherited corneal defects such as corneal dystrophies, retinal detachment and degeneration and atrophy of the iris, and retinal diseases which are secondary to glaucoma and diabetes, such as diabetic retinopathy. In addition, the term ocular disease includes conditions which are not genetically based but still cause ocular disorders or dysfunctions. These include, but are not limited to, viral infections such as Herpes Simplex Virus or cytomegalovirus (CMV) infections, allergic conjunctivitis and other ocular allergic responses, dry eye, lysosomal storage diseases, glycogen storage diseases, disorders of collagen, disorders of glycosaminoglycans and proteoglycans, sphinogolipodoses, mucolipidoses, disorders of amino acid metabolism, dysthyroid eye diseases, anterior and posterior corneal dystrophies, retinal photoreceptor disorders, corneal ulceration and other ocular wounds such as those following surgery.

By "protein useful in the treatment of an ocular disease" herein is meant a protein that is effective to ameliorate one or more symptoms of the ocular disease.

In one embodiment, the ophthalmic composition is delivered to corneal epithelial cells. Corneal epithelial cells are subject to injury, allergic reactions and infections, among others. Thus, proteins that are useful in the treatment of these conditions, and others, may be delivered via the present invention. In another embodiment, the ophthalmic composition is delivered to corneal endothelial cells. This is particularly significant since dysfunction of the corneal endothelial cells causes blindness. This layer is often damaged during cataract extraction, which is currently the most common surgical operation in the United States. In addition, since the corneal endothelium cannot regenerate (since cell division does not occur), the expression of proteins that cause division or regeneration of corneal endothelial cells could be a significant treatment of corneal endothelial damage.

In another embodiment, the ophthalmic composition is introduced into the cells of the trabecular meshwork, beneath the periphery of the cornea. The trabecular meshwork is the outflow tract from the anterior chamber of the eye, which allows aqueous humor (the fluid contained within the eye) to drain from the eye. This is significant since glaucoma is a common cause of visual loss in the U.S., and is a result of increased intraocular pressure. Therefore, the methods of the present invention may be useful to regulate the outflow of aqueous humor and treat or cure glaucoma. In one embodiment, the ophthalmic composition is introduced to cells of the choroid layer of the eye. The choroid layer of the eye is part of the blood supply to the retina, and thus may supply proteins to the retina. For example, BDNF (brain-derived neurotrophic factor) may be delivered in this manner to treat retinal degeneration. In alternative embodiments, the ophthalmic composition is introduced to cells of the retina, sclera or ciliary body. This may be done, for example, for controlling production of aqueous fluid in the treatment or prevention of glaucoma.

The following example is illustrative of the present invention.

EXAMPLE 1 Methods

Vector potency was assessed using a neomycin phosphotransferase (neo¹) titer assay for various mixtures of a vector (Gordon, E. M., et ah, Cancer Res. 60:3343-3347 (2000); Xu F., et al. Int. J. Molec. Med. 8:19-30 (2001)) suspended in either an ophthalmic solution of this invention having the formulation shown in Table 2, or in one of four commercially available ophthalmic solutions (Genteal™ Lubricant Eye Drops; Thera™ Tears; Refresh™ Tears; or Visine™ Tears; Table 3).

Table 2. Ophthalmic formulation of this invention for expression vectors

Table 3. Components of four commercially available ophthalmic solutions

Vectors

High titer vectors were generated using a transient three plasmid cotransfection system (Soneoka Y., et al., Nucleic Acids Res. 23:628-633 (1995)), in which the packaging components gag-pol, a wild type murine leukemia virus-based amphotropic CAE envelope {env) or a chimeric env bearing a von Willebrand factor (vWF) derived collagen-binding (matrix targeting) motif, and a retroviral vector bearing either a nuclear targeted β- galactosidase gene or a dnGl construct expressed from the CMN promoter were placed on separate plasmids, each of which contained the Sv40 origin of replication. For comparisons of in vivo efficacy, a non-targeted retroviral vector bearing an antisense cyclin Gl construct "aGl" (Skotzko, M. J., et al, Cancer Res. 55:5493-5498 (1995); Zhu, N. L., et al, Circulation 96:628-635 (1997); Chen, D. S., et al, Human Gene Ther. 8:1679-1686 (1997); Hung, G., et al, Int. J. Pediatr. Hematol Oncol. 4:317-325 (1997)), and a non-targeted retroviral vector bearing only the neo^r gene (null control) were used. In vitro studies

Viral titers were measured in murine NIH3T3 cells in mixtures of dnGl vector and the inventive ophthalmic solution, as described previously (Skotzko et al, Cancer Res. 55:5493-5498, (1995)). Briefly, 2 x 10⁴ cells in 3 mL D10 (DMEM + 10% FBS) were plated into each uncoated plastic well in 6-well plates, and allowed to attach overnight at 37° C. The cultures were transduced with 1 mL of each mixture of vector: ophthalmic formulation in the presence of polybrene (8 g/mL) at 37° C for 30 min. Viral titer determination was based on expression of the neomycin phosphotransferase (neo^r) gene. Viral titer was expressed as the number of G418-resistant cfu/mL. Figures 1A-E compare the expression efficiencies based on viral titers of murine NIH3T3 cells in mixtures of dnGl vectors in an ophthalmic solution of this invention (A), Genteal™ Lubricant Eye Drops (B), Thera™ Tears (C), Refresh™ Tears (D), or Visine™ Tears (E). In vivo studies

In vivo experiments were conducted with New Zealand adult white rabbits. The mean weight of the animals was 8.5 pounds (SD 0.89). The rabbits were examined at slit lamp after two days in the vivarium, to rule out presence of external ocular disease, such as discharge, conjunctival injection, or corneal scarring. To evaluate the efficiency of gene delivery to laser-treated cornea by topical eye drop application, four rabbits were treated with either a targeted or non-targeted vector bearing a marker gene, the vectors being suspended in the ophthalmic solution of this invention (Table 2) for delivery. To evaluate the efficacy of the anti-proliferative constructs, 20 rabbits were randomly labeled 1 to 20, and assigned to receive either a control or a therapeutic vector. Four additional rabbits were treated with increasing concentrations of the dnGl vector to evaluate retroviral toxicity and vector dissemination to non-target organs. Commercially available eye drop solutions (Table 3) were also employed to suspend the retroviral vectors for comparison. The following experimental procedures were conducted.

One hour after excimer laser PTK surgery was administered to the animals, instillation of topical eye drops of the solution containing a vector was initiated. Investigators performing instillation were masked for the treatment applied. The vials were labeled with letters "A," "B" and "C," according to the vector administered. The topical application sequence was similar to the numeric values assigned to the rabbits, i.e., vector "A" to rabbit 1, vector "B" to rabbit 2, vector "C" to rabbit 3, etc. Each rabbit received one 40 μL eye drop of vector mixed in the inventive ophthalmic solution (Table 2) every 10 minutes for a 2 hour period on the first day, and the same scheme for 1.5 hours on the second day. Rabbits in Vector "A" group received a collagen-targeted retroviral vector bearing a dominant negative cyclin Gl (dnGl) construct (n = 7); the vector "B" group received a retroviral "null" vector bearing on the neo^r gene (n = 7), and the vector "C" group received a retroviral vector bearing an antisense cyclin Gl (aGl) construct (n = 6). The cumulative vector dose for each rabbit in all 3 groups was about 5 x 10⁷ colony forming units (cfu).

Corneal haze monitoring was conducted by an observer who was blinded to the type of vector treatment. Objective corneal haze assessment was performed using a tangential slit light (Model BL900 Slit-lamp, Haag-Streit, Switzerland) focused on the surface of the cornea and oriented at 45° incidence to this plane. The image reflected was recorded by digital photography (DCTRV-8, Sony Corp., Tokio, Japan) at 90° to the corneal surface. Lighting conditions in the room and in the slit lamp source were controlled to be similar to avoid bias at the time of the image analysis. Images were downloaded into a computer by means of a digital image capture software (Studio DV 1.05.303, Pinnacle Systems Inc., Mountain View, CA) at 1500 x 1125 pixel resolution and evaluated by a digital imaging analysis software (Scion Image, 4.0.2., Scion Corp. Frederick, MD). For corneal haze assessment, a modified procedure of objective corneal haze measurement was used (Seitz, B., et al, Am. J. Ophthalmol 126:630-639 (1998); Maldonado, M. J., et al, Am. J. Ophthalmol. 123:31-41 (1997)). Prior calibration/standardization to enable comparisons between rabbits, each image was transformed into a grayscale and three areas within the light reflection (superior, middle and inferior) of 50 x 50 pixel each were measured. The imaging software recognizes different shades of gray in a scale from 1 (white) to 255 (black).

After determining the most efficient group in corneal haze prevention, an additional group of 6 rabbits (3 female, 3 male) were subjected to studies of toxicity with 3 increasing dosages of the dnGl vector. One male rabbit and one female rabbit were selected for each dosage group. The same PTK and eye drop application scheme was used. A modified Draize score was used to address external toxicity (Imayasu, M., et al, CLAO J. 18:260-266 (1992)). This group of animals was sacrificed at two weeks, and histological evaluation of paraformaldehyde-fixed tissue sections, with PAS staining, was conducted to rule out signs of ocular inflammation. Tissue sections were stained with hematoxylin-eosin stain. Under light microscopy, tissue sections were evaluated for integrity of organ architecture, detection of cellular swelling or necrosis, presence of acute or chronic inflammation, hypo/hypercellularity, thrombosis or any other abnormality. Results

In vitro studies. As summarized in Table 4, expression efficiency, as measured by viral titer, was about 6- to 50-fold greater when the vector was suspended in an ophthalmic formulation of this invention (Table 2), compared to four commercially available ophthalmic solutions (Genteal™ Lubricant Eye Drops; Thera™ Tears; Refresh™ Tears; or Visine™ Tears (Table 3)). The viral titer in the mixture of vector and the inventive ophthalmic formulation was higher compared to those of the four commercially available eye drop formulations

Table 4. In vitro expression efficiency

In vivo studies. In vivo, the ophthalmic composition of this invention did not cause any eye irritation or corneal ulceration in the animals, while the commercially available solutions containing serum components caused an inflammatory reaction (data not shown). Furthermore, histologic examination of paraformaldehyde-fixed sections of ocular adnexa (lower and upper eyelids), contralateral eye, nasal mucosa and nasal septum of rabbits, treated with the inventive ophthalmic solution and dnGl vector suspended therein, showed no evidence of inflammation, ulceration, thrombosis or hemorrhage. There was a 6% incidence of infectious keratitis in animals treated with laser surgery and vector or placebo that were not given antibiotic prophylaxis.

The foregoing description is considered as illustrative only of the principles of the invention. Further, since numerous modifications and changes will be readily apparent to those skilled in the art, it is not desired to limit the invention to the exact construction and process shown as described above. Accordingly, all suitable modifications and equivalents may be resorted to falling within the scope of the invention as defined by the claims that follow.

The words "comprise," "comprising," "include," "including," and "includes" when used in this specification and in the following claims are intended to specify the presence of stated features, integers, components, or steps, but they do not preclude the presence or addition of one or more other features, integers, components, steps, or groups thereof.

Claims

What is claimed is:

1. A stable aqueous ophthalmic composition comprising an expression vector and a vector stabilizing agent, said agent being in an amount that maintains the potency of said vector.

2. The ophthalmic composition of claim 1, wherein said vector stabilizing agent is selected from the group consisting of serum albumin, sucrose, and lactose.

3. The ophthalmic composition of claim 2, wherein said albumin is human serum albumin.

4. The ophthalmic composition of claim 3, comprising about 0.5 to about 5 gram percent of said human serum albumin.

5. The ophthalmic composition of claim 4, comprising about 1 to about 2 grams percent of said human serum albumin.

6. The ophthalmic composition of claim 1, having a pH between about 7.1 and 7.4.

7. The ophthalmic composition of claim 1, further comprising at least one amino acid.

8. The ophthalmic composition of claim 2, comprising about 2 to about 4% (w/v) of said sucrose or said lactose.

9. The ophthalmic composition of claim 1, further comprising a therapeutically effective amount of an additional therapeutically active material.

10. The ophthalmic composition of claim 9 wherein said additional therapeutically active material comprises ciprofloxacin, a penicillin, a semisynthetic penicillin, fluoroquinolone, erythromycin, rifampicin, streptomycin, ofloxacin, norfloxacin, cefazolin, tobramycin, gentamycin, an aminoglycoside, amoxicillin, ampicillin, carbenicillin, ticarcillin, mezlocillin, a cephalosporin, vancomycin, chloramphenicol, clindamycin, bacitracin, polymyxin, spectinomycin, a sulfonamide, trimethopri-m, or mixtures thereof.

11. The ophthalmic composition of claim 1, further comprising one or more vitamins or minerals selected from the group consisting of inositol, folic acid, pantothenic acid, nicotinic acid amide, riboflavin, thiamine, pyridoxine, choline, ferric nitrate, and pyruvic acid.

12. The ophthalmic composition of claim 1 , wherein said stabilizing agent is in amount that stabilizes said vector for at least two years when said ophthalmic composition is stored at about -80° C.

13. The ophthalmic composition of claim 1, wherein said stabilizing agent is in amount that stabilizes said vector for at least three days when said ophthalmic composition is stored at about 4° C.

14. The ophthalmic composition of claim 1, wherein said stabilizing agent is in amount that stabilizes said vector for at least eight hours when said ophthalmic composition is stored at about room temperature.

15. The ophthalmic composition of claim 1, wherein said expression vector is a retroviral vector.

16. The ophthalmic composition of claim 15, wherein said retroviral vector is a Moloney murine leukemia virus-based retroviral vector, a human immunodeficiency virus-based lentiviral vector, an adenoviral vector, an adeno-associated virus vector, a herpes virus vector, or a pseudotyped virus.

17. The ophthalmic composition of claim 1, wherein said expression vector is a non- viral vector.

18. A stable aqueous ophthalmic composition comprising an expression vector and a vector stabilizing agent selected from the group consisting of serum albumin, sucrose, and lactose, said solution having a pH between about 7.1 and 7.4.

19. A method of stably storing an expression vector, comprising combining said expression vector with an ophthalmic solution comprising a vector stabilizing agent in an amount that maintains the potency of said vector.

20. A method of delivering an expression vector to a mammal, comprising combining said expression vector with a vector stabilizing agent in an amount that maintains the potency of said vector.

21. The method of claim 20, wherein said combination is titrated to about pH 7.1 to 7.4.

22. The method of claim 20, wherein said combination is sterilized prior to said delivery.

23. A method of ameliorating an ocular disease or disorder in a mammal, comprising administering to said mammal an ophthalmic composition, said composition comprising an expression vector and a vector stabilizing agent in an amount that maintains the potency of said vector, wherein said expression vector expresses a protein which^" ameliorates said disease or disorder.

24. The method of claim 23, wherein said ophthalmic composition is administered in topically.

25. The method of claim 23, wherein said ophthalmic composition is delivered to corneal keratocytes.

26. The method of claim 23, wherein said ophthalmic composition is administered systemically.

27. The method of claim 26, wherein said systemic administration is a method selected from the group consisting of intravitreous, intramuscular, subcutaneous, intraperitoneal, intravenous, intra-arterial, intranasal, sublingual, intrarectal, intrabladder, intravaginal, intracervical, transmembranous, oral, inhalation, sublingual and oral epithelial membrane delivery routes.

28. A stable aqueous ophthalmic composition comprising an expression vector, human serum albumin, L-arginine hydrochloride, L-cystine dihydrochloride, glycine, L-histidine hydrochloride, L-leucine, L-lysine hydrochloride, L-methionine, L-serine, L-valine, inositol, monobasic sodium phosphate, calcium chloride, magnesium sulfate, and sodium bicarbonate, said solution having a pH between about 7.1 and 7.4.