HOF-GON	ZZCC	5 × 10⁸	49	48	3
HOF-GON	QLCC	5 × 10⁸	52	43	5
HOF-GON	TOCC	5 × 10⁸	81	7	12
HOF-GON	XTCC	5 × 10⁸	88	4	7
HOF-GON	ZZCC	5 × 10⁹	67	3	0
HOF-GON	QLCC	5 × 10⁹	79	13	7
HOF-GON	TOCC	5 × 10⁹	91	0	9
HOF-GON	XTCC	5 × 10⁹	90	0	10
HOF-NEP	ZZCC	5 × 10⁸	19	76	5
HOF-NEP	QLCC	5 × 10⁸	21	74	5
HOF-NEP	TOCC	5 × 10⁸	47	37	16
HOF-NEP	ZZCC	5 × 10⁹	52	36	12
HOF-NEP	QLCC	5 × 10⁹	61	26	13
HOF-NEP	TOCC	5 × 10⁹	81	0	19
HOF-SCH	ZZCC	5 × 10⁸	40	53	6
HOF-SCH	QLCC	5 × 10⁸	42	51	8
HOF-SCH	XTCC	5 × 10⁸	83	5	12
HOF-SCH	ZZCC	5 × 10⁹	78	8	15
HOF-SCH	QLCC	5 × 10⁹	83	6	11
HOF-SCH	XTCC	5 × 10⁹	88	0	11

These data were confirmed by a second method,[0014]³H-thymidine incorporation to assess cell proliferation. In this assay a dose response was measured and compared to untreated control cells. These data supported the BrdU incorporation assay in that the p21 and p16 rAd constructs were able to inhibit³H-thymidine incorporation at lower doses than the no transgene or RB56 rAd constructs. The estimated ED₅₀was about 10 fold lower with the p16 and p21 vector constructs than rAd-null or rAd-RB56. Also similar to the observation in the BrdU incorporation assay, there was a dose-dependent inhibition of 3H-thymidine incorporation with the control rAd-null and rAd-RB56 constructs.

Additional experiments were performed comparing the activity of rAd-p56RB, rAd-p21, rAd-p16 and rAd-p53 in additional human ocular fibroblast cells. Treatment of fibroblasts with rAd expressing p21, p16, or p53 resulted in dramatically reduced percentage of cells incorporating BrdU and conversely retained a higher percentage of cells in G0/G1 phase. Based on the GFP analysis it can be estimated that at a dose of 1×10[0015]⁹PN/ml all of the cells were expressing the respective transgene. This was the second lot of TOCC to be tested and cells responded to this lot similarly as to the first lot. Cells treated with QLCC did not respond differently than cells treated with ZZCC. Cells derived from two different human donors gave comparable responses to each of the constructs. It is therefore expected that TOCB and other vector constructs expressing p21 will be effective at inhibition of S phase entry as demonstrated with TOCC.

TABLE 2


Cell cycle analysis of HOF cells treated with rAd and released
from serum starvation.

	Vector	% g0/g1	% s	% g2/m

HOFtol ut	37	60	4
HOFtol gfcb 1e8	39	57	4
HOFtol ftcb 1e8	47	49	4
HOFtol gfcb 1e9	50	45	5
HOFtol ftcb 1e9	78	14	8
HOFtol qlcc 1e8	38	57	4
HOFtol tocc 1e8	65	27	8
HOFtol xtcc 1e8	69	23	8
HOFtol zzcc 1e8	43	52	5
HOFtol qlcc 1e9	44	50	7
HOFtol tocc 1e9	89	0	11
HOFtol xtcc 1e9	91	2	8
HOFtol zzcc 1e9	47	50	3
HOFcar ut	50	41	9
HOFcar gfcb 1e8	52	41	7
HOFcar ftcb 1e8	62	30	8
HOFcar gfcb 1e9	68	25	7
HOFcar ftcb 1e9	88	2	9
HOFcar qlcc 1e8	50	41	8
HOFcar tocc 1e8	82	9	10
HOFcar xtcc 1e8	80	11	9
HOFcar zzcc 1e8	62	31	6
HOFcar qlcc 1e9	69	23	8
HOFcar tocc 1e9	91	1	9
HOFcar xtcc 1e9	89	1	10
HOFcar zzcc 1e9	71	22	7

In summary, the treatment of these cells with QLCC (rAD-RB56) did not demonstrate an effect greater than the control vector, ZZCC. However, these data demonstrate that treatment of human ocular cells with genes expressing cyclin dependent kinase inhibitors such as p16 or p21 is an effective therapy for treatment of ocular diseases.[0016]

B. Cyclin Dependent Kinase Inhibitors[0017]

The term cyclin dependent kinase inhibitors includes the human, p27kip, p57kip2, p15ink4b, p18ink4c, p19ink4d, p16ink4a and p21sdi wild-type proteins, homologous protein sequences from other organisms, as well as any mutations or truncations thereof which display essentially the same function as the wild-type polynucleotide or protein sequence as well as polynucleotide sequences encoding same.[0018]

1. p16[0019]

The term “p16” is meant to refer to a 156 amino acid polypeptide having the amino acid sequence provided below: (Sequence ID NO: 2):[0020]

MEPAAGSSMEPSADWLATAAARGRVEEVRALLEAGALPNAPNSYGRRPIQVM[0021]

MMGSARVAELLLLHGAEPNCADPATLTRPVHDAAREGFLDTLVVLHRAGARL[0022]

DVRDAWGRLPVDLAEELGHRDVARYLRAAAGGTRGSNHARIDAAEGPSDIPD[0023]

The p16 molecule has been referred to in the literature by the following names: CDKN2A, CDK4I, Hs.1174, MLM, p16, INK4, MTS1, CMM2, CDKN2 and cyclin-dependent kinase inhibitor 2A. The human p16 genomic coding sequence is arranged in three exons on human chromosome 9p. The human cDNA coding sequence is well known in the literature (See Okamoto, A. et al., (1994)[0024]Proc. Natl. Acad. Sci. U.S.A.91 (23), 11045-11049) and is available as GenBank Accession Number L27211

2. p21[0025]

The wild type p21 protein is a 164 amino acid protein having cell regulatory functions. The cDNA and protein sequence are desribed in Smith, et al., U.S. Pat. No. 5,302,706 issued Apr. 12, 1994, the entire teaching of which is herein incorporated by reference. p21 is also known in the scientific literature as p21sdi, p21waf1, p21cip1 and p21pic1. The term p21 also includes polynucleotide sequence encoding the human wild-type protein and homologous sequences from other organisms, as well as any mutations, truncations, or anti-sense nucleic acid which displays essentially the same function as the wild-type polynucleotide or protein sequence.[0026]

C. Delivery Systems[0027]

When the method of treatment to be employed is to introduce a nucleotide sequence encoding p16 or p21, it is possible to incorporate the naked plasmid into a cell However, when delivering the p16 or p21 coding sequence to the target cell, it is preferred that the plasmid be incorporated into a viral or non-viral delivery system.[0028]

1. Non-Viral Delivery Systems[0029]

Examples of non viral delivery systems used to introduce the p16 or p21 gene to the target cell 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 containing the cyclin dependent kinase gene 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.[0030]

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. Additional genes, such as those encoding drug resistance, can be included to allow selection or screening for the presence of the recombinant vector. Such additional genes can include, for example, genes encoding neomycin resistance, multi-drug resistance, thymidine kinase, beta-galactosidase, dihydrofolate reductase (DHFR), and chloramphenicol acetyl transferase.[0031]

The expression plasmid containing the therapeutic gene may be encapsulated in liposomes. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. The delivery of DNA sequences to target cells using liposome carriers is well known in the art. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al.[0032]Ann. Rev. Biophys. Bioeng.9:467 (1980), Szoka, et al. U.S. Pat. No. 4,394,448 issued Jul. 19, 1983, as well as U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, incorporated herein by reference. Liposomes useful in the practice of the present invention may be formed from one or more standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. Examples of such vesicle forming lipids include DC-chol, DOGS, DOTMA, DOPE, DOSPA, DMRIE, DOPC, DOTAP, DORIE, DMRIE-HP, n-spermidine cholesterol carbamate and other cationic lipids as disclosed in U.S. Pat. No. 5,650,096. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. Additional components may be added to the liposome formulation to increase serum half-life such as polyethylene glycol coating (so called “PEG-ylation”) as described in U.S. Pat. Nos. 5,013,556 issued May 7, 1991 and 5,213,804 issued May 25, 1993.

In order to insure efficient delivery of the therapeutic gene to a particular tissue or organ, it may be advantageous to incorporate elements into the non-viral delivery system which facilitate cellular targeting. For example, a lipid encapsulated expression plasmid may incorporate modified surface cell receptor ligands to facilitate targeting. Although a simple liposome formulation may be administered, the liposomes either filled or decorated with a desired composition of the invention of the invention can delivered systemically, or can be directed to a tissue of interest, where the liposomes then deliver the selected therapeutic/immunogenic peptide compositions. Examples of such ligands includes antibodies, monoclonal antibodies, humanized antibodies, single chain antibodies, chimeric antibodies or functional fragments (Fv, Fab, Fab') thereof.[0033]

Alternatively, the DNA constructs of the invention can be linked through a polylysine moiety to a targeting moiety as described in Wu, et al. U.S. Pat. No. 5,166,320 issued Nov. 24, 1992 and Wu, et al, U.S. Pat. No. 5,635,383 issued Jun. 3, 1997, the entire teachings of which are herein incorporated by reference.[0034]

2. Viral Delivery Systems[0035]

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, preferably selected from baculoviridiae, parvoviridiae, picornaviridiae, herpesveridiae, poxviridae or adenoviridiae. Chimeric vectors may also be employed which exploit advantageous elements of each of the parent vector properties (See e.g., Feng, et al. (1997)[0036]Nature Biotechnology15:866-870.) Such viral genomes may be modified by recombinant DNA techniques to include the cyclin dependent kinase inhibitor gene and may be engineered to be replication deficient, conditionally replicating or replication competent. In the preferred practice of the invention, the vectors are replication deficient or conditionally replicating. Preferred vectors are derived from the adenoviral, adeno-associated viral and retroviral genomes. In the most preferred practice of the invention, the vectors are replication incompetent vectors derived from the human adenovirus genome. The transgene may also be under control of a tissue specific promoter region allowing expression of the transgene only in particular cell types.

It may be valuable in some instances to utilize or design vectors to achieve introduction of the exogenous transgene in a particular cell type. Certain vectors exhibit a natural tropism for certain tissue types. For example, vectors derived from the genus herpesviridiae have been shown to have preferential infection of neuronal cells. Examples of recombinantly modified herpesviridiae vectors are disclosed in U.S. Pat. No. 5,328,688 issued Jul. 12, 1994.[0037]

In other instances, to insure efficient delivery of the therapeutic gene to a particular tissue or organ, it may be advantageous to incorporate elements into the viral delivery system which facilitate cellular targeting. Viral envelopes used for packaging the constructs of the invention can be modified by the addition of receptor ligands or antibodies specific for a receptor to permit receptor-mediated endocytosis into specific cells (e.g., WO 93/20221, WO 93/14188; WO 94/06923). In some embodiments of the invention, the DNA constructs of the invention are linked to viral proteins, such as adenovirus particles, to facilitate endocytosis. (See, e.g. Curiel, et al. (1991)[0038]Proc. Natl. Acad. Sci. U.S.A.88:8850-8854). Cell type specificity or cell type targeting may also be achieved in vectors derived from viruses having characteristically broad infectivities by the modification of the viral envelope proteins. For example, cell targeting has been achieved with adenovirus vectors by selective modification of the viral genome knob and fiber coding sequences to achieve expression of modified knob and fiber domains having specific interaction with unique cell surface receptors. Examples of such modifications are described in Wickham, et al. (1997)J. Virol.71(11):8221-8229 (incorporation of RGD peptides into adenoviral fiber proteins); Arnberg, et al.(1997)Virology227:239-244 (modification of adenoviral fiber genes to achieve tropism to the eye and genital tract); Harris and Lemoine (1996)TIG12(10) :400-405; Stevenson, et al. (1997)J. Virol.71(6) :4782-4790; Michael, et al. (1995)Gene Therapy2:660-668 (incorporation of gastrin releasing peptide fragment into adenovirus fiber protein); and Ohno, et al.(1997)Nature Biotechnology15:763-767 (incorporation of Protein A-IgG binding domain into Sindbis virus). Also see U.S. Pat. Nos. 5,721,126 and 5,559,099, herein incorporated by reference. Other methods of cell specific targeting have been achieved by the conjugation of antibodies or antibody fragments to the envelope proteins (see, e.g., Michael, et al. (1993)J. Biol. Chem.268:6866-6869, Watkins, et al. (1997)Gene Therapy4:1004-1012; Douglas, et al. (1996)Nature Biotechnology14: 1574-1578. Alternatively, particular moieties may be conjugated to the viral surface to achieve targeting (See, e.g. Nilson, et al. (1996)Gene Therapy3:280-286 (conjugation of EGF to retroviral proteins)).

Conditionally replicating viral vectors are used to achieve selective expression in particular cell types while avoiding untoward broad spectrum infection. Examples of conditionally replicating vectors are described in Bischoff, et al. (1996)[0039]Science274:373-376; Pennisi, E. (1996)Science274:342-343; Russell, S. J. (1994)Eur. J. of Cancer30A(8) :1165-1171. Additionally, the viral genome may be modified to include inducible promoters which achieve replication or expression of the transgene only under certain conditions. Examples of inducible promoters are known in the scientific literature (See, e.g. Yoshida and Hamada (1997)Biochem. Biophys. Res. Comm.230:426-430; Iida, et al. (1996)J. Virol.70(9) :6054-6059; Hwang, et al.(1997)J. Virol.71(9) :7128-7131; Lee, et al. (1997)Mol. Cell. Biol.17(9) :5097-5105; and Dreher, et al. (1997)J. Biol. Chem.272(46); 29364-29371). The transgene may also be under control of a tissue specific promoter region allowing expression of the transgene only in particular cell types.

In some instances, particularly when employing a conditionally replicating or replication competent vector, it may be desirable to include a suicide gene in the viral vector in addition to the therapeutic gene. A suicide gene is a nucleic acid sequence, the expression of which renders the cell susceptible to killing by external factors or causes a toxic condition in the cell. A well known example of a suicide gene is the thymidine kinase (TK) gene (see e.g. Woo, et al. U.S. Pat. No. 5,631,236 issued May 20, 1997 and Freeman, et al. U.S. Pat. No. 5,601,818 issued Feb. 11, 1997) in which the cells expressing the TK gene product are susceptible to selective killing by the administration of gancyclovir. This provides a “safety valve” to the viral vector delivery system to prevent widespread infection due to the spontaneous generation of fully replication competent viral vectors of broad range infectivity.[0040]

In the preferred practice of the invention, the vector is derived from genus adenoviridiae. Particularly preferred vectors are derived from the human adenovirus type 2 or type 5. Such vectors are preferably are replication deficient by modifications or deletions in the E1a and/or E1b coding regions. Other modifications to the viral genome to achieve particular expression characteristics or permit repeat administration or lower immune response are preferred. More preferred are recombinant adenoviral vectors having complete or partial deletions of the E4 coding region, optionally retaining (or deleting) E4 ORF6 and ORF 6/7. The E3 coding sequence has been demonstrated to be nonessential and may be deleted from adenoviral vectors but is preferably retained. In particular, it is preferred that the promoter operator region of E3 be modified to increase expression of E3 to achieve a more favorable immunological profile for the therapeutic vectors. Most preferred are human adenoviral type 5 vectors containing a DNA sequence encoding a cyclin dependent kinase inhibitor under control of the cytomegalovirus promoter region and the tripartite leader sequence having E3 under control of the CMV promoter and deletion of E4 coding regions while retaining E4 ORF6 and ORF 6/7. In the most preferred practice of the invention as exemplified herein, the cyclin dependent kinase inhibitor is p16 or p21.[0041]

3. Protein Delivery Systems[0042]

Alternatively to viral or non-viral delivery of p16 or p21 coding sequences, the p16 or p21 protein may also be administered directly. When the protein is to be administered directly, it is preferred that the protein be incorporated into a formulation which facilitates or enhances the uptake of the protein into the target ocular cell.[0043]

Formulations may include excipients which stabilize polypeptides, such as methionine (U.S. Pat. No. 5,358,708); osmolytes, lyotropic salts, water-soluble synthetic and natural polymers, surfactants, sulfated polysaccharides, proteins, and buffers (U.S. Pat. No. 5,580,856); fatty acids, amino acids, vitamins (U.S. Pat. No. 5,078,997), and so on.[0044]

D. Pharmaceutical Formulation[0045]

The invention further provides pharmaceutical formulations comprising the therapeutic gene, in a viral or non-viral delivery system for administration. The compositions of the invention will be formulated for administration by manners known in the art acceptable for administration to a mammalian subject, preferably a human. In particular delivery systems may be formulated for intramuscular, intravenous, injectable depot type devices or topical administration.[0046]

The compositions of the invention can also be administered in topical formulations or polymer matrices, hydrogel matrices, polymer implants, or encapsulated formulations to allow slow or sustained release of the compositions. A particularly preferred formulation is a suspension or solution of the delivery system in a topical ocular formulation, such as eye drops.[0047]

1. Carriers[0048]

When the delivery system is formulated as a solution or suspension, the delivery system is in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.[0049]

The compositions may 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, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.[0050]

The concentration of the compositions of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.[0051]

2. Delivery Enhancers[0052]

The pharmaceutical formulations of the invention may optionally include one or more delivery-enhancing agents, The term “delivery enhancing agents” includes agents which facilitate the transfer of the nucleic acid or protein molecule to the target cell. Examples of such delivery enhancing agents detergents, alcohols, glycols, surfactants, bile salts, heparin antagonists, cyclooxygenase inhibitors, hypertonic salt solutions, and acetates. Alcohols include for example the aliphatic alcohols such as ethanol, N-propanol, isopropanol, butyl alcohol, acetyl alcohol. Glycols include glycerine, propyleneglycol, polyethyleneglycol and other low molecular weight glycols such as glycerol and thioglycerol. Acetates such as acetic acid, gluconic acid, and sodium acetate are further examples of delivery-enhancing agents. Hypertonic salt solutions like 1M NaCl are also examples of delivery-enhancing agents. Examples of surfactants are sodium dodecyl sulfate (SDS) and lysolecithin,[0053]polysorbate 80, nonylphenoxypolyoxyethylene, lysophosphatidylcholine, polyethylenglycol 400,polysorbate 80, polyoxyethylene ethers, polyglycol ether surfactants and DMSO. Bile salts such as taurocholate, sodium tauro-deoxycholate, deoxycholate, chenodesoxycholate, glycocholic acid, glycochenodeoxycholic acid and other astringents like silver nitrate may be used. Heparin-antagonists like quaternary amines such as protamine sulfate may also be used. Cyclooxygenase inhibitors such as sodium salicylate, salicylic acid, and non-steroidal antiinflammatory drug (NSAIDS) like indomethacin, naproxen, diclofenac may be used.

Detergents include anionic, cationic, zwitterionic, and nonionic detergents. Exemplary detergents include but are not limited to taurocholate, deoxycholate, taurodeoxycholate, cetylpyridium, benalkonium chloride, ZWITTERGENT®3-14 detergent, CHAPS (3-{(3-Cholamidopropyl)dimethylammoniol}-1-propanesulfonate hydrate, Aldrich), Big CHAP, Deoxy Big CHAP, TRITON®-X-100 detergent, C12E8, Octyl-B-D-Glucopyranoside, PLURONIC®-F68 detergent,[0054]TWEEN® 20 detergent, andTWEEN® 80 detergent (CALBIOCHEM® Biochemicals).

The concentration of the delivery-enhancing agent will depend on a number of factors known to one of ordinary skill in the art such as the particular delivery-enhancing agent being used, the buffer, pH, target tissue or organ and mode of administration. The concentration of the delivery-enhancing agent will be in the range of 1% to 50% (v/v), preferably 10% to 40% (v/v) and most preferably 15% to 30% (v/v). Preferably, the detergent concentration in the final formulation administered to the patient is about 0.5-2X the critical micellization concentration (CMC).[0055]

In order to facilitate the improved gene transfer for nucleic acid formulations comprising commercial Big-CHAP preparations, the concentration of Big CHAP will vary based on its commercial source. When the Big CHAP is sourced from CALBIOCHEM®, it is preferred that the concentration be in a range of 2 to 10 millimolar. More preferred is 4 to 8 millimolar. Most preferred is approximately 7 millimolar.[0056]

When the Big CHAP is sourced from Sigma, it is preferred that the concentration of Big CHAP be in a range of 15 to 35 millimolar. More preferred is 20 to 30 millimolar. Most preferred is approximately 25 millimolar.[0057]

In a further embodiment of the invention, delivery-enhancing agents having Formula I are provided:[0058]
wherein n is an integer from 2-8, X1 is a cholic acid group or deoxycholic acid group, and X2 and X3 are each independently selected from the group consisting of a cholic acid group, a deoxycholic acid group, and a saccharide group. At least one of X2 and X3 is a saccharide group. The saccharide group may be selected from the group consisting of pentose monosaccharide groups, hexose monosaccharide groups, pentose-pentose disaccharide groups, hexose-hexose disaccharide groups, pentose-hexose disaccharide groups, and hexose-pentose disaccharide groups. In one preferred embodiment, the compounds of the present invention have the Formula II:[0059]
wherein X1 and X2 are selected from the group consisting of a cholic acid group and a deoxycholic acid group and X3 is a saccharide group.[0060]
These compounds are preferably used in the range of about 0.002 to 2 mg/ml, more preferably about 0.02 to 2 mg/ml, most preferably about 0.2 to 2 mg/ml in the formulations of the invention. Most preferred is approximately 2 mg/ml.[0061]
Phosphate buffered saline (PBS) is the preferred solubilizing agent for these compounds. However, one of ordinary skill in the art will recognize that certain additional excipients and additives may be desirable to achieve solubility characteristics of these agents for various pharmaceutical formulations. For example, the addition of well known solubilizing agents such as detergents, fatty acid esters, surfactants may be added in appropriate concentrations so as to facilitate the solubilization of the compounds in the various solvents to be employed. When the solvent is PBS, a preferred solubilizing agent is[0062]Tween 80 at a concentration of approximately 0.15%.
These delivery-enhancing compounds may be used alone, in combination with each other, or in combination with another delivery-enhancing agent.[0063]
E. Diseases Amenable to Treatment[0064]
The formulations of the present invention are useful in the treatment of ocular diseases. The term ocular diseases includes but is not limited to diseases of the eye associated with the hyperproliferation cells in the eye. Examples of hyperproliferative disorders include glaucoma surgery failure and proliferative vitreoretinopathy. Other ocular diseases associated with excessive angiogenesis such as age related macular degeneration, retinopathy of prematurity, and diabetic retinopathy may also be treated in accordance with the practice of the present invention.[0065]
Proliferative vitreoretinopathy describes a condition whereby the retina of eye is pulled away from the wall of the eye caused by a hyperproliferation of cells of the retinal pigmented epithelium in response to the injury which caused the retinal detachment. Subsequent contraction of the cellular membrane in the time course of proliferative vitreoretinopathy is a primary cause of failure of retinal reattachment surgery.[0066]
Glaucoma results from an abnormally high pressure in the eye. The commonly accepted surgical treatment for glaucoma is to provide a drain for excess vitreous humor from the eye to relieve the excess pressure. A significant complication of this surgery results from the reocclusion of the drain site from the hyperproliferation of the fibroblasts resulting in repeated surgical intervention.[0067]
The present invention provides methods and compositions for the treatment of “glaucoma surgery failure” by administration of p21 or p16 prevent hyperproliferation in these tissues surrounding the drainage site to prevent the occlusion of the drainage duct.[0068]
F. Methods of Administration[0069]
The therapeutic agents of the invention can be introduced into the tissue of interest in vivo or ex vivo by a variety of methods. In some embodiments of the invention, the therapeutic agent is introduced to cells by such methods as liposome fusion, injection, topically or biolistics. In some embodiments of the invention, the compositions of the invention can be administered directly into the eye, such as to the intraophthalmic artery, subretinal, intravitreal or subconjunctival space. The formulations of the present invention may be administered to subconjunctival and scleral tissues at the time of surgery, preferably in the form of topical drop formulations or with depot devices such as a gel foam (Weck cell) sponge. Additionally, the formulations of the present invention may be administered following surgery, by subconjuctival injection. The formulations of the present invention may be administered in a single dose or in multiple doses. Preferably, when doses are administered, the factor determining the frequency of repeat administration is the duration of transgene expression.[0070]
In the case of glaucoma surgery failure and proliferative vitreoretinopathy, the formulations of the present invention are preferably administered at the time of surgery. In the case of angiogenic diseases such as age related macular degeneration and diabetic retinopathy, the formulations of the present invention are administered over a course of treatment ranging from weeks to years. The preferred routes of administration for the treatment of such diseases include ophthalmic artery administration, subretinal injection, intravitreal injection. Sustained release formulations such as implants would also be appropriate for the treatment of such long term disease indications. These formulations may also be administered in combination with other anti-angiogenic agents.[0071]
Subretinal injections for the treatment of retinal proliferative disease, especially proliferative vitreoretinopathy. This procedure may be performed before, during or after surgery, preferably during the surgical procedure.[0072]
Alternatively, angiogenic or proliferative diseases of the eye (especially the cells of the retinal pigment epithelium) may be treated by the intra-arterial, or subretinal, administration of viral or non-viral formulations of the present invention. In particular, viral or non-viral formulations may be delivered via the opthalmic artery to enhance delivery to the target tissue.[0073]
1. Therapy[0074]
In one embodiment of the invention, a DNA sequence encoding a cyclin dependent kinase inhibitor such as p27kip, p57kip2, p15ink4b, p18ink4c, p19ink4d p16ink4a or p21sdi is administered to the eye. In some embodiments the cyclin dependent kinase inhibitor gene or polypeptide is provided in combination with each other and/or one or more of the following genes or proteins: p53, RB, a suicide gene or gene product.[0075]
In some embodiments of the invention, the formulations of the present invention may also be administered in combination with other chemotherapeutic agents. Examples of chemotherapeutic agents useful in the practice of the present invention includes fluorouracil and mitomycin-C. Additional agents, such corticosteroids, may also be administered in the course therapy.[0076]
In some embodiments of the invention, therapeutic polypeptides of the invention are administered directly to a patient in need of treatment. A “therapeutically effective” dose is an amount of therapeutic gene or polypeptide sufficient to prevent or reduce severity of the pathogeneis of the disease.[0077]
In some embodiments of the invention, the compositions of the invention are administered ex vivo to cells or tissues explanted from a patient, then returned to the patient. Examples of ex vivo administration of therapeutic gene constructs include Arteaga et al. (1996)[0078]Cancer Research56(5) :1098-1103; Nolta et al. (1996)Proc. Natl. Acad. Sci. U.S.A.93(6) :2414-9; Koc et al. (1996)Seminars in Oncology23(1) :46-65; Raper et al. (1996)Annals of Surgery223(2) :116-26; Dalesandro et al. (1996)J. Thorac. Cardi. Surg.11(2) :416-22; and Makarov et al. (1996)Proc. Natl. Acad. Sci. U.S.A.93(1) :402-6.
G. Dosage Ranges[0079]
Therapeutically effective amounts of the pharmaceutical composition comprising a therapeutic gene, such as p16 or p21 in a recombinant viral vector delivery system formulated in a buffer comprising a delivery-enhancing agent will be administered in accord with the teaching of this invention. For example, therapeutically effective amounts of the p16 or p21 cyclin dependent kinase inhibitor gene in a recombinant adenoviral vector formulated in a buffer optionally containing a delivery-enhancing agent are in the range of about 1×10[0080]⁸particles/ml to 1×10¹²particles/ml, more typically about 1×10⁸particles/ml to 5×10¹¹particles/ml, most typically 1×10⁹particles/ml to 1×10¹¹particles/ml (PN/ml).

EXAMPLES

The following examples provide the methodology and results of experiments demonstrating the recombinant adenoviruses (rAd) that express the p16 and/or p21 cell cycle control genes to inhibit proliferation of target cells. As will be apparent to those skilled in the art to which the invention pertains, the present invention may be embodied in forms other than those specifically disclosed above, without departing from the spirit or essential characteristics of the invention. The particular embodiments of the invention described below, are therefore to be considered as illustrative and not restrictive.[0081]

The following examples are intended to illustrate, not limit the scope of this invention. In the following examples, “g” means grams, “ml” means milliliters, “mol” means moles, “EC” means degrees Centigrade, “min.” means minutes, “FBS” means fetal bovine serum, and “PN” specifies particle number. All temperatures are in degrees Centigrade unless otherwise specified.[0082]

Example 1Construction of Recombinant Adenoviral Vectors

Recombinant adenoviruses were constructed to express the p16 and p21 coding sequences following infection of primary ocular fibroblasts. The DNA sequence encoding p16 employed in the construction of the present recombinant adenoviral vectors was (Sequence ID No: 1):[0083]


	ATG GAG CCT TCG GCT GAC TGG CTG GCC ACG GCC GCG

	GCC CGG GGT CGG GTA GAG GAG GTG CGG GCG CTG CTG

	GAG GCG GGG GCG CTG CCC AAC GCA CCG AAT AGT TAC

	GGT CGG AGG CCG ATC CAG GTC ATG ATG ATG GGC AGC

	GCC CGA GTG GCG GAG CTG CTG CTG CTC CAC GGC GCG

	GAG CCC AAC TGC GCC GAC CCC GCC ACT CTC ACC CGA

	CCC GTG CAC GAC GCT GCC CGG GAG GGC TTC CTG GAC

	ACG CTG GTG GTG CTG CAC CGG GCC GGG GCG CGG CTG

	GAC GTG CGC GAT GCC TGG GGC CGT CTG CCC GTG GAC

	CTG GCT GAG GAG CTG GCC CAT CGC GAT GTC GCA CGG

	TAC CTG CGC GCG GCT GCG GGG GGC ACC AGA GGC AGT

	AAC CAT GCC CGC ATA GAT GCC GCG GAA GGT CCC TCA

	GAC ATC CCC GAT TGA

The DNA sequence encoding p21 employed in the construction of the present recombinant adenoviral vectors was as follows (Sequence ID No: 3):[0084]


	ATG TCA GAA CCG GCT GGG GAT GTC CGT CAG AAC CCA

	TGC GGC AGC AAG GCC TGC CGC CGC CTC TTC GGC CCA

	GTG GAC AGC GAG CAG CTG AGC CGC GAC TGT GAT GCG

	CTA ATG GCG GGC TGC ATC CAG GAG GCC CGT GAG CGA

	TGG AAC TTC GAC TTT GTC ACC GAG ACA CCA CTG GAG

	GGT GAC TTC GCC TGG GAG CGT GTG CGG GGC CTT GGC

	CTG CCC AAG CTC TAC CTT CCC ACG GGG CCC CGG CGA

	GGC CGG GAT GAG TTG GGA GGA GGC AGG CGG CCT GGC

	ACC TCA CCT GCT CTG CTG CAG GGG ACA GCA GAG GAA

	GAC CAT GTG GAC CTG TCA CTG TCT TGT ACC CTT GTG

	CCT CGC TCA GGG GAG CAG GCT GAA GGG TCC CCA GGT

	CGA CCT GGA GAC TCT CAG GGT CGA AAA CGG CGG CAG

	ACC AGC ATG ACA GAT TTC TAC CAC TCC AAA CGC CGG

	CTG ATC TTC TCC AAG AGG AAG CCC TAA

The DNA sequence encoding p56-Rb employed in the construction of the present recombinant adenoviral vectors was as follows (Sequence ID No: 5):[0085]


	ATG AAC ACT ATC CAA CAA TTA ATG ATG ATT TTA AAT

	TCT GCA AGT GAT CAA CCT TCA GAA AAT CTG ATT TCC

	TAT TTT AAC AAC TGC ACA GTG AAT CCA AAA GAA AGT

	ATA CTG AAA AGA GTG AAG GAT ATA GGA TAC ATC TTT

	AAA GAG AAA TTT GCT AAA GCT GTG GGA CAG GGT TGT

	GTC GAA ATT GGA TCA CAG CGA TAC AAA CTT GGA GTT

	CGC TTG TAT TAC CGA GTA ATG GAA TCC ATG CTT AAA

	TCA GAA GAA GAA CGA TTA TCC ATT CAA AAT TTT AGC

	AAA CTT CTG AAT GAC AAC ATT TTT CAT ATG TCT TTA

	TTG GCG TGC GCT CTT GAG GTT GTA ATG GCC ACA TAT

	AGC AGA AGT ACA TCT CAG AAT CTT GAT TCT GGA ACA

	GAT TTG TCT TTC CCA TGG ATT CTG AAT GTG CTT AAT

	TTA AAA GCC TTT GAT TTT TAC AAA GTG ATC GAA AGT

	TTT ATC AAA GCA GAA GGC AAC TTG ACA AGA GAA ATG

	ATA AAA CAT TTA GAA CGA TGT GAA CAT CGA ATC ATG

	GAA TCC CTT GCA TGG CTC TCA GAT TCA CCT TTA TTT

	GAT CTT ATT AAA CAA TCA AAG GAC CGA GAA GGA CCA

	ACT GAT CAC CTT GAA TCT GCT TGT CCT CTT AAT CTT

	CCT CTC CAG AAT AAT CAC ACT GCA GCA GAT ATG TAT

	CTT TCT CCT GTA AGA TCT CCA AAG AAA AAA GGT TCA

	ACT ACG CGT GTA AAT TCT ACT GCA AAT GCA GAG ACA

	CAA GCA ACC TCA GCC TTC CAG ACC CAG AAG CCA TTG

	AAA TCT ACC TCT CTT TCA CTG TTT TAT AAA AAA GTG

	TAT CGG CTA GCC TAT CTC CGG CTA AAT ACA CTT TGT

	GAA CGC CTT CTG TCT GAG CAC CCA GAA TTA GAA CAT

	ATC ATC TGG ACC CTT TTC CAG CAC ACC CTG CAG AAT

	GAG TAT GAA CTC ATG AGA GAC AGG CAT TTG GAC CAA

	ATT ATG ATG TGT TCC ATG TAT GGC ATA TGC AAA GTG

	AAG AAT ATA GAC CTT AAA TTC AAA ATC ATT GTA ACA

	GCA TAC AAG GAT CTT CCT CAT GCT GTT CAG GAG ACA

	TTC AAA CGT GTT TTG ATC AAA GAA GAG GAG TAT GAT

	TCT ATT ATA GTA TTC TAT AAC TCG GTC TTC ATG CAG

	AGA CTG AAA ACA AAT ATT TTG CAG TAT GCT TCC ACC

	AGG CCC CCT ACC TTG TCA CCA ATA CCT CAC ATT CCT

	CGA AGC CCT TAC AAG TTT CCT AGT TCA CCC TTA CGG

	ATT CCT GGA GGG AAC ATC TAT ATT TCA CCC CTG AAG

	AGT CCA TAT AAA ATT TCA GAA GGT CTG CCA ACA CCA

	ACA AAA ATG ACT CCA AGA TCA AGA ATC TTA GTA TCA

	ATT GGT GAA TCA TTC GGG ACT TCT GAG AAG TTC CAG

	AAA ATA AAT CAG ATG GTA TGT AAC AGC GAC CGT GTG

	CTC AAA AGA AGT GCT GAA GGA AGC AAC CCT CCT AAA

	CCA CTG AAA AAA CTA CGC TTT GAT ATT GAA GGA TCA

	GAT GAA GCA GAT GGA AGT AAA CAT CTC CCA GGA GAG

	TCC AAA TTT CAG CAG AAA CTG GCA GAA ATG ACT TCT

	ACT CGA ACA CGA ATG CAA AAG CAG AAA ATG AAT GAT

	AGC ATG GAT ACC TCA AAC AAG GAA GAG AAA TGA

A viral vector backbone was created based on a human adenovirus type 5 genome comprising deletions of the E1a and E1b and protein IX gene functions and partial deletion of the E4 coding region (retaining the function of the E4 orf 6 and E4 orf 6/7 genes). The recombinant viral vectors for expression of p16 and p21 were constructed as described in Wang, et al. (1997)[0086]Cancer Research57:351-354. The recombinant viral vectors for expression of p56 was constructed as described in Smith, et al. (1997)Circulation96(6) :1899-1905. This sequence was inserted into the viral vector backbone so as to be under control of the CMV promoter element. The resulting vector was designated QLCC.

Example 2Preparation of Target Cells

Human ocular fibroblast were used in in vitro assays as they are the cellular component causing pathology in glaucoma surgery failure. Primary ocular fibroblast cell lines from three different human sources (HOF-gon, HOF-nep and HOF-sch) were obtained from by Drs. S. Schwartz, D. Farber, and S. Ogueta, Jules Stein Eye Institute, University of California Los Angeles. Cells were synchronized in G1 by incubation in low serum containing HAMs F12/DME media (commercially available from Irvine Scientific, Irvine Calif.) containing 0.5% FBS for a period of 3 days.[0087]

Example 3Evaluation of Activity in a Human Ocular Fibroblasts Model

Human ocular fibroblast cells (prepared in substantial accordance with the teaching of Example 2 above) were infected with rAd constructs (prepared in substantial accordance with the teaching of Example 1 above) in low serum media (F12/DME media containing 0.5% FBS) for 20-24 h with a dose of either 5×10[0088]⁸and 5×10⁹adenoviral particles.

The cells were stimulated to enter the cell cycle by incubation in complete media (10% FBS) and assayed 18-24 hours after stimulation. Response to rAd mediated gene expression was measured by 3H-thymidine incorporation or by BrdU incorporation. Plates were rinsed and re-fed with complete media (10% FBS). 16 h after release BrdU was added for 5 h and cells were harvested and analyzed by FACS for BrdU incorporation (DNA synthesis) and PI staining (DNA content). Cells that had incorporated BrdU were stained using a FITC conjugated mAb to BrdU (commercially available from Becton-Dickinson) and detected by FACS analysis.[0089]

Bivariate analysis on DNA content and BrdU positive cells was used to analyze the data. The response to treatment comparing Ad-null (ZZCC) with rAd-p21 (TOCC) are represented in the two-dimensional plot is shown in FIG. 1. The X-axis (FL2-A) corresponds to propidium iodide (PI) staining or DNA content. The Y-axis (FL1-H) corresponds to FITC staining or BrdU incorporation. Therefore, in the lower right quadrant are cells that did not exit g1 phase during labeling with BrdU. The cells in the arc from the lower left to the upper right quadrants represent cells in the process of BrdU incorporation at the time of harvest. Cells in the upper left quadrant had incorporated BrdU during the labeling period and then divided.[0090]

The percent of cells remaining in G1 was determined from the lower left quadrant. The percent of cells in S phase was determined from the both upper quadrants and therefore represent the percent cells in S-phase during labeling. The cells in the lower right quadrant are labeled G2. Two doses of virus were tested, 5×10[0091]⁸and 5×10⁹particles per ml of media. The from the analysis of human ocular fibroblasts (HOF-) from 3 human donors (GON, NEP and SCH) and data is summarized in Table 1 above. Dose response to rAd treatment measured by tritiated thymidine incorporation shown in FIG. 2.

Example 4Construction of Additional Viral Vectors

A viral vector backbone was created based on a human adenovirus type 5 genome comprising deletions of the E1a and E1b and protein IX gene functions and partial deletion of the E3 coding region. Specifically, the deletions of base pairs 355 to 3325 was used to eliminate E1a and E1b functions, deletion of base pairs 3325 to 4021 was used to eliminate protein IX function and deletions of 28592 to 30470 were used to eliminate E3 functions. See Wills, et al. (1994)[0092]Human Gene Therapy5:1079-1088. The DNA sequence encoding the cytomegalovirus immediate early promoter without the presence of the CMV promoter intron was inserted into the rAd viral genome. This vector without an exogenous transgene was used as control vector and was designated ZZCB.

The green fluorescent protein (GFP) coding sequence was obtained as a NheI to BclI restriction endonuclease cleavage fragment from the vector pEGFP-C1 (commercially available from CloneTech). This sequence was inserted into the XbaI to BamHI site of the resulting vector was designated GFCB.[0093]

The recombinant viral vectors for expression of p53 were constructed as described in Wills, et al., supra.[0094]

Example 5Comparison of p53, p56RB and p21 Activity

Human Ocular Fibroblast (HOF) cells obtained from 2 human donors (HOFtol and HOFcar) were prepared in substantial accordance with Example 2 above. The GFCB and FTCB virus constructions prepared in accordance with the teaching of Example 4 were tested in comparison with the previously constructed p16 and p56RB vectors prepared in accordance with Example 1. Two doses of virus were tested, 1×10[0095]⁸and 1×10⁹particles per ml of media 24 h. Plates were rinsed and re-fed with complete media (10% FBS). 16 h after release BrdU was added for 5 h and cells were harvested and analyzed by FACS for BrdU incorporation (DNA synthesis) and PI staining (DNA content). Controls included untreated cells released (ut 10). In addition to cell cycle analysis the percent of gfcb treated cells expressing GFP (green fluorescent protein) transgene was determined by FACS immediately after cells were harvested. The percent of cells that incorporated BrdU during the labeling period (16-21h post-release) is reported as %s.

Results[0096]

Serum starved human ocular fibroblast were treated with rAd for 24 h then rAd was washed. Cells were released from quiescence by feeding cells with complete media and cells were harvested 21 h later for analysis of transgene expression by FACS. Treatment with 1×10[0097]⁸pn/ml resulted in 62% and 79% positive cells in HOF-TOL and HOF-CAR respectively. A dose of 1×10⁹resulted in 100% of the cells expressing GFP transgene for both HOF-TOL and HOF-CAR. Cell cycle analysis of cells is tabulated in Table 2 above.

As will be apparent to those skilled in the art to which the invention pertains, the present invention may be embodied in forms other than those specifically disclosed above, without departing from the spirit or essential characteristics of the invention. The particular embodiments of the invention described above, are, therefore to be considered as illustrative and not restrictive. The scope of the present invention is as set forth in the appended claims rather than being limited to the examples contained in the foregoing description.[0098]