WO2000047218A1 - Regulation of photoreceptor degeneration - Google Patents

Abstract

The present invention provides a method for regulating or preventing retinal degeneration in a patient, wherein the degeneration is apoptotic, and wherein the method comprises administering to a patient experiencing retinal degeneration or having a genetic or disease-based predisposition for retinal degeneration, a therapeutically effective amount of a compound comprising an apoptosis-modulating polypeptide or nucleic acid encoding same. In accordance with the present method, the modulating compound regulates or controls apoptosis of a cell or population of cells in the retina of the patient, wherein the preferred cell or cell population comprises photoreceptor cells of the retina.

Description

Regulation of Photoreceptor Degeneration

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 60/1 19, 792, filed February 1 1 , 1999, which is incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to methods for modulating apoptosis of a cell or population of cells in the eye, particularly for modifying or regulating photoreceptor degeneration in the retina.

BACKGROUND OF THE INVENTION

Retinal degenerations are a major cause of blindness and there is a need for effective treatments (Adler, Arch Ophthalmol 114:79 (1996)). It is estimated that one in 3,500 to 4,000 people is affected by hereditary retinal degenerations (Boughman et al, Am J Hum Genet 32:223-235 (1980)). Age-related macular dystrophy is the leading cause of irreversible blindness in the population above age 50. Mutations of several genes are believed to be responsible for hereditary retinal degenerations, a group of disorders collectively called retinitis pigmentosa (RP). The major cause of retinitis pigmentosa is a slow and progressive loss of photoreceptors.

An intensive search is currently underway to identify genes responsible for macular degeneration. The introduction of mutant rhodopsin genes into the genomes of experimental animals has successfully reconstructed retinitis pigmentosa in mice (Olsson et al, Neuron 9:815-830 (1992)), pigs (Petters et al, Nature Biotech 15:965-970 (1997)), and rats (Steinberg et al, Invest Ophthalmol Vis Sci 37:S698 (1996); Steinberg et al,

Invest Ophthalmol Vis Sci 38:S226 (1997)). The transgenic animals have been shown to effectively mimick the phenotypes of retinitis pigmentosa as observed in humans, thereby providing animal models representative of the human condition.

Studies have shown that photoreceptor degeneration proceeds through an apoptotic process. DNA fragmentation, a hallmark of apoptosis, has been demonstrated in degenerating photoreceptors (Chang et al, Neuron 11 :595-605 (1993); Portera-Cailliau et al, Proc Natl Acad Sci 91 :974-97812 (1994)). Apoptosis is an active, rather than a passive process, resulting in cell suicide. Diseases and conditions in which apoptosis has been implicated fall into two categories, those in which there is increased cell survival (i.e., apoptosis is reduced) and those in which there is excessive cell death (i.e., apoptosis is increased).

Since apoptosis, or programmed cell death, was originally shown to be a cell suicide process with distinct morphological characteristics (Wyllie, J Pathol 153:313-316 (1987)), enormous progress has been made in unraveling the components of the death mechanism. However, limited information about the molecular mechanism of photoreceptor degeneration in vertebrates is currently available

Pioneering studies performed on the nematode C. elegans identified a complement of genes related to cell death, including ced-3, ced-4, and ced-9. The first identified mammalian homologue of CED-3 was the interleukin-1 beta-converting enzyme (ICE) (Yuan et al., Cell 75:641-652 (1993)). Eventually, a search by a number of different investigators for ICE-related proteins revealed an entire family of proteases in mammals, comprising at least twelve members of the caspase family (cystein-containing aspartate- specific protease) (Thornberry et al, Science 281 :1312-1316 (1998); Cohen, Biochem J 326:1-16 (1997)). Among them, activation of caspase-3 has been shown to participate in apoptosis (Thornberry et al, 1998), especially in neurons (Kuida et al, Nature 384:368- 372 (1996); Armstrong et al, J Neurosci 17:553-562 (1997); Yakovlev et al, J Neurosci 17:7415-7424 (1997); Namura et al, J Neurosci 18:3659-3668 (1998; Cheng et al, JClin Invest 101 :1992-1999 (1998)).

Moreover, in the retina, caspase-3 has been associated with ganglion cell death after optic nerve transection (Kermer et al, J Neurosci 18:4656-4662 (1998)). However, it remained unknown whether the cascade of reactions responsible for cell death, once started could be arrested, i.e., whether rescue would be possible for the rods and cones responsible for vision.

When caspase-3 was first cloned and found to share similarities with ICE, it was named CPP32 (Fernandes-Alnemri et al, JBiol Chem 269:30761-30764 (1994)). Subsequently, two other groups independently identified it and named it Yama (Tewari et al, Cell 81 :801-809 (1995)) and apopain (Nicholson et al, Nature 376:37-43 (1995)). Caspases are synthesized as inactive proenzymes. Activation of caspases requires cleavage at specific aspartate sites to release one large and one small subunit to form active proteases. For example, enzymatically-activated caspase-3 comprises two subunits. Pro- caspase-3 (32 kD) is cleaved at Asp-28-Ser-29 and Asp-175-Ser-176 to generate a large subunit of 17 kD and a small subunit of 12 kD (Nicholson et al., Nature 376:37-43 (1995)).

A tetrapeptide aldehyde, Ac-YVAD-CHO, has been synthesized, based on the YVHD recognition sequence in pro-interleukin-lbeta for ICE. It is a potent reversible inhibitor of ICE, and its closest homologue is caspase-4. Ac-YVAD-CHO is one of the most selective ICE inhibitors, with an affinity for ICE that is six orders of magnitude higher than for caspase-3 (Fernandes-Alnemri et al, Cancer Res 55:6045-6052 (1995); Margolin et al, J Biol Chem 272:7223-7228 (1997)).

A similar tetrapeptide aldehyde, Ac-DEVD-CHO, was synthesized based on the PARP cleavage site. It is a reversible inhibitor of caspase-3, and also inhibits caspase-7, caspase-1 and caspase-4 (Fernandes-Alnemri et al, 1995; Margolin et al, 1997). Irreversible caspase inhibitors z-VAD-fmk and z-DEVD-fmk were designed to have increased cell permeability. Both have been used in cellular systems and intact animals (Livingston, J Cell Biochem 64:19-26 (1997)).

Since the discovery of mutations in the rhodopsin gene in humans with retinitis pigmentosa (Farrar et al, Genomics 8:35-40 (1990); Dryja et al, Nature 343:364-366 (1990a); Dryja et al, New EngJMed 323:1302-1307 (1990b)), various mutations of the visual pigment have been introduced into the genomes of animals as representative models of these disorders (Olsson et al, 1992; Petters et al, 1997; Steinberg et al, 1996; 1997). These transgenic animals, particularly transgenic rats with a rhodopsin mutation, are valuable tools for evaluating retinal degeneration. However, prior to the present invention research in the art had failed to identify the causative agent(s) in the death of photoreceptors. Thus, a need remained for a better understanding of photoreceptor degeneration, its cause(s) and, more importantly, its prevention or treatment. SUMMARY OF THE INVENTION

Studies have shown that photoreceptor death during degeneration is apoptotic. Yet, prior to the present invention, little was known about the molecular mechanism of cell death in photoreceptors. In response to this need for a better understanding of the mechanism(s) of photoreceptor cell death, the present invention provides a novel mechanism, e.g., a caspase-3 -dependent mechanism, which plays a critical role in the demise of photoreceptors in the retina. Thus, it is an object of this invention to characterize and understand the effect of caspase inhibition, particularly caspase-3 inhibition in the retina of the eye, thereby permitting the identification of methods of preventing or treating apoptotic retinal degeneration and progressive death of photoreceptors typically seen in human patients, e.g., in patients suffering from retinitis pigmentosa or a genetic predisposition for retinal degeneration.

Discovery in the present invention of the role of caspase inhibition in regulating cell death in photoreceptors in selected transgenic animal models, e.g., S334ter-3 rats, led to the novel conclusion that a common caspase-3 -dependent mechanism is responsible for photoreceptor apoptosis in a variety of different types of retinal degenerations. Three lines of evidence confirmed the findings in the present invention that activation of caspase-3 is a key step in the cell death process in photoreceptors. First, a dramatic increase in the activity of caspase-3 -like proteases was observed in model animals to coincide with the peak of cell death. Second, an increase in the pl2 subunit of the actived form of caspase-3 was found in correlation with an increase in caspase-3 activity. Third, when administered into the eye, a peptide inhibitor of caspase-3, z-DEVD-fmk, protected a population of photoreceptor cells from death.

In accordance with the present invention, different apoptotic stimuli, such as mutations in photoreceptor-specific genes, chronic stress, ischemia, continuous light exposure, excitotoxicity or certain antibodies are known to activate the caspase-3 - dependant death machinery, causing photoreceptor apoptosis.

The present invention provides a method of regulating retinal degeneration in a patient, wherein degeneration is apoptotic, and wherein the method comprises administering to a patient experiencing retinal degeneration a therapeutically effective amount of a compound comprising an apoptosis-modulating compound. Further provided is the method wherein wherein the modulating compound regulates or controls apoptosis of a cell or population of cells in the retina of the patient. Particularly provided is a method, wherein the cell or cell population comprises photoreceptor cells of the retina.

Also provided is a method, wherein apoptosis is inhibited or reduced following the administration of the modulating compound the modulating compound; and a method, wherein apoptosis is enhanced or increased following the administration of the modulating compound.

In addition the method is provided, wherein the modulating compound comprises either an apoptosis inhibiting compound or an apoptosis enhancing compound. The present invention also provides a method of preventing retinal degeneration in a patient, wherein the degeneration is apoptotic, and wherein the method comprises administering to a patient with a genetic or disease-based predisposition for retinal degeneration an effective amount of a compound comprising an apoptosis-modulating compound. Also provided is the method wherein the modulating compound prevents apoptosis of a cell or population of cells in the retina of the patient. Particularly provided is the method, wherein the cell or cell population comprises photoreceptor cells of the retina.

Further provided is a method, wherein apoptosis is prevented by the administration of the modulating compound. In accordance with the provided methods are embodiments wherein the target of the apoptosis modulating or preventing compound comprises an activated caspase, preferably and activated caspase-3. The apoptosis inhibiting compounds comprise z- DEVD-fmk or z-VAD fmk. The patient is a vertebrate, preferably a mammal, more preferably a human. Thus, it is an object of the present invention to provide a method of blocking or inhibiting activation of the apoptotic polypeptide, such a caspase, more particularly caspase-3, to treat or protect the photoreceptor cells, at least to some extent, from degeneration.

The invention, including these and other provisions, will be more fully understood from the following detailed description of preferred embodiments, drawings and examples, all of which are intended to be for illustrative purposes only, and not intended in any way to limit the invention.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 depicts photoreceptor degeneration in S334ter-3 rats models, as seen by light microscopy (superior region), of plastic-embedded sections of their retinas. Sections are depicted from the S334ter-3 rats at postnatal day (PD) 6 in FIG. 1 A; at PD 8 in FIG.

IB; at PD 10 in FIG. IC; at PD 1 1 in FIG. ID; at PD 12 in FIG. IE; at PD 14 in FIG. IF; at PD 16 in FIG. 1G; at PD 18 in FIG. 1H; and at PD 20 in FIG. II. The ONL is indicated in each panel by a white bar. Pyknotic nuclei in FIGs. IB and IC are indicated by white arrow heads. Sections were stained with toluidine blue. Scale bar= 20 μm.

FIG. 2 depicts photoreceptors in normal Sprague-Dawley rats during postnatal development, as seen by light microscopy (superior region), of plastic-embedded sections of their retinas. Sections of retinas from the Sprague-Dawley rats are depicted at PD 6 in FIG. 2A, at PD 8 in FIG. 2B, at PD 10 in FIG 2C, at PD 12 in FIG. 2D, at PD 16 in FIG.

2E, and at PD 20 in FIG. 2F. The ONL is indicated in each panel by a white bar. Sections were stained with toluidine blue. Scale bar = 20 μm.

FIG. 3 depicts DNA fragmentation in degenerating photoreceptors of the S334ter-3 rats. Cryosections (10 μm) from the retina of a PD 11 S334ter-3 rat (FIG. 3 A) and from a wild-type Sprague-Dawley rat (FIG. 3B) were TUNEL labeled with fluorescein to visualize the DNA fragmentation. In the S334ter-3 rat (A), numerous cells in the ONL are labeled. The distribution of labeled cells is notably denser in the proximal, than in the distal ONL. No labeled cells are found in the ONL of the control retina (FIG. 3B). A few cells in the INL are also labeled in the control (FIG. 3B) and the transgenic rat (FIG. 3A). The following abbreviations are used in FIGs. 3A and 3B: rpe = retinal pigment epithelium; onl = outer nuclear layer; inl = inner nuclear layer; and gc = ganglion cell layer. Scale bar = 50 μm.

FIG. 4 graphically depicts for comparison, the activities of the caspase-3 -like and caspase- 1 -like proteases in the retina. Activities of caspase-3 -like or caspase- 1 -like proteases were determined by measuring the cleavage of Ac-DEVD-AMC or Ac-YVAD-

AMC, respectively. Retinas were collected at PD 8, 9, 10, 11, 12, 13, 14, and 16 from S334ter-3 or normal Sprague-Dawley rats. Intensity of fluorescence was measured on a Perkin-Elmer LS-50B Luminescence Spectrometer at 30 second intervals for 900 seconds. The rate of accumulation of fluorescence was calculated as the activity of a given enzyme. Data are presented as Mean ± SD (n=3). *** p<0.00\, * p<0.0\ (Student's t test). FIG. 5 depicts two immunoblots. FIG. 5 A shows the pi 2 subunit band of activated caspase-3 in the retina during photoreceptor degeneration in S334ter-3 rats at PD 8 (P8), PD 10-12 (PI 0-12), PD 14 (PI 4) and PD 16 (PI 6). FIG. 5B shows the 12 kD subunit band of activated caspase-3 in the retina of PD 10 (P10) and 12 (PI 2) age matched Sprague- Dawley rats (SD). FIG. 6 depicts the protective effect of z-DEVD-fmk on the photoreceptors of the test animals. Plastic-embedded sections of retinas from a normal rat (FIG. 6A), and a S334ter-3 rat treated with DMSO (FIG. 6B) or z-DEVD-fmk (FIG. 6C) at PD 20 were examined by light microscopy. The normal retina has well-developed outer and inner segments. The ONL has 12-13 rows of nuclei (FIG. 6A). In the DMSO treated retina, the ONL has only one row of nuclei and inner segments become short stumps (FIG. 6B). In the z-DEVD-fmk treated retina from the same animal (FIG. 6C), the ONL has 4-5 rows of nuclei, and the inner segments are seen to be better preserved, although still shortened and disorganized. Some dislocated cells are seen in the subretinal space next to the RPE. Sections were stained with toluidine blue. The following abbreviations are used in FIGs 3A: RPE = retinal pigment epithelium; OS = outer segment; IS, = inner segment; ONL = outer nuclear layer; OPL = outer plexiform layer; and INL = inner nuclear layer. Scale bar = 20 μm.

FIG. 7 depicts an immunoblot of the pl2 subunit band of activated caspase-3 after intraocular injection of the caspase-3 inhibitor, z-DEVD-fmk (50 μg) in the left eyes of PD 9 S334ter-3 rats (Tg, Ihb +), and vehicle only into their right eyes (Tg, Ihb -). Retinas were collected at PD 11. Retinas of age-matched Sprague-Dawley (SD) rats served as controls (SD, Ihb -).

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a method of modulating, regulating or controlling cellular degeneration or apoptosis in the retina of the eye, in particularly in modulating, regulating or controlling programmed cell death of a cell or selected population of cells, as exemplified by the photoreceptor cells in the retina. In particular, the present invention establishes a role of caspasex activity in such selected populations of cells, more specifically of activated caspase-3 in photoreceptor cell degeneration. The effect is exemplified below in an animal model, specifically in a line of transgenic rats that carry a murine rhodopsin mutation, S334ter (Steinberg et al., 1996; 1997), wherein a clear relationship has been found between the increase in Ac-DEVD-AMC cleavage, the quantitative increase in the 12 kD subunit of caspase-3, and the course of photoreceptor degeneration. Upon activation of the caspase protease, these animals underwent a degenerative course leading to the death of more than 90% of all photoreceptors in less than two weeks. At the peak rate of photoreceptor death, the loss of photoreceptors reached 3-4 rows of nuclei in the ONL in one day, or close to 30% of the photoreceptors in the entire retina (FIG. 1). A significant increase in caspase-3 activation was seen during the rapid phase of photoreceptor death, in which more than 50% of the photoreceptors in these animals died in just two days, postnatal day (PD) 1 1 and 12.

It is estimated that in the trangenic rat retina, one row of nuclei in the ONL represents about one million photoreceptors. Thus, at the peak of degeneration, the death rate is approximately 3-4 million rods per day. The massive photoreceptor cell death seen in these animals provided an opportunity to investigate and understand the biochemistry of photoreceptor degeneration.

Moreover, intraocular administration of a recognized caspase inhibitor, e.g., the irreversible caspase-3 inhibitor z-DEVD-fmk, was shown to offer protection to the photoreceptors from degeneration, providing further evidence that caspase-3 activation promotes or is a cause of the death of photoreceptors. Details of these findings will be presented below, demonstrating that the present invention provides a common mechanism for photoreceptor degeneration, which will permit a greater understanding of photoreceptor cell death, while at the same time providing the basis for a new approach to therapies for retinal degenerative disorders Activation of caspase-3 is important for the initiation of apoptosis in many neuronal classes. In a cell free system, caspase-3 is activated by Apaf-1 in the presence of three factors: pro-caspase-9, cytochrome c. and dATP. Evidence shows that Apaf-1 forms a complex with pro-caspase-9 through a caspase-recruitment domain in Apaf-1 and a same caspase-recruitment domain in the long pro-domain of caspase-9. The binding between Apaf-1 and pro-caspase-9 occurs only in the presence of cytochrome c and dATP. The formation of Apaf-1 /caspase-9 complex leads to the activation of caspase-9, which in turn activates caspase-3 (Li et al, Cell 91 :479-489 (1997)). Moreover, murine caspase-1 1 has been shown to be the upstream regulator of caspase-1 in the ICE pathway (Wang et al, J Biol Chem 271 :20580-20587 (1996); Wang et al, Cell 92:501-509 (1998)).

For instance, in caspase-3 knock-out mice, there is a selective defect in cell death in the central nervous system that leads to a doubling of brain size (Kuida et al., 1996). This finding suggests the critical role of caspase-3 in morphogenetic cell death in the brain. See, e.g., published European patent application 842,665, which examines apoptotic proteases and their enzymes in numerous cell populations ranging from cancer through baldness, but which fails to even suggest an effect on retinal cells or a population of photoreceptor cells .

Supporting findings have been reported subsequent to the present invention by Tezel & Wax, Invest Ophthal & Visual Sci 40:2660-2667 (Oct. 1999).

"Apoptosis" is recognized in the art as an active process of gene-directed cellular self-destruction. The process is also referred to as "programmed cell death." By an "apoptosis-reducing amount of apoptotic polypeptide" is meant an amount of polypeptide sufficient to prevent, inhibit, reduce or neutralize the percentage of cells undergoing apoptosis in a given population of cells known to undergo apoptosis, such photoreceptor cells, relative to a population of untreated control cells of the same type. Preferably, the reduction in apoptosis is at least 10%, more preferably at least 50%, and even more preferably, at least 5-fold, relative to an untreated control cell of the same type. An "apoptosis-inhibiting amount" or "apoptosis blocking amount" would have the same meaning.

For the purposes of this invention, the terms "protein," "peptide" and "polypeptide" are used interchangeably, the definition of which would be recognized by one in the art. An apoptotic protein, or peptide, or polypeptide refers to a compound, such as a neurochemical, which upon activation in the eye causes or accelerates apoptosis of a cell or cell population, such as a population of photoreceptor cells. A "cell population" is a group of the same or similar cells in a particular environment, wherein the cells react in substantially the same way to stimuli.

An "apoptosis-enhancing amount of apoptotic polypeptide" is meant to be an amount of polypeptide sufficient to increase, enhance or stimulate the percentage of cells undergoing apoptosis in a given population of cells known to undergo apoptosis, such as photoreceptor cells, relative to a population of untreated control cells of the same type. An "apoptosis-enhancing amount" refers to a polypeptide effecting an increase, enhancement or stimulation in apoptosis, preferably by at least 10%, more preferably at least 50%, and even more preferably, at least 5-fold, relative to an untreated control cell of the same type. "Apoptosis-associated" genes (and polypeptides) are associated with modulation, regulation or control (enhancement or reduction, respectively) of the apoptosis phenomena in a cell. By "apoptosis-regulating gene" ("apoptotic gene" or "apoptosis gene") is meant a gene (or nucleic acid sequence or fragment or derivative thereof), which modulates, regulates or controls (either enhances or inhibits, respectively) the process of apoptosis or encodes a peptide product that regulates the process of apoptosis. By "apoptosis- regulating polypeptide" is meant a polypeptide (or protein or peptide, or active fragment, derivative, or analog thereof), which modulates, regulates or controls (either enhances or inhibits, respectively) the process of apoptosis or encodes a peptide product that regulates the process of apoptosis. The terms "apoptosis-regulating" peptide and "apoptosis- modulating" peptide, polypeptide or compound are used interchangeably with the terms apoptotic polypeptide or apoptosis polypeptide, as defined above.

The present invention identifies compositions and provides methods for modulating, regulating or controlling apoptosis in a suitable cell or a population of suitable cells, by introducing into the cell or cells an effective amount of protein or polypeptide (an apoptosis regulating polpeptide), or the nucleic acid molecule encoding same, in an amount sufficient to effect measurable modulation, regulation or control of the apoptotic process as compared with a comparable control cell or cell population. In the preferred embodiment, apoptosis is reduced, inhibited or prevented, wherein the introduced protein or polypeptide, or the nucleic acid molecule encoding same, has sufficient anti-apoptosis biological activity to effect a reduction, inhibition or prevention of apoptosis. In this case, reduction, neutralization, inhibition and the like refers to a measurable decrease or reduction in apoptosis or apopototic activity, preferably by at least 10%, more preferably at least 50%, and even more preferably, at least 5-fold, relative to an untreated control cell of the same type. In one preferred embodiment, the introduced apoptosis-regulating polypeptide or apoptosis-regulating gene has the ability to modulate, regulate or control activation of at least one member in the caspase signaling cascade, effecting reduction, inhibition or prevention of apoptosis of, e.g., the photoreceptor cells of the retina.

In accordance with the present invention, down regulation of apoptosis activity enhances survival of photoreceptor cells of the retina. Evaluation of the magnitude and direction of this effect indicates that reduction of the relevant caspase activity, whether accomplished downstream, e.g., as in the present invention, or whether accomplished upstream, e.g., by preventing caspase activation and/or by inhibiting the caspase activators would be equally efficacious.

It is important to note, when reduction, inhibition or prevention of apoptosis is the desired modulation in the present invention, that the method of this invention inhibits apoptosis even in the presence of apoptotic-inducing agents. Accordingly, this method provides an improvement over prior art methods wherein apoptosis can be inhibited by interfering with the induction pathway at the level of ligand induction, such as by providing antibodies or anti-ligand antibodies to interfere with the binding of the ligand to its cell surface receptor. However, this invention can be combined with the use of such prior art methods to inhibit apoptosis.

The terms "preventing," "blocking," "neutralizing," "inhibiting" and the like are intended to interchangeably mean a reduction in cell death or a prolongation in the survival time of the cell. They also are intended to mean a diminution in the appearance of, or a delay in, the appearance of moφhological and/or biochemical changes normally associated with apoptosis. When the delay or reduction in activation or apoptotic activity is permanent, the modulating method is considered to be preventative. Thus, this invention provides compositions and methods to increase survival time and/or survival rate of a cell or population of cells which, absent the use of the method, would normally be expected to die. Accordingly, it also provides compositions and methods to prevent or treat diseases or pathological conditions associated with unwanted cell death in the eye or retina of a subject.

Similarly, the terms "enhancing" or "stimulating" are interchangeably intended to mean an increase in cell death or a reduction in the survival time of the cell. They also are intended to mean an increase in the appearance or a acceleration in the appearance of morphological and/or biochemical changes normally associated with apoptosis, when such apoptosis is desired. In this case, enhancement, stimulation, increase or the like refers to a measurable increase or enhancement in apoptosis or apopototic activity, preferably by at least 10%, more preferably at least 50%, and even more preferably, at least 5-fold, relative to an untreated control cell of the same type.

"Modulation," "regulation" or "control" are terms interchangeably intended to broadly refer to any change in the apoptosis of the selected cell or cell population, or the biological changes associated with apoptosis, regardless of whether the effect is inhibition or enhancement, as defined above. Thus, an "apoptosis-modulating amount" of a compound, peptide, protein, polypeptide, or the nucleic acid molecule or gene encoding same, would mean a sufficient or effective amount to enhance or reduce (depending on the desired effect), apoptosis of a cell or cell population in the eye or retina.

Suitable cells or "target cells" for the practice of the present invention include, but are not limited to, cells in which apoptosis activity or apoptotic effect per se, or on other cells or cell populations, is modulated (inhibited or enhanced) by one or more endogenous or exogenous agents, conditions or circumstances, e.g., mutations, chronic stress, or continuous exposure to light. In one embodiment of the present invention, these cells constitutively and inducibly express receptors for activated caspase, specifically for activated caspase-3. The "target polypeptide," or simply the "target" refers to the protease or similar compound, which is directly or indirectly causing retinal degeneration or apoptosis of a cell or cell population in the eye, preferably a population of photoreceptor cells. To be effective in the present invention, the apoptosis-regulating or -modulating polypeptide or nucleic acid molecule expression product modulates the apoptotic or proteolytic activity of the target (either increasing or reducing the activity, as desired). Thus, it is the target polypeptide upon which the method of the present invention functions. In one embodiment of the present invention an "apoptosis-modulating amount" of a protein, peptide or polypeptide, or active analog, fragment or derivative thereof, is administered to the eye of the patient to modulate (enhance or reduce, respectively) the apopotosis of a cell or cell population in the eye. The preferred protein, peptide or polypeptide is administered as an isolated preparation.

As used herein, the term "an isolated preparation" describes a compound, e.g., a protein or polypeptide, which has been separated from components which naturally accompany it. Typically, a compound is isolated when at least 10%, more preferably at least 20%), more preferably at least 50%, more preferably at least 60%, more preferably at least 75%, more preferably at least 90%, and most preferably at least 99%> of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column chromatography, gel electrophoresis or HPLC analysis. A compound, e.g., a protein, is also considered to be isolated when it is essentially free of naturally associated components, or when it is separated from the native contaminants which accompany it in its natural state.

In a preferred embodiment, the purified preparation of the isolated polypeptide having the ability to enhance or reduce apoptosis in the cell or cell population in the eye, preferably to modulate, regulate or control apoptosis of the photoreceptor cells of the retina, is at least about 60 amino acids in length. More preferably, it is at least 120 amino acids, even more preferably, at least 300 amino acids, yet more preferably, at least 500 amino acids, and even more preferably, at least 700 amino acids in length. In an additional embodiment the polypeptide encodes the full length protein or a regulated version thereof. The present invention also provides for using analogs of proteins or peptides capable of binding to, neutralizing or inhibiting an apoptosis polypeptide, e.g., an activated caspase, such as activated caspase-3. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both.

For example, conservative amino acid changes may be made which, although they alter the primary sequence of the protein or peptide, do not normally alter its function.

Conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine.

Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences, which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

Also included are polypeptides, which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids. The peptides of the invention are not limited to products of any of the specific exemplary processes listed herein.

In addition to substantially full length polypeptides, the present invention provides for enzymatically active fragments of the polypeptides. For, example, a caspase-3 inhibitor or an active fragment thereof is enzymatically active if it binds to, neutralizes or reduces the proteolytic or apoptotic activity of activated caspase-3 in the eye, for example in a manner similar to that of the z-V AD-fmk or z-DEVD-fmk, as described in the examples below. If the presence of the inhibitor, active fragment, derivative or homologue thereof is sufficient to block or prevent activation of the protease or target polypeptide, or to prevent expression of proteolytic or apoptotic activity by the protease or target, the apoptotic modulating compound is an apoptosis-preventing compound.

As used herein, the term "fragment," as applied to a polypeptide, will ordinarily be at least about twenty contiguous amino acids, typically at least about fifty contiguous amino acids, more typically at least about seventy continuous amino acids, usually at least about one hundred contiguous amino acids, preferably at least about five hundred continuous amino acids, more preferably at least about one thousand contiguous amino acids, and most preferably at least about one thousand two hundred to about one thousand nine hundred or more contiguous amino acids in length. An active fragment functions in much the same manner, and expresses essentially the same phenotype as the full-length expression product from which it came.

A mutant, derivative, homologue or fragment of the subject polypeptide or gene is, therefore also one in which selected domains in the related protein or gene share significant homology (at least about 40% homology under at least moderately stringent conditions), with the same domains in the preferred embodiment of the present invention. It will be appreciated that the definition of such an agent or compound may be applied to either amino acid or nucleic acid sequences, and encompasses those amino acid or nucleic acid molecules having at least about 40% homology, in any of the described domains contained therein.

In addition, when the term "homology" is used herein to refer to the domains of the apopotosis-modulating polypeptides or proteins, it should be construed to be applied to homology at both the encoding nucleic acid and the amino acid levels. Significant homology between similar domains in such nucleic acids or their protein products is considered to be at least about 40%), preferably, the homology between domains is at least about 50%), more preferably, at least about 60%, even more preferably, at least about 70%), even more preferably, at least about 80%, yet more preferably, at least about 90% and most preferably, the homology between similar domains is about 99%, of the polypeptides per se or of the nucleic acid encoding the expression products thereof. Homology is determinable by one of ordinary skill by simple side-by-side comparisons of the two sequences, or by any known either manual or electronic technique or method in the art. As applied to this invention, an agonist of the apoptotic compound is a different compound that mimics the action of the apoptosis modulator on the retinal tissue, thereby enhancing or increasing the apoptotic activity. An antagonist of the apoptotic neurochemical is a compound that inhibits, neutralizes, reduces, opposes, prevents or blocks action of the apoptotic compound on the retinal tissue. Both agonists and antagonists of the apoptotic compound are encompassed with the present invention as modulators of the apoptotic process in the selected cell or cell population. In another embodiment of the present invention an "apoptosis-modulating amount" of a gene or nucleic acid molecule, or active analog, fragment or derivative thereof, and which encodes an expression product, which modulates (enhance or reduce, respectively) the apopotosis of a cell or cell population in the eye, is administered to the eye of the patient. The preferred gene or nucleic acid molecule is administered as an isolated or purified preparation. As used herein, the term "an isolated preparation" or a "purified preparation" describes a compound, e.g., a gene or nucleic acid molecule, which has been separated from components which naturally accompany it. Typically, a compound is isolated when at least 10%, more preferably at least 20%), more preferably at least 50%o, more preferably at least 60%), more preferably at least 75%), more preferably at least 90%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method.. A compound, e.g., a gene or nucleic acid molecule, is also considered to be isolated when it is essentially free of naturally associated components or when it is separated from the native contaminants which accompany it in its natural state. According to the present invention, preferably, the isolated nucleic acid encoding the biologically active gene, or nucleic acid molecule, or fragment thereof, as defined above, is full length or of sufficient length to encode a regulated or active apoptosis modulator, such as an inhibitor of activated caspase-3. In one embodiment the nucleic acid is at least about 200 nucleotides in length. More preferably, it is at least 400 nucleotides, even more preferably, at least 600 nucleotides, yet more preferably, at least 800 nucleotides, and even more preferably, at least 1000 nucleotides in length.

The DNA sequence encoding the apoptosis modulator of the present invention can be duplicated using a DNA sequencer and methods well known to those of skill in the art. For example, the sequence can be chemically replicated using PCR (Perkin-Elmer) which in combination with the synthesis of oligonucleotides, allows easy reproduction of DNA sequences. A DNA segment of up to approximately 6000 base pairs in length can be amplified exponentially starting from as little as a single gene copy by means of polymerase chain reaction (PCR). In this technique, a denatured DNA sample is incubated with two oligonucleotide primers that direct the DNA polymerase-dependent synthesis of new complementary strands. Multiple cycles of synthesis each afford an approximate doubling of the amount of target sequence. Each cycle is controlled by varying the temperature to permit denaturation of the DNA strands, annealing the primers, and synthesizing new DNA strands. The use of a thermostable DNA polymerase eliminates the necessity of adding new enzyme for each cycle, thus permitting fully automated DNA amplification. Twenty-five amplification cycles increase the amount of target sequence by approximately 10⁶ -fold. The PCR technology is the subject matter of U.S. Pat. Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202.

The nucleic acid can be duplicated using a host-vector system and traditional cloning techniques with appropriate replication vectors. A "host-vector system" refers to host cells, which have been transfected with appropriate vectors using recombinant DNA techniques. The vectors and methods disclosed herein are suitable for use in host cells over a wide range of eukcaryotic organisms. This invention also encompasses cells transformed with the novel replication and expression vectors described herein.

Indeed, a gene encoding the modulating nucleic acid, such as the nucleic acid sequence encoding an activated caspase-3 inhibitor, can be duplicated in many replication vectors, such as the vaccinia virus as described in Pickup et al., al., Proc. Natl. Acad. Sci. 83:7698-7702 (1986)), and isolated using methods described in Sambrook et al, 1989. The selected gene, made and isolated using the above methods, can be directly inserted into an expression vector, such pcDNA3 (Invitrogen) and inserted into a suitable animal or mammalian cell such as a guinea pig cell, a rabbit cell, a simian cell, a mouse, a rat or a human cell.

In the practice of one embodiment of this invention, the modulating gene, such as the purified nucleic acid molecule encoding e.g., an apoptosis inhibitor, such as a caspase inhibitor or an inhibitor of activated caspase-3, is introduced into the cell and expressed. Thus, cell death is aborted, prevented, reduced or inhibited. A variety of different gene transfer approaches are available to deliver the gene or gene fragment encoding the modulating nucleic acid into a target cell, cells or tissues. Among these are several non- viral vectors, including DNA/liposome complexes, and targeted viral protein DNA complexes. To enhance delivery to a cell, the nucleic acid or proteins of this invention can be conjugated to antibodies, or binding fragments thereof, which bind cell surface antigens. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention. This invention also provides the targeting complexes for use in the methods disclosed herein.

Non-viral techniques may include, but are not limited to colloidal dispersion, asialorosonucoid-polylysine conjugation, or, less preferably, microinjection under surgical conditions

The nucleic acid molecule encoding the modulator also can be incoφorated into a "heterologous DNA" or "expression vector" for the practice of this invention. The term "heterologous DNA" is intended to encompass a DNA polymer such as viral vector DNA, plasmid vector DNA or cosmid vector DNA. Prior to insertion into the vector, it is in the form of a separate fragment, or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in "substantially pure form," i.e., free of contaminating endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the segment and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector. As used herein, "recombinant" is intended to mean that a particular DNA sequence is the product of various combination of cloning, restriction, and ligation steps resulting in a construct having a sequence distinguishable from homologous sequences found in natural systems. Recombinant sequences can be assembled from cloned fragments and short oligonucleotides linkers, or from a series of oligonucleotides. As noted above, one means to introduce the nucleic acid into the cell of interest is by the use of a recombinant expression vector. "Recombinant expression vector" is intended to include vectors, which are capable of expressing DNA sequences contained therein, where such sequences are operatively linked to other sequences capable of effecting their expression. It is implied, although not always explicitly stated, that these expression vectors must be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA.

Accordingly, "expression vector" is given a functional definition, and any DNA sequence which is capable of effecting expression of a specified DNA sequence disposed therein is included in this term as it is applied to the specified sequence. Suitable expression vectors include viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids and others. Adenoviral vectors are a particularly effective means for introducing genes into tissues in vivo because of their high level of expression and efficient transformation of cells both in vitro and in vivo.

Expression levels of the gene or nucleotide sequence inside a target cell are capable of providing gene expression for a duration and in an amount such that the nucleotide product therein is capable of providing a therapeutically effective amount of gene product or in such an amount as to provide a functional biological effect on the target cell.

By "gene delivery" is meant transportation of a composition or formulation into contact with a target cell so that the composition or formulation is capable of being taken up by means of a cytotic process (i.e., pinocytosis, endocytosis, phagocytosis, ect.) into the interior or cytoplasmic side of the outermost cell membrane of the target cell where it will subsequently be transported into the nucleus of the cell in such functional condition that it is capable of achieving gene expression.

By "gene expression" is meant the process, after delivery into a target cell, by which a nucleotide sequence undergoes successful transcription and translation such that detectable levels of the delivered nucleotide sequence are expressed in an amount and over a time period that a functional biological effect is achieved. "Gene therapy" encompasses the terms gene delivery and gene expression. Moreover, treatment by any gene therapy approach may be combined with other, more traditional therapies.

Replication-incompetent retroviral vectors also can be used with this invention. As used herein, the term "retroviral" includes, but is not limited to, a vector or delivery vehicle, many of which are known in the art, having the ability to selectively target and introduce the coding sequence into dividing cells. As used herein, the terms "replication- incompetent" is defined as the inability to produce viral proteins, precluding spread of the vector in the infected host cell. As would be understood by those of skill in the art, the nucleic acid introduced with a retroviral vector would be a ribonucleic acid (RNA). The methodology of using replication-incompetent retroviruses for retroviral -mediated gene

By "patient" or "subject" is meant any vertebrate or animal, preferably a mammal, most preferably a human, with apoptosis or degeneration of at least some portion of the photoreceptor cells of the eye, or with a suseptibility to, or genetic predisposition to such apoptosis or cellular degeneration in the retina. Thus, included within the present invention are animal, bird, reptile or veterinary patients or subjects, the intended meaning of which is self-evident. Despite notable differences in anatomy between the eyes of primates and those of rodents or birds, photoreceptor death among other animals closely resembles that in the primate, as shown by studies made on rats, chicks and young monkeys. All are encompassed by the methods of the present invention. A line of transgenic rats that carry a murine rhodopsin mutation, S334ter

(Steinberg et al., 1996; 1997), have proven to be an accepted animal model demonstrating the retinal degeneration phenotype of human patients exhibiting photoreceptor degeneration of, e.g., retinitis pigmentosa or a genetic predisposition for retinal degeneration. In accordance with the present invention, preliminary experiments have indicated that caspase-3 is involved in photoreceptor death in other animal models as well, (unpublished results). Thus, the therapeutic effect of administering an effective amount of an apoptosis-regulating polpeptide to modulate, regulate or control apoptosis of a cell or population of cells, such as the photoreceptor cells of the eye, is effective across and among species, and need not be limited to only the demonstrated effect in rhodopsin mutants.

In the ordinary visual function of the eye of an animal, light forming an image passes through the lens and is received by the retina, a neural tissue embryologically related to the brain. The retina transmits this information to the optic nerve, which sends it on to the brain. Without wishing to be bound to any particular theory or mode of action, it is believed that the compositions and methods of the invention act on proteases or other polypeptide apoptotic compounds, which directly or indirectly cause cellular degeneration or death of a cell type or population of cells in the retina of the eye. Retinal neurochemicals ( . e. , neuro-active chemical compounds) are key ingredients in the vision process. Specifically, light forming images are sensed by the light receptors, the rods and cones, of the retina. These photoreceptors act as transducers, changing light energy into electrical and/or chemical signals. Thus, it is a pu ose of this invention to preserve or restore the transducing capability of the photoreceptor cells in the retina of a patient by modulating the apoptotic activity of the compound or polypeptide affecting the retinal cells. In the regular process of transmitting the image information to the brain, retinal nerve cells release neurochemicals to pass information to adjacent retinal cells as parts of networks in the retina leading to the formulation and qualities of the signals that are then transmitted to the brain via the optic nerve. The caspase proteases are activated in a signaling cascade, much like the blood clotting cascade, wherein each protease resides in an inactive pro-form until it is acted upon by another enzyme or protease in the cascade, which then cleaves the pro-enzyme into its active form. Once activated, that enzyme or protease then signals, acts upon or activates the next polypeptide in the signaling cascade, etc., until apoptosis is triggered in the target cell population. Therefore, if a link in the signaling cascade is blocked or sufficiently inhibited as to be ineffective in the activation of the next polypeptide in the signaling cascade, apoptosis will be reduced, inhibited or prevented. Similarly, if the signaling cascade has been blocked, but enhanced apoptosis is desired, this can be achieved by replacing the missing or ineffective polypeptide, as in the blood clotting cascade. See, e.g., Wilson, Exp Eye Res 69:255-266 (1999), published after the present invention, and herein incorporated by reference.

Although the present invention exemplifies directly modulating (inhibiting) the apoptotic activity of the proteolytic compound (e.g., caspase-3), the target may also include compounds acting upon or causing the activation of the proteolytic compound. Thus, the link in the cascade sequence leading to activation may be broken, and cellular degeneration modulated, although the action is indirect, rather than directly on the target proteolytic or apoptotic compound. The bcl-2 family of proteins represent adapters needed for the activation of caspases, and exemplify an apoptosis-target activation-assisting polypeptide that could be modulated to effect an indirect change in the activation of the target compound or polypeptide. The bcl-2 family includes both apoptosis-promoting (e.g., bax and bad) and apoptosis-inhibiting (e.g., bcl-2 and bcl-xL) members For example, when retinal cell apoptosis was induced, Tezel & Wax, 1999 reported that bcl-2 expression decreased and bax expression increased.

In additional embodiments of the invention, the agents which modulate apoptosis in the cell population in the eye, such as those which act upon caspase-3, specifically on activated caspase-3, or its proteolytic derivatives, include neutralizing monoclonal antibodies to the apoptosis-associated compound or apoptosis polpeptides, e.g., activated caspase-3, or proteolytic derivatives, and fragments of said antibodies. These include the binding domain, as well as active derivatives of said antibodies, and fragments and allelic forms thereof. Antibodies may be raised against the apoptosis associated compound or apoptosis polpeptide by following standard procedures available in the art for the generation of antibodies which are described, for example, in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.).

By "purified antibody" is meant an antibody which is at least 60%>, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%_>, and most preferably at least 99%, by weight, antibody, e.g., a caspase-3-specific antibody. A purified antibody may be obtained, for example, by affinity chromatography using recombinantly-produced protein or conserved motif peptides and standard techniques. The invention can employ not only intact monoclonal or polyclonal antibodies, but also an immunologically-active antibody fragment, such as a Fab' or (Fab')2 fragment, or a genetically engineered Fv fragment (Ladner et al, U.S. Pat. No. 4,946,788). By "specifically binds" is meant an antibody, which recognizes and binds a specified protein, but which does not substantially recognize and bind other molecules in a sample, e.g., a biological sample, which naturally includes protein.

The above-described antibodies, since they are of use in therapy, may be "humanized" using for recombinant DNA technology. Specifically, the tail region of a non-human antibody in accordance with the invention may be exchanged for that of a human antibody. For a more complete humanization, the framework regions of the non- human antibody may be exchanged for human framework regions as is known in the art. These exchange processes may be carried out at the DNA level using recombinant techniques. Such procedures have the effect of increasing the characteristic features of the antibodies, which are specific to human proteins and thus reducing the possible occurrence of harmful hypersensitivity reactions.

Neutralizing the target polypeptide, by e.g., anti-caspase-3, would also be of use to inhibit the apoptotic or proteolytic compound, e.g., caspase-3, and thereby to modulate apoptosis in a range of non-transformed as well as transformed cell types; such an approach could be useful in the treatment of, for example, autoimmune diseases which arise from a defect in apoptosis. Such diseases may be of genetic origin, and patients may be treated because they carry a genetic predisposition to a disease, but do not yet exhibit symptoms. Diabetic retinopathy and glaucomatous optic neuropathy, including retinal ganglion cell apoptosis, exemplify such disease states.

In another embodiment of the present invention, the apoptosis-modulating or apoptosis-regulating compound is designed by mimetics for synthetic production. The designing of mimetics to a pharmaceutically active compound, such as the apoptosis- associated polypeptide, is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g., peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Similarly, the acidic environment of the vitreous of the eye can also degrade therapeutic peptide compounds before they have had an opportunity to perform. Mimetic design, synthesis and testing is generally used to avoid randomly screening large number of molecules for a target property. There are several steps commonly taken in the design of a mimetic from a compound having a given target property. First, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, such as caspase-3, this can be done by systematically varying the amino acid residues in the peptide, e.g., by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its "pharmacophore."

Once the pharmacophore has been found, its structure is modeled to according its physical properties, e.g., stereochemistry, bonding, size and/or charge, using data from a range of sources, eg spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.

In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modeled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this the design of the mimetic. A template molecule is then selected, onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit such properties. Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing, and which can then be administered to the eye or retinal cells to modulate apoptosis of a retinal cell or population of cells.

The invention further features an isolated preparation of a nucleic acid, which is antisense in orientation to a portion or all of nucleic acid molecule encoding the apoptosis- associated polypeptide in the eye (the target gene), and which is sufficient to bind to, inhibit, reduce or prevent the activation of or activity in the target. The antisense nucleic acid must be of sufficient length as to inhibit expression of the target gene of interest. The actual length of the nucleic acid may vary based upon a number of factors, including e.g., the nature of the target gene, and the region targeted within the gene. Typically, such a preparation will be at least about 15 contiguous nucleotides, more typically at least 50 or even more than 50 contiguous nucleotides in length. As used herein, a sequence of nucleic acid is considered to be antisense when the sequence being expressed is complementary to, and essentially identical to the non-coding DNA strand of the nucleic acid molecule encoding the apoptosis-associated polypeptide in the eye, but which does not encode the target apoptosis-associated polypeptide per se as found in the eye. "Complementary" refers to the subunit complementarity between two nucleic acids, e.g. , two DNA molecules. When a nucleotide position in both molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are said to be complementary to each other. Thus two nucleic acids are complementary when a substantial number (at least 50%>) of the corresponding positions in each of the molecules are occupied by nucleotides which normally base-pair with each other (e.g., A:T and G:C nucleotide pairs). The invention further provides an assay for determining agents, which bind to. neutralize or inhibit an apoptotic polypeptide in the eye, such as activated caspase-3, thereby reducing, modulating or preventing apoptosis in a cell or cell population in the eye. such as photoreceptor cells. Such assay comprises administering an agent under test to e.g.. photoreceptor cells at low cell density, and monitoring apoptotic death in the cells. A further assay for determining agents, which bind to, neutralize or inhibit an apoptotic polypeptide in the eye, such as activated caspase-3, thereby reducing, inhibiting or preventing apoptosis in photoreceptor cells according to the invention comprises administering the agent under test to photoreceptor cells at high cell density, and monitoring apoptotic death in said cells. Agents may thus be selected which effectively reduce, inhibit or prevent apoptosis. The agents which are thus selected, are also intended to be a part of the present invention

The invention further provides an assay for determining compounds or compositions, which enhance, stimulate or increase the apoptotic polypeptide in the eye, such as activation of caspase-3, thereby stimulating, increasing or enhancing apoptosis in a cell or cell population in the eye, such as photoreceptor cells. Such assay comprises administering an compound or composition under test to photoreceptor cells at low cell density, and monitoring apoptotic death in the cells. A further assay for determining compounds or compositions, which will enhance, stimulate or increase an apoptotic polypeptide in the eye, such as activated caspase-3, thereby stimulating, increasing or enhancing apoptosis in photoreceptor cells according to the invention, comprises administering the agent under test to photoreceptor cells at high cell density, and monitoring apoptotic death in said cells. Modulating compounds or polypeptides may thus be selected which effectively stimulate, increase or enhance apoptosis. The compounds or compositions thus selected are also intended to be a part of the present invention.

Novel compounds or compounds identified using these assays form yet a further aspect of the invention, as does the use of known reagents identified using these assays in the modulation or control of apoptosis in a cell or cell population in the eye, such as a population of photoreceptor cells. In accordance with the present invention, the apoptosis modulating polypeptides or compounds, as described above, when used in therapy, for example in the treatment of retinal degeneration, such as an anti-caspase-3 compound, can be administered to a patient either alone or as part of a pharmaceutically acceptable composition, and optionally with a preservative, diluent, and the like. They may further be administered in the form of a composition in combination with a pharmaceutically acceptable carrier or excipient, and which may further comprise pharmaceutically acceptable salts. Examples of such carriers include both liquid and solid carriers, such as water or saline, various buffer solutions, cyclodextrins and other protective carriers or complexes, glycerol and prodrug formulations. Combinations may include other pharmaceutical agents, such as dopaminergics, adrenergics, cholinergics or growth factors for administration to the eye. The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. In fact, combined therapeutics may advantageously confer selectivity in situations within the eye, in which cells other than fully-differentiated photoreceptor cells may be encountered, e.g., iris or cornea.

Various methods of administration of the therapeutic or preventative agent can be used, following known formulations and procedures. Although ocular administration is described herein and is generally preferred, systemic or targeted administration may also be employed under suitable circumstances. The compositions can be administered to vertebrates, including patients, such as humans and animals (or other veterinary patients), parenterally (e.g., intraocularly by injection), or locally by powders, ointments, salve or drops. Similar methods of administration may be used when the preferred embodiment of the invention introduces apoptosis-enhancing compositions

Compounds or compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, saline, buffered saline, dextrose, ethanol, glycerol, polyols, and the like, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions may also contain adjuvants, such as preserving, wetting, emulsifying, and dispensing agents.

Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. Prolonged absoφtion of the injectable pharmaceutical form can be brought about by the use of agents delaying absoφtion, for example, aluminum monostearate and gelatin.

Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Repetition rates for dosing can be readily estimated based upon measured residence times and concentrations of the drug in bodily fluids or tissues. The actual dosage of the modulating compound or poylpeptide, such as a caspase- 3 inhibitor, caspase-3 -binding or apoptosis-neutralizing protein, depends on a number of factors, including the size and health of the individual patient, but, generally, from 0.01 μg to 100 g per kg of body weight, inclusive are administered per day to an adult in any pharmaceutically-acceptable formulation. Optimum dosages may vary depending on the relative potency of individual polypeptides, and can generally be estimated based on EC₅₀s found to be effective in either in vitro or in vivo animal models. Doses may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Dosages of oligonucleotides or nucleic acid molecules for gene therapy will be understood by one of ordinary skill in the field of gene therapy.

Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein doses are maintained at levels ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years. In a preferred embodiment of the invention, the method of treatment involves direct targeting (e.g., topical) application of the therapeutic agent to the eye or more specifically to the vitreous of the eye, preferably to the photoreceptor cells. It may also involve the use of targeting systems, such as antibody or cell specific ligands introduced into the eye. Targeting may be desirable for a variety of reasons; for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells. The amount of modulating compound or polypeptide, such as a caspase-3- inhibiting or neutralizing polypeptide or analog, to be administered in the present method, will vary according the species of the individual, the amount of orbital tissue present in the eye and the distance the peptide will have to diffuse from the site of administration, and the penetrability of the eye, as well as the targeted cells.

Instead of administering these modulating polypeptides or compounds directly, they can also be produced in the target cells by expression from an encoding gene introduced into the cells, e.g., in a viral vector. The vector could be targeted to the specific cells to be treated, or it could contain regulatory elements, which are switched on more or less selectively by the target cells.

Alternatively, the modulating polypeptide or compound of the present method could be administered in a precursor form, for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. This former type of approach involves targeting the activating or modulating polypeptide or compound to the cells by conjugation to a cell-specific antibody, while the latter involves producing the activating or modulating agent, e.g., an enzyme, in a vector by expression from encoding DNA in a viral vector.

In one embodiment, the invention provides a method of treating a patient having or at risk of having a genetic or chronic disease, wherein the disease has an etiology associated with degeneration of the photoreceptors or another apoptitic condition in the eye, wherein the method comprises administering to the patient a therapeutically effective amount of a composition which modulates the expression of the apoptosis gene or polypeptide in the eye or affecting the photoreceptor cells, such that the disease is ameliorated. Diseases subject to treatment by the inventive method include, but are not limited to, diabetic retinopathy and glaucomatous optic neuropathy, including retinal ganglion cell apoptosis.

By "therapeutically effective" as used herein, is meant that amount of composition that is of sufficient quantity to ameliorate the cause of or effect of the retinal or macular degeneration or death of the photoreceptor cells. By "ameliorate" is meant a lessening or reduction or prophylactic prevention of the detrimental effect of the disorder in the patient receiving the therapy. The subject of the invention is preferably a human, however, it can be envisioned that any animal with a apoptotic condition of the eye or destruction of the retina or photoreceptor cells can be treated in the method of the present invention. It is further envisioned that under selected circumstances, controlled stimulation or enhancement of apoptosis of the target activity may be desired, in which case the term "ameliorate" will be directed to the increased or enhanced expression of the apoptosis- regulating gene or the apoptotic protein or peptide, e.g., activated caspase-3.

The compositions used in the methods of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional compatible pharmaceutically-active materials such as, e.g., antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the invention.

The present invention is further described in the following examples. These examples are not to be construed as limiting the scope of the appended claims. Reference is made to standard textbooks of molecular biology that contain definitions and methods and means for carrying out basic techniques, encompassed by the present invention. See, for example, Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982) and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989) and the various references cited therein. These references and the previously cited publications, as well as those cited in the ensuing Examples, are expressly incoφorated by reference into this specification.

EXAMPLES

All experiments were carried out using heterozygous S334ter-3 rats. These rats were produced by mating homozygous breeders of the line 3 of transgenic rats that carry a murine rhodopsin mutant S334ter (S334ter-3), with wild type Sprague-Dawley rats. The homozygous breeders were from the laboratory of M.M. LaVail of the University of California, San Francisco. Experimental controls were age-matched Sprague-Dawley rats. Animals were kept in a 12: 12-hr lightidark cycle at an in-cage illumination of < 10 foot- candles (1 ft-c=10.76 lux). The in-cage temperature was maintained at 20-22°C. The animals were killed by CO₂ overdose, immediately followed by vascular perfusion with mixed aldehydes (LaVail, Exp Eye Res 21 : 167-192 (1975)), herein incoφorated by reference. Their eyes were excised and embedded in an Epon/Araldite mixture, sectioned at 1 μm thickness to display the entire retina along the vertical meridian (LaVail, 1975). Retinal sections were examined by light microscopy. All experiments were repeated 3 times to verify the consistency of the results.

Example 1 - Photoreceptor Degeneration in Heterozygous S334ter-3 rats

The progressive photoreceptor degeneration in the retinas of heterozygous S334ter- 3 rats follows a pattern described by LaVail (M.M. LaVail, personal communications). Twenty-seven (27) transgenic animals from three litters were used for experiments.

Intraocular injections were given directly into the vitreous of the eye by 32 gauge needles. Representative light micrographs of heterozygous S334ter-3 rat retinas from postnatal day (PD) 6 to 20 are shown in FIG. 1. The degeneration was shown from examinations of the S334ter-3 rats at postnatal day (PD) 6 in FIG. 1A; at PD 8 in FIG. IB; at PD 10 in FIG. IC; at PD 1 1 in FIG. ID; at PD 12 in FIG. IE; at PD 14 in FIG. IF; at PD 16 in FIG. 1G; at PD 18 in FIG. 1H; and at PD 20 in FIG. II. The ONL is indicated in each panel by a white bar. Pyknotic nuclei in FIGs. IB and IC are indicated by white arrow heads.

Twelve (12) wild-type Sprague-Dawley rats were used as control animals for experiments, the results of which are shown in FIG. 2. The overall appearance of the retina of a transgenic animal at PD 6 (FIG. 1A) was similar to an age-matched control (FIG. 2 A), differing slightly in stage of postnatal development. Typically, 30-50 pyknotic nuclei were seen in the outer nuclear layer (ONL). However, by comparison in the retina of a PD 8 S334ter-3 rat, the loss of photoreceptors became evident at PD 8, by which point only one or two pyknotic nuclei were seen in the control ONL (not shown). Pyknotic nuclei were found mainly in the proximal half of the ONL at PD 8 (FIG. IB), PD 10 (FIG. I C). and 1 1 (FIG. ID). In the trangenic animals, some photoreceptor nuclei were found to be displaced to the subretinal space (FIGs. IB- IE).

By PD 10, the retinas of the transgenic rats were seen to be very different from that of the control: Not only were many pyknotic nuclei found in the ONL in the transgenic rats, but the inner segment of photoreceptors had become disorganized (FIG. IC). The peak of photoreceptor death occurred at PD 11 and 12. By PD 1 1, a substantial loss of photoreceptors had occurred (FIG. ID). By PD 12, the ONL in the transgenic animals contained only 6-7 rows of nuclei, in marked contrast to 12-13 rows in normal controls (FIG. 2C). The inner segments were even more disorganized, as shown in FIG. IE.

By PD 14, the rows of nuclei in the ONL had decreased to only 3-4 in the S334ter- 3 rats and the remaining inner segments were little more than short stumps (FIG. IF). The nuclei in the ONL had further declined to 2-3 rows at PD 16. Finally by PD 20, the photoreceptors had degenerated in the transgenic animals to the extent that only one row of nuclei remained with residual inner segments (FIGs. IG, IH, and II). No outer segments ever developed in the heterozygous S334ter-3 rats.

For comparison, FIG. 2 shows representative light micrographs of normal Sprague- Dawley rat retinas from PD 6 to 20. At PD 6, the overall moφhological appearance of Sprague-Dawley retina (FIG. 2A) was similar to that of the transgenic rats (FIG. 1A). By PD 8, the ONL (FIG. 2B) had thickened and the inner segments had become well organized (FIG. 2B). By PD 10 the outer segments of rod photoreceptors had begun to develop in the control animals (FIG. 2C). At PD 10, the rod outer segments had developed to about half of the length of those in mature retina (FIG. 2D). The photoreceptors in PD 16 (FIG. 2E) and PD 20 (FIG. 2F) control animals were substantially the same as those found in mature normal animals. Only one or two residual pyknotic nuclei were observed in the ONL of any of the control animals from PD 10 through PD 20 (not shown).

Histological analyses showed that in the early stages of photoreceptor degeneration in the S334ter-3 rats (PD 8 through 1 1), the pyknotic nuclei were distributed mainly in the proximal half of the ONL (FIGs. IB, IC, and ID). As the degeneration progressed in the transgenic animals, they were seen in the distal ONL as well (FIG. ID and IE). The displacement of photoreceptor nuclei to the subretinal space (FIG. 1) is likely to be a phenomenon related to the degenerative process, as it is observed only in the retinas of transgenic animals undergoing photoreceptor degeneration.

During degeneration, some photoreceptor nuclei became dislocated in the transgenic animals to the subretinal space next to the retinal pigment epithelial (RPE) cells (FIGs. IB- IE). However, no corresponding dislocation was observed in control animals (FIG. 2).

To determine whether the photoreceptor death in the S334ter-3 rats was apoptotic, the TUNEL (terminal dUTP nick end labeling) method (Gavrieli et al, J Cell Biol 1 19:493-501 (1992)) was used to detect DNA fragmentation. Eyes were removed from 4% paraformaldehyde perfused animals (three transgenic and three control rats), cryoprotected with 20%) sucrose, frozen in Tissue-Tek OCT compound (Miles Inc., Elkhart, IN) in powdered dry ice, and stored at -80°C. Cryosections of 10 μm were cut through the entire retina along the vertical meridian, and thaw-mounted onto Super Frost Plus glass slides (Fisher Scientific, Pittsburgh, PA). TUNEL labeling was carried out using an Apoptosis Detection System (Fluorescein) (Promega , Madison, WI), according to manufacturer's instructions.

As shown in FIG. 3, many cells in the ONL of the PD 11 transgenic rats were TUNEL-positive, indicating significant DNA fragmentation. The distribution of TUNEL- labeled cells was denser in the proximal, than in the distal, ONL (FIG. 3A). This is consistent with the distribution of pyknotic nuclei shown in FIG. ID.

In the age-matched control animals (FIG. 3B), no TUNEL-labeled cells were found in the ONL. In the INL (inner nuclear layer), a few cells were TUNEL positive in both the control (FIG. 3B) and transgenic animals (FIG. 3A). The reason why cells in the proximal ONL are more susceptible to degeneration is not clear, although it is possible that these cells are simply more advanced in development than those in the distal portion of the ONL, and therefore die sooner.

Example 2 - Activation and Measurement of Caspase Activity

To confirm the effect of caspase- 3 on the human retina, representative transgenic animal models were injected intraocular ly with the caspase-3 inhibitor, z-DEVD-fmk

(purchased from Enzyme Systems Products, Dublin, CA). The animals were sacrificed as described and the retinas were dissected, snap-frozen in powdered dry ice, and stored at - 80°C. Total protein was obtained by homogenizing the pooled retinas in a lysis buffer that contained 100 mM HEPES (pH 7.5), 10% sucrose, 1 mM EDTA, 20 mM EGTA, 0.2% CHAPS, 10 mM dithiothreitol, 10 μg leupeptin, 2 μg/ml pepstatin, 2 μg/ml aprotinin, 1 mM PMSF, and 0.03% digitonin. The amount of total protein of each sample was determined by the BCA protein assay (Pierce, Rockford, IL), and the samples were stored at -80°C.

Total protein (100 μg) from each sample was electrophoresed on polyacrylamide gels and transferred to nitrocellular membranes (Bio-Rad Labs, Hercules, CA). Blots were stained briefly with Ponceau S for visual inspection of transfer efficiency. Then, immunoblotting analyses were performed using polyclonal antibodies against the 12 kD subunit of the active form of caspase-3 (Santa Cruz Biotechnology, Santa Cruz, CA). Signals were visualized using an ECL kit (Amersham, Arlington Heights, IL), and recorded on Hyperfilm (Amersham). Caspase activity (caspase-3 or caspase-1) was assessed by measuring the cleavage of the fluorogenic substrates, Ac-DEVD-AMC or Ac-YVAD-AMC, respectively, using a Luminescence Spectrometer (LS-50B, Perkin-Elmer, Norwalk, CT). Cleavage of Ac- DEVD-AMC or Ac-YVAD-AMC was measured for 900 seconds for each sample of 100 μg total protein (accumulation of fluorescence was linear for at least 2 hr). The rate of fluorescence accumulation was calculated as the activity of a given enzyme. Experiments were repeated three times with samples from three litters of transgenic or control animals. Retinal samples were collected from transgenic and age-matched Sprague-Dawley control animals at PD 8, 9, 10, 1 1, 12, 13, 14, and 16. A substantial increase in Ac- DEVD-AMC cleavage was observed in transgenic animals at PD 11, 12, 13, and 14. By PD 11 and PD 12, the cleavage was close to 6-fold that of control level at PD 11 and PD 12, then declined to about 2-fold that of the control level at PD 13 and 14 (FIG. 4A).

Recognizing that Ac-DEVD-AMC is also cleaved by caspase-1, the possibility that the observed caspase activity was that of caspase-1, rather than that of caspase-3, was eliminated by using a caspase-1 specific fluorogenic substrate, Ac-YVAD-AMC. Substrates with a sequence of YVAD, based on the recognition sequence for caspase-1 NVHD, have high selectivity for caspase-1 over caspase-3. In fact, the tetrapeptide aldehyde Ac-YVAD-CHO is highly selective, possessing an affinity for caspase-1 that is six orders of magnitude higher than for caspase-3 (Fernandes-Alnemri et al., 1995; Margolin et al, 1997). As shown in FIG. 4B, no significant alteration in Ac-YVAD-AMC cleavage was observed from PD 8 to PD16 in the S334ter-3 rats, as compared with the control animals, indicating that caspase-1 is not significantly activated during photoreceptor degeneration.

As previously noted, caspases-3 is synthesized as a 32 kD inactive proenzyme, which upon activation is cleaved at Asp-28-Ser-29 and Asp-175-Ser-176 to generate a subunit of 17 kD, and a smaller one of 12 kD upon activation. Accordingly, to assess the amount of active caspase-3 during photoreceptor degeneration, the amount of the 12 kD subunit was measured in the retinas of the S334ter-3 rats by immunoblotting analysis. FIG 5 A displays the relative amounts of the 12 kD subunit during photoreceptor degeneration. As shown, significant increases were found at PD 10, 11, and 12 in the transgenic rats, coincident with the rapid phase of photoreceptor loss in their retinas. Moreover, the amount of the 12 kD subunit in the transgenic rats during the rapid degeneration phase was much higher than the levels seen in the age-matched Sprague-Dawley control rats (FIG. 5B).

Example 3 - Protection of Photoreceptors by Caspase Inhibitor, z-DEVD-fmk To test the importance of caspase-3 activity to photoreceptor death in the S334ter-3 rats, the subject animals were intraocular ly injected with an irreversible inhibitor for caspase-3, z-DEVD-fmk. Six S334ter-3 rats (litter mates) were injected at PD 9 with 50 μg of z-DEVD-fmk (in 1 μl of DMSO) into each left eye and 1 μl of DMSO into each right eye. The animals were sacrificed and the eyes were collected at PD 20. The retinas were examined by light microscopy.

As shown in FIG. 6, the retinas of the PD 20 normal animals were fully developed and intact. The ONL contained 12-13 rows of nuclei, and the outer and inner segments were well organized (FIG. 6A).

The retinas of the S334ter-3 rats, treated with DMSO (FIG. 6B), corresponded to those without any treatment (FIG. II), i.e., only a single row of nuclei remain in the ONL (FIG. 6B). By comparison, the ONL of the z-DEVD-fmk treated retinas from the other eye of each same S334ter-3 animal, contained 4 to 5 rows of pyknotic nuclei (FIG. 6A). In all six animals, the ONL in the control eyes had 1-2 rows of nuclei, whereas the inhibitor- treated eyes had 3-5 rows.

To verify that it was actually the z-DEVD-fmk causing the inhibition of caspase-3 activation in the retinas of S334ter-3 rats, the 12 kD caspase-3 subunit was measured after inhibitor injection of the inhibitor. Three transgenic rats at PD 9 were injected in their left eyes with 50 μg of z-DEVD-fmk (in 1 μl of DMSO), and with 1 μl of DMSO (vehicle only) into their right eyes. Age-matched Sprague-Dawley rats served as controls. The animals were sacrificed and their retinas were collected at PD 1 1 and immunoblotting analyses were performed to detect the pi 2 subunit of the active form of caspase-3, using polyclonal antibodies against the pi 2 subunit of caspase-3. As shown in FIG. 7, the amount of the 12 kD subunit in the retinas treated with z-DEVD-fmk (Tg, Ihb +) was lower than in the retinas treated only with DMSO (Tg, Ihb -). Nevertheless, the 12 kD subunit was present in the transgenic animals at a higher level than in the normal Sprague-Dawley rats (SD, Ihb -).

In sum, intraocular administration of an effective amount of an anti-apoptosis inibitor, such as the disclosed caspase-3 inhibitor, z-DEVD-fmk, or z-VAD-fmk, modulates, regulates and may prevent apoptotic degeneration of the photoreceptor cells of the retina, thereby restoring vision or preventing or reducing the loss of vision caused by retinal degeneration, such as in the case of a patient genetically predisposed to of actively exhibiting the symptoms of retinitis pigmentosa.

While the foregoing specification has been described with regard to certain preferred embodiments, and many details have been set forth for the puφose of illustration, it will be apparent to those skilled in the art without departing from the spirit and scope of the invention, that the invention may be subject to various modifications and additional embodiments, and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention. Such modifications and additional embodiments are also intended to fall within the scope of the appended claims.

Claims

What is claimed is:

1. A method of regulating retinal degeneration in a patient, wherein degeneration is apoptotic, and wherein the method comprises administering to a patient experiencing retinal degeneration a therapeutically effective amount of a compound comprising an apoptosis-modulating compound.

2. The method of claim 1 , wherein the modulating compound regulates or controls apoptosis of a cell or population of cells in the retina of the patient.

3. The method of claim 2, wherein the cell or cell population comprises photoreceptor cells of the retina.

4. The method of claim 2, wherein apoptosis is inhibited or reduced following the administration of the modulating compound the modulating compound.

5. The method of claim 2, wherein apoptosis is enhanced or increased following the administration of the modulating compound.

6. The method of claim 4, wherein the modulating compound comprises an apoptosis inhibiting compound.

7. The method of claim 5, wherein the modulating compound comprises an apoptosis enhancing compound.

8. A method of preventing retinal degeneration in a patient, wherein the degeneration is apoptotic, and wherein the method comprises administering to a patient with a genetic or disease-based predisposition for retinal degeneration an effective amount of a compound comprising an apoptosis-modulating compound.

9. The method of claim 8, wherein the modulating compound prevents apoptosis of a cell or population of cells in the retina of the patient.

10. The method of claim 9, wherein the cell or cell population comprises photoreceptor cells of the retina.

11. The method of claim 9, wherein apoptosis is prevented by the administration of the modulating compound

12. The method of claims 1-11, wherein the target of the apoptosis modulating or preventing compound comprises an activated caspase.

13. The method of claim 12, wherein the caspase is caspase-3.

14. The method of claims 1-6 and 8-13, wherein the apoptosis inhibiting compound is z-DEVD-fmk or z-VAD fmk.

15. The method of claims 1-14, wherein the patient is a vertebrate.

16. The method of claims 15, wherein the vertebrate is mammalian.

17. The method of claim 16, wherein the mammal is human.

18. A method of regulating apoptosis in a cell or cell population in the retina of the eye, where the method comprises treating the cell or cell population with an effective amount of apoptosis-modulating compound, such that apoptosis is modulated.

19. The method of claim 18, wherein the cell or cell population comprises one or more photoreceptor cells.

20. The method of claim 20, wherein the modulating compound prevents apoptosis of photoreceptor cells.

21. The method of claim 20, wherein the modulating compound inhibits or reduces apoptosis of photoreceptor cells.

22. The method of claim 20, wherein the modulating compound stimulates or enhances apoptosis of photoreceptor cells.

23. The method of claim 21 , wherein the modulating compound is an apoptosis inhibiting compound.

24. The method of claim 22, wherein the modulating compound is an apoptosis enhancing compound.

25. The method of claims 20-24, wherein activated caspase is the target of the apoptosis modulating compound.

26. The method of claim 25, wherein the caspase polypeptide is caspase-3.

27. The method of claim 23, wherein the apoptosis inhibiting compound is z-DEVD- fmk.

28. The method of claims 20-27, wherein the photoreceptor cell or cells are human.

29. The method of claims 1-19, wherein the retinal degeneration in the patient is symptomatic of retinitis pigmentosa.

30. The method of claims 1-29, wherein the modulating compound is an antibody.

31. The method of claim 30, wherein the antibody is a monoclonal antibody.

32. The method of claims 1-29, wherein the modulating compound is recombinantly or synthetically produced.

33. The method of claim 32, wherein the modulating compound is a synthetically produced peptidomimetic compond.

34. The method of claims 1-29, wherein the degeneration is treated by gene therapy by introducing or activating a nucleic acid sequence encoding the modulating compound, and where the modulating compound is the expression product of the introduced or activated gene.

35. The method of claims 1-29, wherein the degeneration is treated by introducing or activating a nucleic acid antisense sequence.