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REPORT ON ANIMAL MODELS OF RETINAL DEGENERATION

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REPORT ON ANIMAL MODELS OF RETINAL DEGENERATION Powered By Docstoc
					ANIMAL MODELS FOR STUDYING INHERITED DEGENERATIVE RETINAL DISEASE

February, 2000

The Foundation Fighting Blindness 11350 McCormick Road Executive Plaza I, Suite 800 Hunt Valley, MD 21031-1014 800-683-5555 WWW.BLINDNESS.ORG

Table of Contents

I. II.

INTRODUCTION AGE-RELATED MACULAR DEGENERATION (AMD) A. RODENT MODELS FOR NEOVASCULAR AMD 1. Rhodopsin/VEGF Transgenic Mouse 2. Laser-Induced Rodent Models B. PRIMATE MODELS FOR AMD 1. Argon Laser-Induced Monkey 2. Caribbean Primate Colony

III.

LEBER CONGENITAL AMAUROSIS (LCA) A. AVIAN MODEL FOR LCA 1. rd Chicken MOUSE MODEL FOR LCA (See Section V.A.2) 1. RPE-65 Knock Out Mouse CANINE MODEL FOR LCA (See Section V.C.8) 1. Briard (RPE-65) Dog

B.

C.

IV.

RETINTITIS PIGMENTOSA - AUTOSOMAL DOMINANT - (ADRP) A. RODENT MODELS FOR ADRP 1. Rhodopsin Knock-Out Mouse 2. Pro-23-His Rhodopsin Mouse 3. Pro-23-His Rhodopsin Rat 4. VPP Mouse 5. Q344ter and S334ter Rhodopsin Mice 6. S334ter Rat 7. rds Mouse 8. Crx Knock Out Mouse PORCINE MODEL FOR ADRP 1. Pro-237-Leu Transgenic Pig

B.

V.

RETINITIS PIGMENTOSA - AUTOSOMAL RECESSIVE - (ARRP) A. RODENT MODEL FOR ARRP 1. rd Mouse 2. RPE-65 Knock Out Mouse 3. Tubby-Like Mouse ii

4. RCS Rat B. FELINE MODEL FOR ARRP 1. Abyssinian Cat CANINE MODELS FOR ARRP 1. Cone Degeneration (cd) Dog 2. Progressive rod-cone degeneration (prcd) Dog 3. Early Retinal Degeneration (erd) Dog 4. Rod-cone dysplasia 1 (rcd1) Dog 5. Rod cone dysplasia2 (rcd2) Dog 6. Rod cone dysplasia3 (rcd3) Dog 7. Photoreceptor dysplasia (pd) Dog 8. Briard (RPE-65) Dog

C.

VI.

RETINITIS PIGMENTOSA - X-LINKED - (XLRP) A. RODENT MODEL FOR XLRP 1. XLRP-3 Knock Out Mouse CANINE MODEL FOR XLRP 1. X-linked progressive retinal atrophy (XLPRA)

B.

VII.

STARGARDT DISEASE A. RODENT MODEL FOR STARGARDT DISEASE 1. ABCR Knock Out Mouse

VIII. USHER SYNDROME (US) A. RODENT MODEL FOR US 1. Shaker Mouse

iii

I.

INTRODUCTION Naturally occurring degenerative retinal disease in laboratory and domesticated animals represent a wealth of different pathological manifestations which are important for understanding the basis for human disease. The study of animal models with naturally occurring degenerative retinal disease has provided numerous candidate genes, some of which have led to the identification of new disease genes in humans. Animals with degenerative retinal disease, homologous to that found in humans, make useful models for investigation of treatment by cell transplantation, pharmaceutical and/or gene therapy. Furthermore, the ability to generate gene-specific transgenic and -targeted (knock out / knock in) mice, allow the creation of new models of human retinal disease and facilitate the elucidation of gene dysfunction.

II.

AGE-RELATED MACULAR DEGENERATION (AMD) Age-related macular degeneration (AMD) is the most common cause of vision loss in individuals over the age of fifty-five. In the United States more than 12 million individuals are affected with the disease and at least 6 million have severe visual impairment. There are two types of AMD: dry and wet. Dry AMD accounts for about 90 percent of all cases, but is less devastating than the wet form. Dry AMD is characterized by the presence of lipid-containing deposits called drusen beneath the retinal pigment epithelium (RPE) in the macular region. The dry form may progress to the wet form. The wet form of AMD accounts for only 10 percent of all AMD cases. However, those 10 percent make up the majority of severe vision impairment in AMD. Wet AMD is characterized by choroidal neovascularization (CNV). The abnormal blood vessel growth begins in the vascular choroid and eventually, the vessels break through the basement membrane (Bruch’s membrane) of the RPE and invade the outer retina. The blood vessels are not of the “continuous” type and leak blood and fluid which secondarily damage the photoreceptor cells. Wet AMD tends to progress rapidly and can cause severe damage to central vision. Although there are many proposed animal models for AMD, only a few of the more widely used models are listed here. The fact that primates are the only laboratory mammals having a “true” macula, limits the availability of “ideal” animal models for this disease. Animal models that are currently available mimic some, but not all aspects of the pathology of AMD. It is important to keep in mind that the available animal models involve artificial elicitation of disease in relatively “young” animal eyes, whereas authentic human AMD occurs only after decades have passed.

A.

RODENT MODELS FOR NEOVASCULAR AMD 1. Rhodopsin/VEGF Transgenic Mouse

1

A transgenic mouse containing the vascular endothelial growth factor (VEGF) gene placed under the regulation of the rhodopsin promoter was recently created by Campochiaro and colleagues. Whole mounts of transgenic retinas perfused with fluorescein-labeled dextran showed a sprouting of new blood vessels from the deep retinal capillary bed by post natal day 14. By postnatal day 18, these vessels reached the subretinal space. The subretinal neovascularization was progressively engulfed by the retinal pigmented epithelium. However, invasion of blood vessels from the choroid was not observed. An attempt is currently being made to produce a choroidal neovascularization model by placing the VEGF gene behind the expression of an RPEspecific promoter. Okamoto N, Tobe T, Hackett SF, Ozaki H, Vinores MA, LaRochelle W, Zack DJ and Campochiaro PA. Transgenic mice with increased expression of vascular endothelial growth factor in the retina: a new model of intraretinal and subretinal neovascularization. Am J Pathol. 1997 Jul;151(1):281-91. Tobe T, Okamoto N, Vinores MA, Derevjanik NL, Vinores SA, Zack DJ and Campochiaro PA. Evolution of neovascularization in mice with overexpression of vascular endothelial growth factor in photoreceptors. Invest Ophthalmol Vis Sci. 1998 Jan;39(1):180-188.

2.

LASER-INDUCED RODENT MODELS FOR NEOVASCULAR AMD

Frank and colleagues observed subretinal neovascularization following krypton laser photocoagulation lesions in the posterior retinas of pigmented rats. They observed foci of subretinal neovascularization that was histopathologically similar to that occuring in the wet form of AMD. Choroidal neovascularization occurs in approximately 60% of laser lesions, as evidenced by growth of capillaries through breaks in Bruch's membrane. In addition, approximately 30% of the lesions studied by fluorescein angiography demonstrated leakage. The laser photocoagulation damaged only the choriocapillaris, the retinal pigment epithelium (RPE), and the photoreceptor layer and the neovascularization was surrounded by multiple layers of RPE cells. Similar histopathologic findings have been reported in some human eyes with subretinal neovascularization in age-related macular degeneration. However, like the primate CNV model, the CNV in the krypton-induced CNV rat model regresses with time. Nonetheless, laser-induced rupture of Bruch’s membrane in rodents provides a very reliable model of choroidal neovascularization that can be used to investigate the efficacy of various therapies. Frank RN, Das A and Weber ML. A model of subretinal neovascularization in the pigmented rat. Curr Eye Res. 1989 Mar;8(3):239-247. Dobi ET, Puliafito CA, Destro M. A new model of experimental choroidal neovascularization in the rat. Arch Ophthalmol. 1989 Feb;107(2):264-269.

2

Tobe T, Ortega S, Luna JD, Ozaki H, Okamoto N, Derevjanik NL, Vinores SA, Basilico C and Campochiaro PA. Targeted disruption of the FGF2 gene does not prevent choroidal neovascularization in a murine model. Am J Pathol. 1998 Nov;153(5):1641-6. Seo MS, Kwak N, Ozaki H, Yamada H, Okamoto N, Yamada E, Fabbro D, Hofmann F, Wood JM and Campochiaro PA. Dramatic inhibition of retinal and choroidal neovascularization by oral administration of a kinase inhibitor. Am J Pathol. 1999 Jun;154(6):1743-53.

B.

PRIMATE MODEL FOR AMD 1. Argon Laser-Induced Monkey Model for AMD

The laser-induced AMD model in primates has provided important information related to progression of choroidal neovascularization (CNV) and the role of the RPE in reestablishing the blood-retinal-barrier. However, the laser-induced AMD model may not be ideal because it is a wounding model and may not mimic the pathogenesis of CNV in AMD. The model differs from clinical CNV in that the CNV regresses as evidenced by decreased vessel leakage and envelopment by RPE cells. Nonetheless, it provides a model of neovascularization originating from the choroid. Verteporfin administration has been shown to prevent angiographic leakage for at least 4 weeks in this monkey model. Husain D, Kramer M, Kenny AG, Michaud N, Flotte TJ, Gragoudas ES, Miller JW. Effects of photodynamic therapy using verteporfin on experimental choroidal neovascularization and normal retina and choroid up to 7 weeks after treatment. Invest Ophthalmol Vis Sci. 1999 Sep;40(10):2322-2331. Miller H, Miller B, Ryan SJ. The role of retinal pigment epithelium in the involution of subretinal neovascularization. Invest Ophthalmol Vis Sci. 1986 Nov;27(11):1644-1652. Virdi PS and Hayreh SS. Ocular neovascularization with retinal vascular occlusion. I. Association with experimental retinal vein occlusion. Arch Ophthalmol. 1982 Feb;100(2):331-341. 2. Caribbean Primate Colony In the hope of identifying an animal model for age-related macular degeneration (AMD) several groups have examined aged rhesus monkeys from a seminatural colony at the Caribbean Primate Research Center (CPRC) of the University of Puerto Rico. Approximately 75% of the aged animals examined had drusen in the posterior pole. Ultrastructural analysis revealed drusen resembled AMD-afflicted human retinas. Abnormalities in all layers of Bruch's membrane were observed with deposits of heterogeneous material, comprised of membranous, granular, and cellular components in both the inner collagenous zone (ICZ) and the outer collagenous zone (OCZ). Although the changes 3

resembled changes observed in human aging and AMD, none of the animals exhibited the exudative form of AMD or disciform scarring. Engel HM, Dawson WW, Ulshafer RJ, Hines MW and Kessler MJ. Degenerative changes in maculas of rhesus monkeys. Ophthalmologica. 1988;196(3):143-50. Ulshafer RJ, Engel HM, Dawson WW, Allen CB and Kessler MJ. Macular degeneration in a community of rhesus monkeys. Ultrastructural observations. Retina. 1987 Fall;7(3):198-203.

III.

LEBER CONGENITAL AMAUROSIS (LCA) Leber congenital amaurosis (LCA) is the designation for a group of autosomal recessive retinal dystrophies that represent the most common genetic causes of congenital visual impairment of retinal origin in infants and children. LCA is characterized by moderate to severe visual impairment identified at or within a few months of birth, infantile nystagmus, sluggish pupillary responses (and occasionally a paradoxical pupil response), and absent or poorly recordable electroretinographic responses early in life. LCA has been shown to be caused by mutations in three different genes: guanylate cyclase, CRX (see section IV.A.5) and RPE-65 (see section V.A.2).

A.

AVIAN MODEL FOR LCA 1. RD Chicken (guanylate cyclase mutant)

The retinal degeneration (rd) chicken is a natural mutant animal with disease homologous to human LCA. At hatching, the retina of the rd chicken is fully developed and possesses normal morphology, but fails to respond to light stimulation. However, a decrease in the number of outer segments is apparent by Day 10 and by six months, most of the photoreceptor inner segments and nuclei are absent, with the exception of a few randomly scattered cone cells. Analyses of retinal cGMP, the internal second messenger of phototransduction, shows that the amount of cGMP in predegenerate, fully developed rd/rd photoreceptors is 5-10 times less than that seen in normal photoreceptor cells. . Although the retinas are fully developed and possess normal morphology at hatching, they fail to respond to light stimulation. The low levels of cGMP in the rd chicken retina are a consequence of a null mutation in the photoreceptor guanylate cyclase (GC1) gene. The GC1 gene has also been shown to be mutated in Leber congenital amaurosis, making the rd chicken an ideal animal model for this condition. 4

B.

MOUSE MODEL FOR LCA 1. RPE-65 Knock Out Mouse (See Section V.A.2) CANINE MODEL FOR LCA 1. Briard (RPE-65) Dog (See Section V.C.8) Semple-Rowland SL, Lee NR, Van Hooser JP, Palczewski K, and Baehr W. A null mutation in the photoreceptor guanylate cyclase gene causes the retinal degeneration chicken phenotype. Proc Natl Acad Sci U S A. 1998 Feb 3;95(3):1271-1276. Ulshafer RJ, Allen CB. Hereditary retinal degeneration in the Rhode Island Red chicken: ultrastructural analysis. Exp Eye Res. 1985 Jun;40(6):865-877.

C.

IV.

RETINTITIS PIGMENTOSA - AUTOSOMAL DOMINANT (ADRP) Retinitis pigmentosa (RP) denotes a group of hereditary retinal dystrophies, characterized by the early onset of night blindness followed by a progressive loss of the visual field. RP is very heterogeneous, both phenotypically and genetically. The primary defect underlying RP affects the function of the rod photoreceptor cell, and, subsequently, mostly unknown molecular and cellular mechanisms trigger the apoptotic degeneration of these photoreceptor cells. Autosomal dominant inheritance accounts for approximately 10% of the RP inheritance pattern. It is estimated that in the United States 200,000 individuals are affected with the some form of this disease. Mutations in five genes: (rhodopsin, peripherin/RDS, Neural Retina Leucine/NRL, RP1 protein and CRX) have been shown to result in the dominant form of RP. Many other autosomal dominant RP loci have been identified but the gene causing mutations have not been identified and characterized.

A.

RODENT MODELS FOR ADRP 1. Rhodopsin Knock Out Mouse

Mutations in the rhodopsin gene account for more than 10% of all cases and represent the most common cause of autosomal dominant retinitis pigmentosa. Humphries and colleagues generated mice carrying a targeted disruption of the rhodopsin (Rho) gene. The rod outer segements of Rho-/- mice do not develop fully and entire photoreceptors are lost over a 3 month period. No rod ERG response is present in 8-week-old animals. Rho+/- animals retain the majority of their photoreceptors although the inner and outer segments of these cells display some structural disorganization, the outer segments becoming shorter in older mice. The rhodopsin KO animals provide a useful genetic background on which to express other mutant opsin transgenes as well as assessing the therapeutic potential of re-introducing functional rhodopsin genes into degenerating retinal tissues. Humphries MM, Rancourt D, Farrar GJ, Kenna P, Hazel M, Bush RA, Sieving PA, Sheils DM, McNally N, Creighton P, Erven A, Boros A, Gulya K, Capecchi MR, Humphries P. 5

Retinopathy induced in mice by targeted disruption of the rhodopsin gene. Nat Genet. 1997 Feb;15(2):216-219. Lem J, Krasnoperova NV, Calvert PD, Kosaras B, Cameron DA, Nicolo M, Makino CL, Sidman RL. Morphological, physiological, and biochemical changes in rhodopsin knockout mice. Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):736-41.

2.

Pro-23-His Rhodopsin Mouse

The P23H mutation is the most prevalent mutation in human ADRP patients. Twelve percent of American patients with autosomal dominant retinitis pigmentosa (ADRP) carry a substitution of histidine for proline at codon 23 (P23H) in their rhodopsin gene, resulting in photoreceptor cell death from the synthesis of the abnormal gene product. Transgenic mice containing the P23H mutation appear to develop normal photoreceptors, but their light-sensitive outer segments never reach normal length. With advancing age, both rod and cone photoreceptors are reduced progressively in number. The degeneration of the transgenic retina is associated with a gradual decrease of light-evoked electroretinogram responses. Naash MI, Hollyfield JG, al-Ubaidi MR, Baehr W. Simulation of human autosomal dominant retinitis pigmentosa in transgenic mice expressing a mutated murine opsin gene. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5499-5503.

3.

Pro-23-His Rhodopsin Rat

A P23H mutant rhodopsin transgenic rat has also been created by LaVail and colleagues which is similar to the P23H transgenic mouse. The transgenic rat offers larger eyes necessary for some experimental purposes. Three different rates of degeneration are present in three different lines of the P23H rats due to different levels of expression of the mutant rhodopsin. Recent experimental therapeutic studies show that neurotrophic factors injected into the eyes can slow the rate of photoreceptor degeneration, and that ribozymes targeting the P23H mRNA sequence in the rat model considerably slow the rate of degeneration for at least three months. Steinberg RH, Flannery JG, Naash M, Oh P, Matthes MT, Yasumura D, Lau-Villacorta C, Chen J, LaVail MM. Transgenic rat models of inherited retinal degeneration caused by mutant opsin genes. Invest Ophthalmol Vis Sci. 1996; 37:S698. Steinberg RH, Matthes MT, Yasumura D, Lau-Villacorta C, Nishikawa S, Cao W, Flannery JG, Naash M, Chen J, LaVail MM. Slowing by survival factors of inherited retinal degenerations in transgenic rats with mutant opsin genes. Invest Ophthalmol Vis Sci. 1997; 38:S226. 6

Lewin AS, Drenser KA, Hauswirth WW, Nishikawa S, Yasumura D, Flannery JG, LaVail MM. Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa. Nat Med. 1998 Aug;4(8):967-971.

4.

The VPP Mouse

The VPP mouse is a transgenic strain carrying three mutations (V20G, P23H, P27L) near the N-terminus of opsin, the apoprotein of rhodopsin, the rod photopigment. These animals exhibit a slowly progressive degeneration of the rod photoreceptors, and concomitant changes in retinal function that mimic those seen in humans with autosomal dominant retinitis pigmentosa resulting from a point mutation (P23H) in opsin. Naash MI, Hollyfield JG, Al-Ubaidi MR, Baehr W. Simulation of human autosomal dominant retinitis pigmentosa in transgenic mice expressing a mutated murine opsin gene. Proc Natl Acad Sci USA. 1993; 90:5499-5503.

5.

Q344ter and S334ter Rhodopsin Mice

Of the many rhodopsin mutations that lead to ADRP in human patients, those resulting in truncation of the rhodopsin molecule at the carboxy terminus typically have a more severe clinical phenotype than those with mutations at other parts of the molecule. Transgenic mice with truncation mutations that remove the last 5 amino acids (Q344ter) and the last 15 amino acids (S334ter) from the carboxy terminus are available. In the case of the S334ter mice, all of the phosphorylation sites, and thus all of the arrestin binding sites, are removed from the rhodopsin molecule leading to prolonged photoresponses following light exposure. Sung C-H, Makino C, Baylor D, Nathans J. A rhodopsin gene mutation responsible for autosomal dominant retinitis pigmentosa results in a protein that is defective in localization to the photoreceptor outer segment. J Neurosci. 1994; 14:5818-5833. Chen J, Makino CL, Peachey NS, Baylor D, Simon MI. Mechanisms of rhodopsin inactivation in vivo as revealed by COOH-terminal truncation mutant. Science. 1995; 267:374-377.

6.

S334ter Rhodopsin Rat

Like the S334ter rhodopsin mouse, S334ter rhodopsin transgenic rats have been produced. Five lines of the rats are available with different rates of degeneration corresponding to different levels of mutant rhodopsin expression. These range from the fastest degeneration among all animal models to the slowest. Like in the P23H transgenic rats, photoreceptor degeneration in rats with S334ter truncated rhodopsin has been slowed by the application of neurotrophic factors. 7

Steinberg RH, Flannery JG, Naash M, Oh P, Matthes MT, Yasumura D, Lau-Villacorta C, Chen J, LaVail MM. Transgenic rat models of inherited retinal degeneration caused by mutant opsin genes. Invest Ophthalmol Vis Sci. 1996; 37:S698. Steinberg RH, Matthes MT, Yasumura D, Lau-Villacorta C, Nishikawa S, Cao W, Flannery JG, Naash M, Chen J, LaVail MM. Slowing by survival factors of inherited retinal degenerations in transgenic rats with mutant opsin genes. Invest Ophthalmol Vis Sci. 1997; 38:S226.

7.

rds Mouse

Peripherin/rds is a transmembrane glycoprotein expressed in vertebrate photoreceptors. It is located at the rim of the disc membranes of the photoreceptor outer segments, where it is thought to play an important role in folding and stacking of the discs. Mutations in the gene encoding peripherin have been implicated in the pathogenesis of both autosomal dominant retinitis pigmentosa as well as various forms of macular dystrophy. Initially, the identification of a mutation in the rds mouse model defined the role of this gene in hereditary retinal dystrophies. The rds mouse is a non-transgenic, natural mutant with a mutation in the rds/peripherin gene similar to that found in human RP families. In the homozygous rds mice, the receptor layer remains rudimentary and the outer segment does not develop. However, the other retinal layers show a normal trend of growth during the first 2 weeks after birth. Thereafter the morphological layers containing visual cell structures--the receptor, the outer nuclear, and the outer plexiform layers begin to thin. The loss of visual cells is readily marked by the reduction of the outer nuclear layer and is first evident at 2 weeks after birth. Degeneration is more rapid up to the age of 2-3 months, when the outer nuclear layer is reduced to half of its original thickness; thereafter degeneration progresses more slowly. At 9 months, photoreceptor cells in the peripheral retina are lost and by 12 months, the entire retina is completely lacking of in photoreceptor cells. The inner parts of the retina, including inner nuclear, inner plexiform, and ganglion cell layers, remain morphologically unaffected until irregular vascularization follows total loss of visual cells. Several studies have shown that administration of certain neurotrophic factors results in a delay in the rate of retinal degeneration. These findings raise the potential for pharmaceutical / genetic intervention in treating degenerative retinal disease.

Cayouette M, Behn D, Sendtner M, Lachapelle P, Gravel C. Intraocular gene transfer of ciliary neurotrophic factor prevents death and increases responsiveness of rod photoreceptors in the retinal degeneration slow mouse. J Neurosci. 1998 Nov 15;18(22):9282-9293. LaVail MM, Yasumura D, Matthes MT, Lau-Villacorta C, Unoki K, Sung CH,Steinberg RH. Protection of mouse photoreceptors by survival factors 8

in retinal degenerations. Invest Ophthalmol Vis Sci. 1998 Mar;39(3):592602. Travis GH, Brennan MB, Danielson PE, Kozak CA, Sutcliffe JG. Identification of a photoreceptor-specific mRNA encoded by the gene responsible for retinal degeneration slow (rds). Nature. 1989 Mar 2;338(6210):70-73. Sanyal S, De Ruiter A, Hawkins RK. Development and degeneration of retina in rds mutant mice: light microscopy. J Comp Neurol. 1980 Nov 1;194(1):193-207.

8.

Crx Knock Out Mouse

Crx, an Otx-like homeobox gene, is expressed specifically in the photoreceptors of the retina and the pinealocytes of the pineal gland. Crx has been proposed to have a role in the regulation of photoreceptor-specific genes in the eye and of pineal-specific genes in the pineal gland. Mutations in human CRX are associated with the retinal diseases, conerod dystrophy-2, retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), which all lead to loss of vision. Mice carrying a targeted disruption of Crx do not elaborate photoreceptor outer segments and lacked rod and cone activity as assayed by electroretinogram (ERG). Expression of several photoreceptor- and pineal-specific genes are also reduced in Crx mutants. Furukawa T, Morrow EM, Li T, Davis FC, Cepko CL. Retinopathy and attenuated circadian entrainment in Crx-deficient mice. Nat Genet. 1999 Dec;23(4):466-470.

B.

PORCINE MODEL FOR ADRP 1. Pro-237-Leu Transgenic Pig Model for ADRP

Investigators at Duke University and North Caroline State University have produced transgenic pigs expressing a Pro347Leu mutation in the rhodopsin gene. The Pro346Leu mutation, like the Pro23H, is one of the more common autosomal dominant mutations. There are 2 lines of Pro347Leu pigs, one of which demonstrates a very rapid retinal degeneration. Like retinitis pigmentosa (RP) patients with the same mutation, transgenic pigs have early and severe rod loss; initially their cones are relatively spared, but the surviving cones slowly degenerate. By age 20 months, there is only a single layer of morphologically abnormal cones and the cone electroretinogram is markedly reduced. The rhodopsin transgenic pig retina shares many cytologic features with human retinas with retinitis pigmentosa and provides an opportunity to examine the earliest stages in photoreceptor degeneration, about which little is known in humans. The pigs are produced over a 3-month breeding cycle and despite the high cost and the problems engendered in dealing with a 250 pound animal, there is much to recommend the pig as a model animal for assessment for retinal degeneration.

9

The retina itself is well constructed and has an ample supply of cones. Even thought the pig retina lack a foveal-macular region, in many aspects the pig eye resembles the primate eye. It has a well-formed sclera and choroid and tolerates surgical procedures well. For these reasons, the pig eye is well suited for experimental, surgical and/or therapeutic treatments. Given the strong similarities in phenotype to that of RP patients, the transgenic pigs provide a large animal model for study of the protracted phase of cone degeneration found in RP. In addition, these animals are invaluable for pre-clinical treatment trials. Li ZY, Wong F, Chang JH, Possin DE, Hao Y, Petters RM, Milam AH. Rhodopsin transgenic pigs as a model for human retinitis pigmentosa. Invest Ophthalmol Vis Sci. 1998 Apr;39(5):808-819 Petters RM, Alexander CA, Wells KD, Collins EB, Sommer JR, Blanton MR, Rojas G, Hao Y, Flowers WL, Banin E, Cideciyan AV, Jacobson SG, Wong F. Genetically engineered large animal model for studying cone photoreceptor survival and degeneration in retinitis pigmentosa. Nat Biotechnol. 1997 Oct;15(10):965-970.

III.

RETINITIS PIGMENTOSA -AUTOSOMAL RECESSIVE (ARRP) Autosomal recessive retinitis pigmentosa (arRP) accounts for the many forms of the disease. Although the phenotype varies, in general the progression of autosomal recessive disease is more rapid than the dominant form. At the present time, mutations in at least seven genes (e.g. cGMP phosphodiesterase (alpha and beta subunits), RPE-65, crumbs homolog 1, cGMP-gated cation channel, tubby-like protein 1 and cellular retinaldehyde-binding protein/CRALBP) have been shown to cause arRP.

A.

RODENT MODELS FOR ARRP 1. rd Mouse

Natural mutant mice homozygous for the rd mutation display hereditary retinal degeneration and the classic rd lines serve as a model for human retinitis pigmentosa. In affected animals the retinal rod photoreceptor cells begin degenerating at about postnatal day 8, and by four weeks no photoreceptors are left. In all regions of the eye, a rapid rod degeneration precedes a much slower cone degeneration. Only about 2% of the rods remain in the posterior region at postnatal day 17, and none by the day 36. By contrast, at least 75% of the cone nuclei remain at day 17. Degeneration is preceded by accumulation of cyclic GMP in the retina and is correlated with deficient activity of the rod photoreceptor cGMP-phosphodiesterase. Scientists first identified the biochemical problem in the retina of the rd mouse as a deficit in the ROS enzyme phosphodiesterase, (PDE) that leads to an abnormal accumulation of cyclic GMP and subsequent photoreceptor cell degeneration and death. The lesion is manifest early in the postnatal period of development. Further studies 10

localized the molecular defect to a mutation in the gene coding for the beta subunit of the PDE enzyme. A number of studies have shown that gene therapy replacement of the PDE gene in the rd mouse retina delays the course of the degeneration. In addition, it has recently been reported that administration of D-cis-diltiazem, a calcium-channel blocker that also acts at light-sensitive cGMP-gated channels, rescued photoreceptors and preserved visual function in the rd mouse. The administration of several different neurotrophic factors such as fibroblast growth factor (FGF), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) are also able to slow the rate of retinal degeneration in the rd mouse. Frasson M, Sahel JA, Fabre M, Simonutti M, Dreyfus H, Picaud S. Retinitis pigmentosa: rod photoreceptor rescue by a calcium-channel blocker in the rd mouse. Nat Med. 1999 Oct;5(10):1183-1187. LaVail MM, Yasumura D, Matthes MT, Lau-Villacorta C, Unoki K, Sung CH,Steinberg RH, Protection of mouse photoreceptors by survival factors in retinal degenerations. Invest Ophthalmol Vis Sci. 1998 Mar;39(3):592-602. Bennett J, Tanabe T, Sun D, Zeng Y, Kjeldbye H, Gouras P, Maguire AM, Photoreceptor cell rescue in retinal degeneration (rd) mice by in vivo gene therapy. Nat Med. 1996 Jun;2(6):649-654. Bowes C, Li T, Danciger M, Baxter LC, Applebury ML, Farber DB, Retinal degeneration in the rd mouse is caused by a defect in the beta subunit of rod cGMPphosphodiesterase. Nature. 1990 Oct 18;347(6294):677-680. Carter-Dawson LD, LaVail MM, Sidman RL, Differential effect of the rd mutation on rods and cones in the mouse retina. Invest Ophthalmol Vis Sci. 1978 Jun;17(6):489-498.

2.

RPE-65 Knock Out Mouse

Mutations in the RPE65 gene can cause severe blindness from birth or early childhood. Although its exact function is unknown, the RPE65 protein is associated with retinal pigment epithelium (RPE) vitamin A metabolism. Rpe65-deficient mice exhibit changes in retinal physiology and biochemistry. Outer segment discs of rod photoreceptors in Rpe65-/- mice are slightly disorganized compared with those of Rpe65+/+ and Rpe65+/mice. Rod function, as measured by electroretinography, is abolished in Rpe65-/- mice, although cone function remains. Rpe65-/- mice lack rhodopsin, but not the opsin apoprotein. Furthermore, all-trans-retinyl esters over-accumulate in the RPE of Rpe65-/mice, whereas 11-cis-retinyl esters are absent. Disruption of the RPE-based metabolism of all-trans-retinyl esters to 11-cis-retinal thus appears to underlie the Rpe65-/phenotype, although cone pigment regeneration may be dependent on a separate pathway. Therefore, the RPE-65 knock out mouse is a model for both retinitis pigmentosa and Lebers congenital amaurosis. 11

Redmond TM, Yu S, Lee E, Bok D, Hamasaki D, Chen N, Goletz P, Ma JX, Crouch RK, Pfeifer K. Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nat Genet. 1998 Dec;20(4):344-351.

3.

Tubby-Like Mouse

A recessive mutation in the tub gene causes obesity, deafness and retinal degeneration in tubby mice. The tub gene is a member of a family of tubby-like genes (TULPs) that encode proteins of unknown function. Members of this family have been identified in plants, vertebrates and invertebrates. The TULP proteins share a conserved carboxyterminal region of approximately 200 amino-acid residues. The human gene TULP1, is expressed specifically in the retina. Upon analysing 162 patients with nonsyndromic recessive retinitis pigmentosa (RP) and 374 simplex cases of RP, Hagstrom and colleagues found two patients that were compound heterozygotes for TULP1 mutations that co-segregated with disease in the respective families. Three of the mutations were missense changes affecting the conserved C-terminal region; the fourth mutation affected a splice donor site upstream of this region. This data suggest that mutations in TULP1 are a rare cause of recessive RP and indicate that TULP1 has an essential role in the physiology of photoreceptors. Hagstrom SA, North MA, Nishina PL, Berson EL, Dryja TP, Recessive mutations in the gene encoding the tubby-like protein TULP1 in patients with retinitis pigmentosa. Nat Genet. 1998 Feb;18(2):174-176.

4.

RCS Rat

The Royal College of Surgeons (RCS) rat model of retinal degeneration has been known for many years. It has been worked on extensively and is maintained in a number of laboratories internationally. Congenic (genetically similar) strains are available that serve as genetic controls and offer both dystrophic and normal animals with different pigmentation types and rates of degeneration. It has been known for some time that the retinal pigment epithelium (RPE) fails to phagocytize photoreceptor rod outer segments which leads to the accumulation of a layer of membranous debris between the retinal photoreceptors and the RPE in the developing retina. Experiments with experimental chimeras have demonstrated that the genetic defect is in the RPE cell, which secondarily results in photoreceptor cell death. The gene mutation in the RCS rat has recently been found to be in the receptor tyrosine kinase Mertk gene. Studies demonstrating that the RCS phenotype can be rescued by transplanation of normal RPE cells into the interphotoreceptor space led to the development of the fields of RPE and retinal transplantation. Moreover, the finding that administration of neurotrophic factors temporarily arrests the progression of photoreceptor degeneration in 12

RCS rats has led to the development of the field of pharmaceutical therapy for retinal degenerative diseases. LaVail MM, Sidman RL, Gerhardt CO. Congenic strains of RCS rats with inherited retinal dystrophy. J Hered. 1975 Jul-Aug;66(4):242-244. Faktorovich EG, Steinberg RH, Yasumura D, Matthes MT, LaVail MM. Photoreceptor degeneration in inherited retinal dystrophy delayed by basic fibroblast growth factor. Nature. 1990; 347:83-86. Sauve Y, Klassen H, Whiteley SJ, Lund RD. Visual field loss in RCS rats and the effect of RPE cell transplantation. Exp Neurol. 1998 Aug;152(2):243-250. D'Cruz PM, Yasumura D, Weir J, Matthes M, Abderrahim H, LaVail MM, Vollrath D. Mutation of the receptor tyrosine kinase Mertk in the retinal dystrophic RCS rat. Hum Mol Genet. 2000; 9:645-651.

B.

FELINE MODEL FOR ARRP 1. Abyssinian Cat

The retinal disease in the Abyssinian cats is a heritable recessive disorder, primarily affecting the photoreceptors. However, the mutation and gene responsible for the retinal degeneration has not yet been identified. In most cases, the retina is ophthalmoscopically normal until the age of 1.5-2 years. The retinal changes that appear are slowly progressive and lead to a generalized retinal atrophy within 2-4 years. Similar to generalized retinitis pigmentosa in humans, the midperiphery/periphery is most severely affected at the earlier stages, and with progression of disease alterations become generalized, the central retina being the best preserved area until the very late stage. Rods are affected prior to cones, but later in the disease there is an involvement of both rods and cones. The earliest histological change is the disorientation of rod outer segment discs, with a high frequency of immature appearing rod outer segment discs in affected animals. Cones are unaffected at this age. At around six months of age, there is disintegration of the rod discs, vacuolization and clumping of disc material and the formation of debris. Subsequently, there is a drop-out of rods. Cones cells remain normal until the age of 2 or 3 years. Currently, the Abyssinian cat is being used in trials on retinal transplantation to see if the disease course can be slowed or stopped. Ivert L, Gouras P, Naeser P, Narfstrom K. Photoreceptor allografts in a feline model of retinal degeneration. Graefes Arch Clin Exp Ophthalmol. 1998 Nov;236(11):844-52. Narfstrom K, Nilsson SE. Hereditary rod-cone degeneration in a strain of Abyssinian cats. Prog Clin Biol Res. 1987; 247:349-368. PMID: 3685034; 13

C.

CANINE MODELS FOR AARP Dogs are unique in having more distinct primary retinal degeneration loci (7 autosomal recessive and one X-linked) than any other animal model of RP. Until now, the disadvantage of using dogs for genetic studies of RP was considerable; they are a "genome poor" species, and approaches to identifying retinal disease loci have been limited to candidate gene studies, a laborious and often unrewarding approach. Within the past 1-2 years, progress in the dog genetics community has provided the tools necessary to take advantage of this unique resource for retinal research. Several pre-clinical trials are currently being conducted in the dog RD models. Table 1 provides a brief synopsis of the various inherited retinal degenerations in the canine. In addition to the 4 major disease strains, 3 other non-allelic diseases are maintained in the colony, which represent unique retinal degeneration loci and may have potential to serve as homologues for specific human RP disorders.

Table 1 Abbreviation / Name Cd Cone degeneration Breed Alaskan malamute Inheritance Simple recessive Pathology Loss of cone outer segment in first 6 months, extrusion of cone nucleus into the inner segment, and displacement into IPM. Postnatal development of photoreceptors is normal. Visual cells show vesicular membranous fragments of outer segment, which progress, and photoreceptor degeneration follows. Rods are more severely affected. Rod photoreceptors fail to differentiate normally and then degenerate. Cone degeneration is a later event in the disease process.

prcd

Progressive rod-cone degeneration (prcd)

Miniature Poodle

Simple recessive

Erd

Early retinal degeneration

Norwegian Elkhound

Simple recessive

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rcd1

Rod-Cone dysplasia

Irish Setters

Simple recessive

Photoreceptor degeneration commences 25 days after birth. Elevated levels of cyclic guanosine monophosphate (cGMP) in both dogs and humans is caused by mutations in the gene for the beta subunit of cGMP phosphodiesterasephosphodieserase (PDE). Pathology is identical to the Irish setter with elevated levels of cGMP, but PDE subunits are normal. Pathology is thought to be similar to that described for rcd1. Mutations in the gene for the alpha subunit of cGMP PDE is thought to be responsible for the disease. Both rods and cones are arrested in their development by 4-5 weeks of age. Degeneration begins at that time; initially, about 50-60% of the photoreceptors are lost by 6 months, but the remaining cells persist over a 34 year period. Disease begins in the periphery and affects rods initially at around 9 months. Carrier females have a 50% reduction in rods and scattered patches of retinal degeneration due to random X-inactivation. (See VI.B.1) Congenital night blindness by 5-6 weeks of age and rod outer segment disc abnormalities are evident. Cones are less severly affected. Lipofuscin accumulation in the RPE. Disease in humans and dogs caused by mutation in the RPE-65 gene.

rcd2

Rod-cone dysplasia 2

Collie

Simple recessive

rcd3

Rod-cone dysplasia 3

Cardigan Welsh corgis

Simple recessive

pd

Photoreceptor dysplasia

Minature Schnauzer

Simple recessive

XL PRA

X-linked Siberian progressive Husky retinal atrophy

X-linked recessive

RPE65

Retinal Degeneration

Briard

Simple recessive

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1.

Cone Degeneration (cd) Dog

Cone Degeneration (cd) is a recessively inherited disorder discovered in 1972 in the Alaska malamute. The disorder has been transferred to a beagle / mongrel background. Affected animals have characteristic day blindness coming sometime after 1 month of age with normal night vision. All cones are lost within the first year of life while rods continue to maintain normal number and morphology, and function throughout life. In effect, cone loss is highly selective. As such, the cd mutant represents the only animal model of rod monochromatism. The parallel with rod monochromatism in man includes an unusual pathological sequence: Unlike other models of photoreceptor degeneration where nuclear pyknosis and apoptotic cell death follow disease and degeneration of the outer and inner segments, this process does not occur, at least initially, in the cd retina. Instead, diseased cones undergo slow extrusion of the nucleus into the inner segment, then displacement into the interphotoreceptor space, and finally placement subjacent to the RPE. The gene for the cd animal has not been identified, but the disease has been mapped to chromosal region homologous to human 8q. This makes the disease a locus homolog for human achromotopsia. Aguirre GD, Rubin LF. Pathology of hemeralopia in the Alaskan malamute dog. Invest Ophthalmol. 1974 Mar;13(3):231-235. Gropp KE, Szel A, Huang JC, Acland GM, Farber DB, Aguirre GD. Selective absence of cone outer segment beta 3-transducin immunoreactivity in hereditary cone degeneration (cd). Exp Eye Res. 1996 Sep;63(3):285-96. 2. Progressive rod-cone degeneration (prcd) Dog Model for ARRP

The prcd strain was identified originally in the miniature poodle. It has been cross-bred to beagles to enhance reproductive performance. The strain is similar in physical appearance and genetic background to other mutant strains similarly cross-bred. This allows comparative studies to be portioned among strains. It also offers an economic advantage: the maintenance of a single line of homozygous dog as controls for all mutant animals in the colony. The prcd affected retina undergoes normal development for rods as well as cones. In early adulthood, the rods begin to degenerate. Later the cones as well manifest a slow degeneration. Two phenotypic forms of the disorder now named prcd (fast) and prcd (slow) exist. Although a search through candidate genes has been performed with many excluded, a causative gene mutation is yet to be found. The disease has been mapped to CFA9, which is homologous to human 17q, therefore this model, this animal is a locus homolog for locus 17. Presently inexplicable, this disease is associated with significantly reduced plasma levels of docosahexaenoic acid (DHA, 22:63). Significantly, a similar abnormality is reported in certain human patients affected with RP and/or Usher syndrome. DHA is essential for normal retinal development and function in humans and experimental animals, and is the major fatty acid of rod outer segments. The significance of the association between low 16

plasma DHA with prcd in dogs, and RP in humans, is undetermined as yet. Furthermore, low DHA levels have been reported for forms of RP that are clearly caused mutations at defined genetic loci. This suggests that the low-DHA phenotype may represent a factor common to a spectrum of retinal degenerations. It is unlikely that it results from the degenerative disorder. Instead, it may well be modulatory, affecting severity in expression. The gene responsible for the low-DHA phenotype could thus represent the prcd mutation itself, a mutation at an autosomal locus linked to the prcd locus, or even a mutation at a locus not linked to prcd. Studies using the prcd model to investigate the relationship of retinal degeneration to abnormal DHA metabolism offer compelling advantages over investigations in humans. Affected dogs and nonaffected controls are reared and bred under precisely the same diet and environmental conditions with closely similar genetic backgrounds and controlled breedings are undertaken as required. Aguirre GD, Acland GM, Maude MB, Anderson RE. Diets enriched in docosahexaenoic acid fail to correct progressive rod-cone degeneration (prcd) phenotype. Invest Ophthalmol Vis Sci. 1997 Oct;38(11):2387-2397. Aguirre G, Alligood J, O'Brien P, Buyukmihci N. Pathogenesis of progressive rod-cone degeneration in miniature poodles. Invest Ophthalmol Vis Sci. 1982 Nov;23(5):610-630. Acland GM, Ray K, Mellersh CS, Gu W, Langston AA, Rine J, Ostrander EA, Aguirre GD. Linkage analysis and comparative mapping of canine progressive rod-cone degeneration (prcd) establishes potential locus homology with retinitis pigmentosa (RP17) in humans. Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):3048-53. 3. Early Retinal Degeneration (erd) Dog Model for ARRP

Early retinal degeneration (erd) is characterized by aberrant structural development both of rod outer segments and of rod and cone synapses. Rapid degeneration of both photoreceptors follows in affected dogs, after 3 months of age. Aberrant morphogenesis of the retina is expressed morphologically as loss of coordinated development of the photoreceptor inner and outer segments, resulting in cells having highly variable dimensions, and by the arrested development of synapses in the outer plexiform layer. Electroretinographically, this stage of the disease can be recognized by the highly characteristic negative waveform, as well as by reduced amplitudes of responses. Because rod and cone synapses develop abnormally, the ERG b-wave, which represents post-synaptic ionic flow, is essentially absent. In older affected dogs, the photoreceptors degenerate and eventually die. This process is very rapid at first (10-12 weeks of age), but more gradual after 6 months of age and is much faster in rods than in cones. The genetic basis for the erd disease is unknown, but it has been mapped to a chromosomal region homologous to human chromosome 12 P. Acland GM, Aguirre GD. Retinal degenerations in the dog: IV. Early retinal degeneration (erd) in Norwegian elkhounds. Exp Eye Res. 1987 Apr;44(4):491-521. 17

Acland GM, Ray K, Mellersh CS, Langston AA, Rine J, Ostrander EA, Aguirre GDA. Novel retinal degeneration locus identified by linkage and comparative mapping of canine early retinal degeneration. Genomics. 1999 Jul 15;59(2):134-42. 4. Rod-cone Dysplasia 1 (rcd1) Dog Model for ARRP

This rcd1 strain has been in progressive development for 25 years. After first undertaking multiple breedings in the Irish setter to prepare homozygous recessive heterozygotes and a breed matched strain of homozygous normal controls, rcd 1 has been outbred to beagles and thus is maintained as a crossbred line. Inadequate cyclic GMP catabolism leads to a many-fold higher level of cyclic GMP. This in turn causes a failure of photoreceptor differentiation plus a very rapid photoreceptor degeneration. In the second month of life rod vision is lost. Substantial deterioration of cone vision usually occurs within one year. Intense investigation first of biochemistry and then of molecular genetics over 25 years has revealed the true defect in this disorder to be a nonsense mutation of codon 807 of a gene to the  subunit of rod cyclic GMP phosphodiesterase. As such, the rcd 1 dog is the homologue for the rd mouse and it has been shown that a number of humans affected with autosomal recessive retinitis pigmentosa, also bear a mutation in the gene encoding the  subunit of rod cyclic GMP phosphodiesterase. This model has been used in several trials with neurotropininc factors and there has been a dramatic rescue of photoreceptors when treatment is started in early stages of disease. Suber ML, Pittler SJ, Qin N, Wright GC, Holcombe V, Lee RH, Craft CM, Lolley RN, Baehr W, Hurwitz RL Irish setter dogs affected with rod/cone dysplasia contain a nonsense mutation in the rod cGMP phosphodiesterase beta-subunit gene. Proc Natl Acad Sci U S A. 1993 May 1;90(9):3968-3972. Barbehenn E, Gagnon C, Noelker D, Aguirre G, Chader G. Inherited rod-cone dysplasia: abnormal distribution of cyclic GMP in visual cells of affected Irish setters. Exp Eye Res. 1988 Feb;46(2):149-159.

5.

Rod cone dysplasia2 (rcd2)

The disease in the Collie is clinically and pathologically indistinguishable from rod-cone dysplasia 1 in the Irish setter. In addition, the disease also has a similar biochemical abnormality. This is, there is deficient activity of cGMP-PDE and a 10-fold elevation of cGMP. The time course of the biochemical changes is similar to that in the rod-cone dysplasia affected Irish setter. Unlike the setter, however, the PDEB gene is not involved, as shown by crossbreeding experiments between affected setters and collies, and, more recently, by sequencing the PDEB coding region. Also excluded as causally associated with the disease are the gamma, delta and alpha subunits of PDE (by linkage and/or direct sequencing of the coding region), and the transducin alpha 1 gene (by linkage). Other phototransduction genes are presently being examined. Thus, rcd2 may represent a novel

18

phototransduction gene defect whose effect on cGMP metabolism results in photoreceptor degeneration. Wang W, Acland GM, Ray K, Aguirre GD. Evaluation of cGMP-phosphodiesterase (PDE) subunits for causal association with rod-cone dysplasia 2 (rcd2), a canine model of abnormal retinal cGMP metabolism. Exp Eye Res. 1999 Oct;69(4):445-453. 6. Rod cone dysplasia 3 (rcd3) Dog Model for ARRP

The pathology and progression of rcd3 in the Cardigan Welsh corgi dog is thought to be similar to rcd1 in the Irish Setter. Mutations in the gene encoding the alpha subunit of rod cyclic GMP phosphodiesterase was recently shown to be responsible for causing the disease. Similar to rcd1, a number of humans affected with autosomal recessive retinitis pigmentosa, have mutations in the gene encoding the alpha subunit of rod cyclic GMP phosphodiesterase. This is the only animal model available with a mutation in this gene. Petersen-Jones SM, Entz DD, Sargan DR. cGMP phosphodiesterase-alpha mutation causes progressive retinal atrophy in the Cardigan Welsh corgi dog. Invest Ophthalmol Vis Sci. 1999 Jul;40(8):1637-1644. 7. Photoreceptor dysplasia (pd) Dog Model for ARRP

Photoreceptor dysplasia (pd) is one of a group of at least six distinct autosomal and one X-linked retinal disorders identified in dogs which are collectively known as progressive retinal atrophy (PRA). It is an early onset retinal disease identified in miniature schnauzer dogs, and pedigree analysis and breeding studies have established autosomal recessive inheritance of the disease. Diseased photoreceptors begin to degenerate soon after retinal development is completed (6 weeks of age). Unlike other diseases, the rate of degeneration is biphasic. Initially rods and cones degenerate rapidly, and the outer nuclear layer is reduced by 50% within 5-6 months. Thereafter, cell loss occurs much more slowly, and the remaining photoreceptor cells are lost within a 3-4 year period. The genetic basis for photoreceptor dysplasia is unknown. Zhang Q, Baldwin VJ, Acland GM, Parshall CJ, Haskel J, Aguirre GD, Ray K. Photoreceptor dysplasia (pd) in miniature schnauzer dogs: evaluation of candidate genes by molecular genetic analysis. J Hered. 1999 Jan-Feb;90(1):57-61. 8. Briard (RPE-65) Dog Model for ARRP and LCA

The Briard dog is affected with a recessively inherited retinal disorder characterized by congenital night blindness with various degrees of visual impairment under photopic illumination. Vision in affected dogs ranges from normal day vision to profound day blindness. The disease was initially described in Swedish dogs as a stationary disorder analogous to human congenital stationary night blindness. More recently, the disease has been described as having a progressive component, and has been termed hereditary retinal dystrophy. 19

Along with the visual impairment, affected dogs have an abnormal electroretinogram (ERG); in general, the recorded responses are normal in waveform, but show a marked diminution of response amplitudes, similar to a "Riggs type" ERG in man. The ERG recorded under DC conditions shows complete absence of the a-, b-, and c-waves, with the latter waveform being replaced by a very slow negative potential which develops when the stimulus intensity is greater than 3 log units above the normal b-wave threshold. Electron microscopy studies of the retina from Briard dogs demonstrate large inclusions, seemingly lipid in nature, mainly in the central and tapetal areas of the retina. Small, membrane bound, electron-dense inclusions are found to be scattered in the RPE cytoplasm and forty to fifty percent of the rod outer segments in the tapetal area show disorientation of the disc membranes. Gal and colleagues identified a homozygous 4-bp deletion (485delAAGA) in the Briard RPE65 gene, which encodes a 65-kDa microsomal protein expressed exclusively in retinal pigment epithelium (RPE). Mutations in the human RPE65 gene are also present in patients with autosomal recessive, Leber congenital amaurosis. Clinical features of the canine disease are quite similar to those described in human. Therefore this form of canine retinal dystrophy provides an attractive animal model of two corresponding human disorders with immediate significance for various therapeutic approaches, including RPE transplantation. Veske A, Nilsson SE, Narfstrom K, Gal. A Retinal dystrophy of Swedish briard/briardbeagle dogs is due to a 4-bp deletion in RPE65. Genomics. 1999 Apr 1;57(1):57-61. Aguirre GD, Baldwin V, Pearce-Kelling S, Narfstrom K, Ray K, Acland GM. Congenital stationary night blindness in the dog: common mutation in the RPE65 gene indicates founder effect. Molecular Vision 1998 Oct 30;4:23. Wrigstad A, Narfstrom K, Nilsson SE. Slowly progressive changes of the retina and retinal pigment epithelium in Briard dogs with hereditary retinal dystrophy. A morphological study. Doc Ophthalmol. 1994;87(4):337-354.

VI.

RETINITIS PIGMENTOSA (X-LINKED) X-linked retinitis pigmentosa (XLRP) is perhaps the most devastating form of RP because of the severity and early onset of the disease, and may account for as much as 25% of RP families. Most males with XLRP show early onset of visual symptoms with night blindness before the age of 20 and are totally blind by the age of forty. At the present time, mutations in 2 different genes (RP-3 and RP-2) have been shown to be responsible for the disease.

A.

RODENT MODEL FOR XLRP 1. RPGR Knock Out Mouse 20

The X-linked RP3 locus codes for retinitis pigmentosa GTPase regulator (RPGR), a protein of unknown function with sequence homology to the guanine nucleotide exchange factor for Ran GTPase. Hong and colleagues created an RPGR-deficient murine model by gene knockout. The knockout mice exhibit cone photoreceptors with ectopic localization of cone opsins in the cell body and synapses and rod photoreceptors with a reduced level of rhodopsin. Both cone and rod photoreceptors degenerate within 6 months of birth. Studies on the wild type mouse, revealed that the RPGR protein is localized to the connecting cilia of rod and cone photoreceptors. These data point to a role for RPGR in maintaining the polarized protein distribution across the connecting cilium by facilitating directional transport or restricting redistribution. The function of RPGR may be essential for the long-term maintenance of photoreceptor viability. Hong DH, Pawlyk BS, Shang J, Sandberg MA, Berson EL and Li T. A retinitis pigmentosa GTPase regulator (RPGR)-deficient mouse model for X-linked retinitis pigmentosa (RP3). Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):364954. B. CANINE MODEL FOR XLRP (RP-3) 1. X-linked progressive retinal atrophy (XLPRA)

XLPRA is an example of an animal model in which a mutation of an X-chromosome gene results in a progressive disease resulting in selective degeneration of the photoreceptors and blindness. Because of conservation of genes on the X-chromosome between mammals, this model lends itself to positional cloning to locate the XLPRA locus, and identify the gene and mutation. At present, the XLPRA locus has been mapped to the RP3 interval and the disease shows tight linkage, LOD score greate than 11 and no recombiniations with RPGR. However, no disease causing mutations have been identified in the gene. is within a 15 cM region on either side of the androgen receptor gene which suggests that it can be a homologue of choroideremia or the RP2 locus. The XLPRA colony represents the largest multi-generational pedigree of an X-linked retinal degeneration in either man or animals in which all have the same gene defect. Acland GM, Blanton SH, Hershfield B, Aguiree GD. XLPRA: a canine retinal degeneration inherited as an X-linked trait. Am J Med Genet. 1994 Aug 1;52(1):27-33.

VII.

STARGARDT DISEASE Stargardt disease is the most common inherited “juvenile” form of macular degeneration. The disease exhibits an autosomal recessive pattern. Children typically begin experiencing central vision loss between 6 and 12 years of age. Although peripheral vision remains unaffected, individuals with Stargardt disease usually experience rapid and 21

severe central vision impairment. Mutations in the ATP-Binding Cassette Retina (ABCR) gene product, “Rim Protein” (RmP), is responsible for the disease. The ABCR gene is expressed exclusively and at high levels in the retina, in rod but not cone photoreceptors. The ABCR gene may also be responsible for some forms of age-related macular degeneration as well. A. RODENT MODEL FOR STARGARDT DISEASE 1. ABCR Knock Out Mouse Model for Stargardts Disease

Mice lacking RmP show delayed dark adaptation, increased all-trans-retinaldehyde (alltrans-RAL) following light exposure, elevated phosphatidylethanolamine (PE) in outer segments, accumulation of the protonated Schiff base complex of all-trans-RAL and PE (N-retinylidene-PE), and striking deposition of a major lipofuscin fluorophore (A2-E) in retinal pigment epithelium (RPE). Data suggests that RmP functions as an outwardly directed flippase for N-retinylidene-PE. Delayed dark adaptation is likely due to accumulation in discs of the noncovalent complex between opsin and all-trans-RAL. Finally, ABCR-mediated retinal degeneration may result from "poisoning" of the RPE due to A2-E accumulation, with secondary photoreceptor degeneration due to loss of the RPE support role. Therefore, the ABCR knock out mouse may be a useful animal model for studying both Stargardts disease as well as age-related macular degeneration. Weng J, Mata NL, Azarian SM, Tzekov RT, Birch DG and Travis GH. Insights into the function of Rim protein in photoreceptors and etiology of Stargardt's disease from the phenotype in abcr knockout mice. Cell. 1999 Jul 9;98(1):13-23. Azarian SM and Travis GH. The photoreceptor rim protein is an ABC transporter encoded by the gene for recessive Stargardt's disease (ABCR). FEBS Lett. 1997 Jun 9;409(2):247-52. Allikmets R, Singh N, Sun H, Shroyer NF, Hutchinson A, Chidambaram A, Gerrard B, Baird L, Stauffer D, Peiffer A, Rattner A, Smallwood P, Li Y, Anderson KL, Lewis RA, Nathans J, Leppert M, Dean M and Lupski JR. A photoreceptor cell-specific ATPbinding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy. Nat Genet. 1997 Mar;15(3):236-246. VIII. USHER SYNDROME Usher syndrome is an autosomal recessive disease that is characterized by moderate to profound hearing impairment and progressive vision loss due to retinitis pigmentosa (RP). It is the major cause of deaf blindness. Approximately 10-15,000 people in the United States have Usher syndrome. There are three known forms of Usher syndrome. Individuals with Usher syndrome type I are born profoundly (completely) deaf and experience problems with balance. In adolescence, they usually begin to exhibit the first signs of RP: night blindness and loss of peripheral vision. Individuals with Usher syndrome type II experience moderate to severe hearing impairment at birth, but they do not have balance problems. Symptoms of RP develop 22

later in adolescence. With Usher syndrome type III, hearing loss and vision loss due to RP are both progressive. At the present time, two genes (myosin VIIA and Usher IIa protein) have been shown to be responsible for Usher syndrome Ib and IIa.

A.

RODENT MODEL FOR USHER SYNDROME 1. Shaker Mouse

Mutations in the murine myosin VIIA gene lead to the shaker-1 phenotype, which manifests cochlear and vestibular dysfunction. However, there is no obvious degeneration of the retina in these animals. The FFB is currently providing support to Dr. David Williams, USC, to develop an animal model for Usher syndrome Ib. Weil D, Blanchard S, Kaplan J, Guilford P, Gibson F, Walsh J, Mburu P, Varela A, Levilliers J, Weston MD. Defective myosin VIIA gene responsible for Usher syndrome type 1B. Nature. 1995 Mar 2;374(6517):60-61. Liu X, Ondek B, Williams DS. Mutant myosin VIIa causes defective melanosome distribution in the RPE of shaker-1 mice. Nat Genet. 1998 Jun;19(2):117-8.

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