Leber Congenital Amaurosis Type 2: The Lucky Ones Presented by Emili Watts.

Leber Congenital AmaurosisType 2: The Lucky Ones

Leber Congenital AmaurosisType 2: The Lucky Ones

Presented by Emili Watts

LCA(2)LCA(2)

• This disease was discovered by Dr. Theodor Karl Gustav von Leber (1840-1917).

• He was a german opthalmologist.

• Also defined Leber’s Optic Atrophy.

• Photo courtesy of http://www.mrcophth.com/ww/leber.html

LCA(2): What is it?LCA(2): What is it?

• Leber Congential Amaurosis is a classification of individuals who are all at the most severe end of a spectrum measuring autosomal recessive early onset retinal degeneration. • LCA is nearly always inherited as an autosomal recessive

disease.• Mutant CRX gene acting dominant.

• Lesser severe cases have been termed “autosomal recessive childhood-onset retinal dystrophy” and “juvenile retinitis pigmentosa.”

• Common characteristics of LCA are:• Nystagmus, poor pupilary reflexes, diminished or extinguished

ERG responses, and eye poking.

Types of LCATypes of LCA

• There are currently 7 well characterized forms of LCA9:• LCA(1): mutation in GUCY2D on 17p13.1; accounts for 21.2% of known LCA cases.• LCA(?): mutation in CRB1 on 1q31; 10% of known LCA cases.• LCA(2): mutation in RPE65 on 1p31; 6.1% of known LCA cases.• LCA(6): mutation in RPGRIP on 14q11; 4.5% of known LCA cases.• LCA(4): mutation in AIPL1 on 17p13.1; 3.4% of known LCA cases.• LCA(?): mutation in TULP1 on 6q21.3; 1.7% of known LCA cases.• LCA(?):mutation in CRX on 19q13.3; 0.6% of known LCA cases.

• This presentation will focus on LCA type 2.• Leroy et al. report that LCA affects 10-20% of blind children. • Lai et al. reports that 10-15% of LCA cases are of LCA(2) origin.

• 57.9% of mutations in the RPE65 gene are null mutations. • Most mutations in RPE65 result in partial or complete loss of function of

[the protein] RPE65, whose function is essential in the Visual Cycle Pathway.

But first…Eye A&P 101But first…Eye A&P 101

Source: http://www.retinaaustralia.com.au/images/eye6.gif

Eye Anatomy Key TermsEye Anatomy Key Terms• Photoreceptor Cells

• Inclusive of rod or cone bipolar neurons. Rod Neurons

Studded with rhodopsin Responsible for vision in reduced light.

Cone Neurons Responsible for day vision and color differentiation.

Rhodopsin Composed of Vitamin A derivative 11-cis-retinal chromophore and the opsin apoprotein. It is the key molecule in the Visual Cycle Pathway.

ElectroRetinoGraph (ERG) When the retina of the eye is stimulated by a flash of light, there is a characteristic sequence

of electrical potentials generated within the retina. The clinical electroretinogram (ERG) is a recording of these potentials.

Visual Cycle Pathway Process by which 11-cis-retinal is converted (by exposure to light) to all-trans-retinal (under

dark conditions), and back again26. Note: RPE65 mediates the metabolism of all-trans-retinal esters to 11-cis-retinoids24.

The Retinal Pigment EpitheliumThe Retinal Pigment Epithelium

• The Retinal Pigment Epithelium is the epithelial layer that separates the retina from the choroid tissue in the eye.

• This is the site of recycling of all-trans-retinal to 11-cis-retinal.

• See handout for Visual Cycle Pathway (Xue et al.)29.

• Why this pathway is important:• 11-cis-retinal is what allows rhodopsin to absorb light

in the visible range; without it’s regeneration, we lose the ability to see light.

RPE65 Fun Facts RPE65 Fun Facts

• The RPE65 gene is 23kb long, encoding 14 exons.

• cDNA length is 3.15kb long, and encodes the 533 amino acid RPE65 protein.

• RPE65 is only found in the RPE as confirmed by Northern Blot analysis of RPE, retina, iris, brain, liver, lung, heart, kidney, and small intestine7.

RPE65 Fun Facts29RPE65 Fun Facts29

• The RPE65 protein is present in 2 distinct forms in vivo, which are indicative of the proteins regulation:• mRPE65- The membrane associated form, which performs

two functions1. Retinoid Binding Protein: Acts as a chaperone for all-trans-retinyl

esters and prepares them for processing by isomerhydrolase (IMH), which converts them to 11-cis-retinol.

2. In the presence of LRAT, mRPE65 acts as a palmitoyl donor for 11-cis-retinol.

• sRPE65- The soluble, unpalmitoylated form of the protein.• This form functions by steriospecifically binding vitamin A, making

it available for LRAT.

• Note: LRAT (lecithin:retinol acyltransferase) catalyzes the esterification of retinol.

Cloning of the gene7Cloning of the gene7

• Hamel et al. (1993)

• Protein purification:• Isolated fresh bovine RPE cells• Ran an immunoblot using RPE9 antibody to

detect RPE65.• Digested the protein with trypsin, chymotrypsin

and Edman Reagent to obtain fragments.

Cloning of the gene, cont7.Cloning of the gene, cont7.

• Screening of cDNA library• 10ug of fresh bovine RPEpoly(A)+ RNA was

used to generate a cDNA library in lambda phage Zap II.

• An 84 nucleotide guessomer was created based upon the amino acid composition of fragment CH-72 obtained by chymotrypsin digestion.

Cloning of the gene, cont7.Cloning of the gene, cont7.

• Screening of the cDNA library and sequence determination.• Three clones were isolated (pPE1, pPE2, and pPE3), and

DNA sequenced to determine authenticity; pPE2 was found to be a foreign cDNA.

• pPE3 was found to hybridize with two other oligonucleotide fragments when ran on an ethidium bromide 1% agarose gel.

• RACE was performed to yield 5’ end clones pPE4 and pPE5, and 3’end clone pPE6.

• Restriction mapping of clones was performed to obtain the RPE65 cDNA sequence (See Handout: Hamel et al.).

• The actual amino acid sequence matched the obtained cDNA sequence.

Gene Localization8Gene Localization8

• Hamel et al. (1994)• Used a human-hamster somatic cell hybrid to place and

FISH to refine RPE65 to the short arm of chromosome 1, loci 31 in humans.• Human-Hammy HindIII digested DNA panels hybridized to

pPE3.• RPE65 genomic clone (pPE275) was labeled and used to find

the RPE65 locus.

• Used interspecific backcross analysis to map the RPE65 mouse homologue to the distal arm of mouse chromosome 3.• RFLPs were used to follow the segregation of the RPE65

locus in backcross mice.

Potential for diagnosisPotential for diagnosis

• Hanein et al. developed a flowchart to aid in LCA type diagnosis.• See handout (Hanein et al.).• Used genotype-phenotype survey of 179

unrelated patients to develop.

Potentials for TreatmentPotentials for Treatment

• Drugs• ???

• Transplant therapies• RPE transplant• Retinal implants

• Viral vectors for gene therapy22

• Adenovirus vectors• Adeno-associated viral vector • Herpes Simplex Vector• Lentivirus Vector

Potentials for Viral VectorsPotentials for Viral Vectors

• Adenovirus Vector (BSL 2)• Favorable because of high transduction efficiency, broad

host range, ability to infect non-dividing cells, • Encoded viral genes can elicit an immune response,

defeating initial delivery, and sabotaging future attempts.

• Adeno-associated Viral Vector (BSL2)• Better candidate because it doesn’t cause any known

disease, thus doesn’t trigger an immune response. • Replication of AAV depends upon presence of wild-type

adenovirus or herpesvirus; if absent, AAV will stably integrate into the host chromosome28.

• Potential problem: AAV integrates via non-homologous recombination in absence of Rep gene.

rAAV-RPE65 in MouserAAV-RPE65 in Mouse

• Gene therapy attempts with recombinant adenovirus as early as 2000 in lab rodents13.

• Lai et al. were published in April 2004 for using rAAV-RPE65 to help restore vision in a mouse model.

• Viral induced RPE65 expression was detectable for up to 18 months post subretinal injection.

• Results: Though the viral delivery was able to induce restarting of the visual cycle and phototransduction in the remaining photoreceptors in RPE65-/- mice, it was unable to slow or halt the photoreceptor degeneration.

• Some vision was recovered in the treated mice, as shown by improved ERG responses.

Photo courtesy of http://www.cals.wisc.edu/sciencereport/02SR-gallery/Building%20on%20the%20Basics/images/03-Wide%20mouse.jpg

•Great for blind mice, but what about the bigger animal?

rAAV-RPE65 in Dogs (p1)rAAV-RPE65 in Dogs (p1)

• Acland et al. (2001) used a naturally occuring large animal model (the RPE65-/- Swedish Briard dog) to explore the possibilities of rAAV-RPE65 gene therapy.

• Tested the effects of both intravitreal and subretinal injection of the vector.

• Qualitative visual assessments were performed 4 months following injection:• ERG, pupillometry and

behavior tests were used to analyze results

Photo courtesy of http://www.dreamscape.com/blueribbonprintwear/briard.jpg

rAAV-RPE65 in Dogs, cont.rAAV-RPE65 in Dogs, cont.

• Results • Intravitral injection was not that successful: no change in

ERG response was observed. • Subretinal injection showed improved results in both

behavioral and physiological fields.• Behavior testing: under dim red light, subretinally injected eyes were

able to consistently avoid objects, whereas untreated or intravitreally injected eyes showed no perception of objects placed in their way.

• ERG responses were improved in eyes that had been subretinally injected.

• Pupillometry showed partial recovery of pupillary response to suprathreshold intensity stimulus.

• These are very encouraging results!!!

LCA type 2 Patients Are “Lucky” (relatively speaking)

LCA type 2 Patients Are “Lucky” (relatively speaking)

• The success with gene therapy in the murine and canine models are very encouraging for those with type two LCA.

• Thus LCA type 2 victims are most likely to be the first treated.

THE END!!!

GlossaryGlossary

• Apoprotein: A polypeptide that combines with a prosthetic group to form a conjugated protein.

• BSL: Guidelines endorsed by the NIH and CDC used for characterization of substance and handling procedures.

• Chromophore: A chemical group capable of selective light absorption resulting in the coloration of certain organic compounds.

• Nystagmus: A rapid, involuntary, oscillatory motion of the eyeball.

• Palmitoylated: Addition of a palmitoyl group. • RACE: rapid amplification of cDNA ends; Used for cloning full-

length 5' or 3' ends of a cDNA. An adapter sequence is added to either 5' or 3' ends of cDNA. The two PCR primers are either specific to the adapter or specific to known sequences of cDNA.

References (page 1)References (page 1)

1. Acland, G.M; Aguirre, G.D.; Ray, J.; Zhang, Q.; Aleman, T.S.; Cideciyan, A.V.; Pearce-Kelling S.E.; Anand, V.; Zeng, Y.; Maguire, A.M.; Jacobson, S.G.; Hauswirth, W. W.; Bennett, J.: Gene therapy restores vision in a canine model of childhood blindness. Nature Genetics 28:92-95, 2001.

2. Aguirre, G. D.; Baldwin, V.; Pearce-Kelling, S.; Narfstrom, K.; Ray, K.; Acland, G. M. : Congenital stationary night blindness in the dog: common mutation in the RPE65 gene indicates founder effect. Molecular Vision 4: 23, 1998.

3. Bavik, C.-O.; Busch, C.; Eriksson, U. : Characterization of a plasma retinol-binding protein membrane receptor expressed in the retinal pigment epithelium. The Journal of Biological Chemistry 267: 23035-23042, 1992.

4. Cremers, F. P. M.; van den Hurk, J. A. J. M.; den Hollander, A. I. : Molecular genetics of Leber congenital amaurosis. Human Molecular Genetics 11: 1169-1176, 2002.


5. Entrez Gene. RPE65 retinal pigment epithelium-specific protein 65kDa. Article can be found at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&dopt=Graphics&list_uids=89826

6. Gu, S.; Thompson, D. A.; Srikumari, C. R. S.; Lorenz, B.; Finckh, U.; Nicoletti, A.; Murthy, K. R.; Rathmann, M.; Kumaramanickavel, G.; Denton, M. J.; Gal, A. : Mutations in RPE65 cause autosomal recessive childhood-onset severe retinal dystrophy. Nature Genetics 17: 194-197, 1997.

7. Hamel, C. P.; Tsilou, E.; Pfeffer, B. A.; Hooks, J. J.; Detrick, B.; Redmond, T. M. : Molecular cloning and expression of RPE65, a novel retinal pigment epithelium-specific microsomal protein that is post-transcriptionally regulated in vitro. The Journal of Biological Chemistry 268: 15751-15757, 1993.

8. Hamel, C. P.; Jenkins, N. A.; Gilbert, D. J.; Copeland, N. G.; Redmond, T. M. : The gene for the retinal pigment epithelium-specific protein RPE65 is localized to human 1p31 and mouse 3. Genomics 20: 509-512, 1994.


9. Hanein, S.; Perrault, I.; Gerber, S.; Tanguy, G.; Barbet, F.; Ducroq, D.; Calvas, P.; Dollfus, H.; Hamel, C.; Lopponen, T.; Munier, F.; Santos, L.; Shalev, S.; Zafeiriou, D.; Dufier, J.-L.; Munnich, A.; Rozet, J.-M.; Kaplan, J. : Leber congenital amaurosis: comprehensive survey of the genetic heterogeneity, refinement of the clinical definition, and genotype-phenotype correlations as a strategy for molecular diagnosis. Human Mutation 23: 306-317, 2004.

10. Johnston, N.: Update on gene therapy. Modern Drug Discovery 4: 43-48, 2001.

11. Lai, C. H.; Yu, M. J. T.; Brankov, M.; Barnett, N. L.; Redmond, T. M.; Narfstron, K.; Rakoczy, P. E.: Recombinant adeno-associated virus type 2-mediated into the Rpe65-/- knockout mouse eye results in limited rescue. Genetic Vaccines Therapy 2: 3, 2004. Also available at http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=15109394

12. Leroy, J. J.; Dharmaraj, S.: Leber congenital amaurosis. Orphanet http://www.orpha.net/data/patho/GB/uk-LCA.pdf , 2003.


13. Li, T.; Davidson, B. L.: Phenotype correction in retinal pigment epithelium in murine mucopolysaccharidosis VII by adenovirus-mediated gene transfer. Proceedings of the National Academy of Science 92: 7700-7704, 1995.

14. Marlhens, F.; Bareil, C.; Griffoin, J.-M.; Zrenner, E.; Amalric, P.; Eliaou, C.; Liu, S.-Y.; Harris, E.; Redmond, T. M.; Arnaud, B.; Claustres, M.; Hamel, C. P. : Mutations in RPE65 cause Leber's congenital amaurosis. (Letter) Nature Genetics 17: 139-141, 1997.

15. Morimura, H.; Fishman, G. A.; Grover, S. A.; Fulton, A. B.; Berson, E. L.; Dryja, T. P. : Mutations in the RPE65 gene in patients with autosomal recessive retinitis pigmentosa or Leber congenital amaurosis. Proceedings of the National Academy of Science 95: 3088-3093, 1998.

16. NCBI Entrez Gene. Retrieved Oct. 4th, 2004. Article can be found at www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=retrieve&dopt=graphics&list_uids=6121


17. NCBI Map Viewer. Retrieved Oct. 4th 2004. Article can be found at www.ncbi.nlm.nih.gov/mapview/maps/cgi?ORG=hum&CHR=1&maps=loc-r.morbid.gene&R1=on&query=RPE65&VERBOSE=ON&Zoom=3

18. Nicoletti, A.; Wong, D. J.; Kawase, K.; Gibson, L. H.; Yang-Feng, T. L.; Richards, J. E.; Thompson, D. A. : Molecular characterization of the human gene encoding an abundant 61 kDa protein specific to the retinal pigment epithelium. Human Molecular Genetics 4: 641-649, 1995.

19. OMIM. Leber Congenital Amaurosis, Type 1; LCA1. Retrieved Oct. 4th 2004. Article can be found at http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=204000

20. OMIM. Leber Congential Amaurosis, Type 2; LCA2. Retrieved Oct. 4th 2004. Article can be found at http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=204100

21. OMIM. Retinal Pigment Epithelium-Specific Protein, 65-KD; RPE65. Retrieved Oct. 4th 2004. Article can be found at http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=180069


22. Pleyer, U.: Gene therapy in hereditary retinal degeneration and the tower of Babel. British Journal of Opthalmology 85: 341-344, 2001.

23. Redmond, T. M.; Harris, E. W.; Yu, S.; Liu, S. Y.; Kapsis, A.; Hamel, C. P.: Analysis of the Human Gene for the Retinal Pigment Epithelium-Specific Protein RPE65. Investigative Opthalmology and Visual Science 36: S598, 1995.

24. Redmond, T. M.; Yu, S.; Lee, E.; Bok, D.; Hamasaki, D.; Chen, N.; Goletz, P.; Ma, J.-X.; Crouch, R. K.; Pfeifer, K. : Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nature Genetics 20: 344-351, 1998.

25. Rutledge, E. A.; Russell, D. W.: Adeno-Associated Virus Vector Integration Junctions. Journal of Virology 71: 8429-8436, 1997.

26. Saladin, K. S. “Anatomy and Physiology: The Unity of Form and Function” pp.580-591, 1998.

27. University of Pennsylvania Health System: Gene Therapy Program: Vector Core. 2003. Article can be found at www.uphs.upenn.edu/penngen/gtp/vcore_av.html#top


28. Vector Development Lab: Material Safety Data Sheet-Adeno-Associated Virus Serotype 2 Recombinant Vectors. Retrieved Oct. 4th 2004. Article can be found at http://medicine.ucsd.edu/gt/AAV.html

29. Xue, L.; Gollapalli, D. R.; Maiti, P.; Jahng, W. J.; Rando, R. R. : A palmitoylation switch mechanism in the regulation of the visual cycle. Cell 117: 761-771, 2004.

Leber Congenital Amaurosis Type 2: The Lucky Ones Presented by Emili Watts.

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