Transcript Leber Congenital Amaurosis Type 2

Leber Congenital Amaurosis
Type 2: The Lucky Ones
Presented by Emili Watts
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?
• 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 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 101
Source: http://www.retinaaustralia.com.au/images/eye6.gif
Eye 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-transretinal (under dark conditions), and back again26.
 Note: RPE65 mediates the metabolism of all-trans-retinal esters to 11-cisretinoids24.
The 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:
•
•
•
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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
• 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 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 functions
1. 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 11cis-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 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.
• 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.
• 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 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 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 Treatment
• Drugs
• ???
• Transplant therapies
• RPE transplant
• Retinal implants
• Viral vectors for gene therapy22
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•
•
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Adenovirus vectors
Adeno-associated viral vector
Herpes Simplex Vector
Lentivirus Vector
Potentials 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 wildtype 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 Mouse
•
•
•
•
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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/02SRgallery/Building%20on%20the%20Basics/images/03Wide%20mouse.jpg
•Great for blind mice, but what
about the bigger animal?
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.
• 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)
• 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!!!
Glossary
• 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 fulllength 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)
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.
References (page 2)
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=Retrie
ve&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 epitheliumspecific protein RPE65 is localized to human 1p31 and mouse 3.
Genomics 20: 509-512, 1994.
References (page 3)
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&p
ubmedid=15109394
12. Leroy, J. J.; Dharmaraj, S.: Leber congenital amaurosis. Orphanet
http://www.orpha.net/data/patho/GB/uk-LCA.pdf , 2003.
References (page 4)
13. Li, T.; Davidson, B. L.: Phenotype correction in retinal pigment
epithelium in murine mucopolysaccharidosis VII by adenovirusmediated 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&do
pt=graphics&list_uids=6121
References (page 5)
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&ma
ps=locr.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: 641649, 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
References (page 6)
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
References (page 7)
28. Vector Development Lab: Material Safety Data Sheet-AdenoAssociated 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.