Lecture #20

1. Lecture #20 Treatment of eye disease 4/11/13

2. Next week • Create your first page • Introduction to topic and why it is important • Doesn’t have to be final version • Picture to illustrate your topic • List of 3-5 references that will be key to your project

3. Finding references • Pubmed • Select Reviews • Filter for free availability • Some of the journals will be available through our library • Request any you need from ILL - NOW

4. Naming pages uniquely • Name your pages with a unique name (perhaps including your initials) • References subpage • If everyone makes a page called “References” they will write over each other

5. Wiki - Table of contents • If at least four headers on a page • table of contents (TOC) appears in front of the first header (or after introductory sections). • Putting __TOC__ anywhere forces the TOC to appear (that is two underscores _ _ before and after) • Putting __NOTOC__ anywhere forces the TOC to disappear.

6. Sources • Use primary references • Try not to use other web pages • Telephone game • Ill try to help find a few papers for each topic

7. For Thursday next week • Make your introductory page • How organize home page • Identify 3-5 references • Put on intro page or stubs for other pages

8. Gene therapy The future promise of curing all disease

9. Gene therapy • Many diseases of the eye are the result of single gene mutations • Over 200 genes now known • Rhodopsin, phototransduction pathway, visual cycle • If you could insert a gene to repair damage - the disease would be cured

10. Ideal scenario for gene therapy • Know the mutated gene to replace • Have good copy of gene • Understand biology • Know tissue and how gene is acting • Know that adding gene back will solve problem • The eye is a contained organ so therapy does not impact other organs

11. Ocular gene therapy • Three kinds of therapies • Introduce a gene to make a protein which alleviates some of symptoms • Introduce a replacement gene to fix mutation • Introduce a gene to knock out faulty gene (RNAi)

12. What is needed to make this happen? • Gene expressed to sufficient extent • Make enough “stuff” to provide relief • Gene expressed for long time • Retinal cells don’t divide so doesn’t need to integrate with DNA • But does need strong promoter so expressed in tissue of interest • No cause of inflammatory or immunogenic response

13. Vectors for gene delivery - Viruses • Pros • Existing method for getting DNA into cells • May be engineered to target particular cells • Can be modified so don’t replicate and destroy cell • Cons • Virus can only handle a gene up to certain size • May trigger immune response making person sick • Person may develop immunity so virus gets destroyed

14. Getting the stuff to the right place Alqawlaq et al 2012

15. Getting gene to tissue of interest • In vivo • Add directly to tissue in the body • Ex vivo • Remove cells of interest • Culture cells • Add vector containing gene of interest • If gene integrates, add back to body

16. Advantages of viruses • Common in humans • 43 different types • Rapidly infect many kinds of human cells with high gene transfer rate • Low pathogenicity • Can hold up to 7.5 kb of DNA • Viral DNA is stable with no rearranging • Viral DNA is easy to manipulate

17. Gene therapy with adenovirus

18. Nature Genetics 28: 92 (2001)

19. Visual cycle (lecture 16) RPE 65 is key isomerase in RPE to convert all trans retinal ester to 11-cis retinol Mutations cause Leber congenital amaurosis

20. Dog model : Swedish Briard • Have mutation in RPE65 • 4 bp deletion • Congenital stationary night blindness • Congenital - from birth • Stationary - stable • Night blindness - affects rods • Can also have some degeneration with time

21. First show can treat RPE cells with adeno-associated virus containing RPE65 to rescue mutant RPE65-/- WT RPE cels RPE65-/- AAV treated WT retina RPE65-/- Nuclei are orange from propidium iodide staining RPE65 antibody glows green

22. In vivo treatment - Divide eye into retinal quadrantsInject AAV-RPE65 into TS NS Nasal superior TS Temporal-superior NI Nasal inferior TI Temporal inferior

23. PCR of DNA from wild type and mutant RPE65 Wild type 109 bp Mutant w/ 4 bp deletion 105 bp

24. Use PCR to screen for expressed gene after innoculate with AAV - RPE65 RPE65 Individual dogs: WT, hetero- and homozygous mutant Cultured RPE cells WT, mutant pre- and post-treatment Persistent expression of new RPE65 form (99 days after inject)

25. Use PCR to screen for expressed gene after innoculate with AAV - RPE65 RPE65 R=retina P=RPE In vivo treatment. See injected functional RPE65 in TS region only Not in other 3 eye quadrants

26. Use PCR to screen for expressed gene after innoculate with AAV - RPE65 RPE65 genomic RT PCR / cDNA Get expressed cDNA only in RPE of quadrant where injection occurred.

27. Electroretinograms show improvement resulting from AAV-RPE65 Downward a wave from photoreceptors Upward b wave from bipolars, oscillations from amarcrine

28. Injections must be subretinal Intravitreal injections don’t work

29. Video by Acland et al showing dog behavior

31. Subretinal injection of AAV-RPE65 gene

32. Retinal photoreceptor distributions before treatment PR thickness

33. Light sensitive area increases as does light sensitivity by:P1 10xP2 100xP3 1000xTime after injection1 month2 months3 months

34. Enhanced light sensitivity to that expected based on number of photoreceptors they have So 11-cis retinal supply is much better!

35. Retinal gene therapy • Is well on its way!

36. Transgenic animals to enhance color processing

37. Jeremy Nathans • Professor, Johns Hopkins and HHMI • BS Chemistry and Biology MIT • PhD Biochemistry and MD, Stanford • Sequenced the bovine rhodopsin gene • Sequence the human rod and cone opsin genes

38. Gerald Jacobs • Professor, UCSB • BA U Vermont • PhD Indiana U • Asst Prof UT Austin • UCSB starting in 1969 • Electrophysiology, psychophysics • Human and primate red/green vision

39. Normal mouse visual pigments UV cone - 360 nm Green cone - 508 nm

40. Engineered a new mouse where green gene replaced by red UV cone - 365 nm Red cone - 565 nm

41. If cross mice will get homozygous..

42. …and heterozygous mice

43. Just like primates • Different alleles on X chromosome • Females can be heterozygous • X inactivation will result in some cones expressing green gene and some expressing the red gene • Does this enable enhanced color vision?????

44. Test spectral sensitivity of different mice 512 nm n=12 556 nm n=17 n=87 • Use electroretinogram to measure sensitivity of entire retina • For heterozygous mice can estimate the fraction of cones which have L pigment

45. Heterozygous mice • Get range of L:M cone ratios in heterozygotes • Differences in X inactivation and / or expression?

46. Behavioral testing Operant training - mouse gets drop of soy milk as reward for choosing the light that differs from the other two

47. Do L cones contribute to light detection? • Compare thresholds at which can see difference from achromatic backgrounds • Add different amounts of either 500 nm or 600 nm light • What is threshold needed to distinguish it from white?

48. Compare threshold at 500nm and 600nm

49. Mice with M+L cones more sensitive to 600 nm light than those with only M How much more red light vs green light is needed to get behavioral response Threshold difference report as Log 500 nm/600nm If log x = 1 then x = 101

50. Mice with M+L cones more sensitive to 600 nm light than those with only M So if only M cones, response requires 101.3=20x more 600 nm than 500 nm light If M+L cones, response requires only 10.85= 7x more 600 nm light than 500 nm light So L cone makes more sensitive to 600 nm light