1 / 74

Lecture #20

Lecture #20. Treatment of eye disease 4 /11/13. 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. Finding references. Pubmed

lukas
Download Presentation

Lecture #20

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

  30. Subretinal injection of AAV-RPE65 gene

  31. Retinal photoreceptor distributions before treatment PR thickness

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

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

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

  35. Transgenic animals to enhance color processing

  36. 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

  37. 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

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

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

  40. If cross mice will get homozygous..

  41. …and heterozygous mice

  42. 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?????

  43. 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

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

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

  46. 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?

  47. Compare threshold at 500nm and 600nm

  48. 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

  49. 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

More Related