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Presenter: Juan Mo

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Presenter: Juan Mo. Learning objectives. What is gene therapy? What is a Dox-regulatable system and how can gene expression be regulated by Dox? What is the difference between TetON and TetOFF? What is the role of RPE65? What is electroretinography (ERG)?. Introduction.

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Presenter: Juan Mo

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  1. Presenter: Juan Mo

  2. Learning objectives • What is gene therapy? • What is a Dox-regulatable system and how can gene expression be regulated by Dox? • What is the difference between TetON and TetOFF? • What is the role of RPE65? • What is electroretinography (ERG)?

  3. Introduction • Gene therapy: the use of DNA as a pharmaceutical agent to treat disease. • Parameters when choosing a gene therapy vector: (1) size limitations for insertion of transgenes, (2) purity and titer of the vector, (3) transduction efficiency, (4) ability to infect dividing and/or quiescent cells, (5) long-term expression of transgenes, (6) integration into the host genome, (7) the need for cell-type specificity or targeted delivery, (8) vector-associated toxicity and immunogenicity.

  4. Virus Vector • Adenovirus • Adeno-associated virus • Herpes simplex virus type 1 • retrovirus

  5. Regulatory System • Doxycycline: is a member of the tetracycline antibiotics group. Doxycycline is used in "Tet-on" and "Tet-off" tetracycline-controlled transcriptional activation to regulate transgene expression in organisms and cell cultures. tTA (Tet-off) or rtTA (Tet-on) : Tet-controlled transcriptional activator TRE: tetracycline response element

  6. tTA (Tet-off) or rtTA (Tet-on) : Tet-controlled transcriptional activator Tet-OFF Regulatable Switch TRE: tetracycline response element

  7. Tet-ON Regulatable Switch tTA (Tet-off) or rtTA (Tet-on) : Tet-controlled transcriptional activator TRE: tetracycline response element

  8. RPE65: The retinal pigment epithelium-specific 65 kDa protein is located in the retinal pigment epithelium and is involved in the conversion of all-trans retinyl ester to 11-cis retinol during phototransduction, which is then used in visual pigment regeneration in photoreceptor cells.

  9. The ability of the Dox-regulatable system (Tet-OFF and Tet-ON) to regulate retinal function in RPE65-deficient dogs??

  10. transactivator Results and Methods cDNA promoter promoter A1 to A3 transactivator promoter promoter cDNA A4 and A5 A6

  11. Subretinal Injection • http://www.jove.com/video/4286/subretinal-injection-gene-therapy-vectors-stem-cells-perinatal-mouse

  12. AAV2/4TetOff.rpe65 (A2) AAV2/4TetOn.rpe65 (A4) CT CT 6mpi 6mpi Zone of the bleb AAV2/4CMV.rpe65 (A6) OCT scanning path CT 6mpi Retinal vessel Fundus photography Optical coherence tomography (OCT) image

  13. immunogenicity AAV2/4TetOff.rpe65 (A2) AAV2/4TetOn.rpe65 (A4) AAV2/4CMV.rpe65 (A6) Mac: macaque, positive control

  14. Electroretinography (ERG) • measures the electrical responses of various cell types in the retina, including the photoreceptors (rods and cones), inner retinal cells (bipolar and amacrine cells), and the ganglion cells. • If a flash ERG is performed on a dark-adapted eye, the response is primarily from the rod system. • Flash ERGs performed on a light-adapted eye will reflect the activity of the cone system. • Sufficiently bright flashes will elicit ERGs containing an a-wave (initial negative deflection) followed by a b-wave (positive deflection).

  15. Sibling of RP patient RP patient

  16. Right eye Left eye

  17. ERG Recording of TetOff Vector Treated Dogs

  18. AAV2/4TetOff.rpe65 in RPE65-deficient dogs treated eye A1: Volume: 600ul untreated eye A2: Volume: 350ul A3: Volume: 590ul

  19. ERG Recording of TetOn Vector Treated Dogs

  20. AAV2/4TetOn.rpe65 in RPE65-deficient dogs A4: Volume: 540ul treated eye untreated eye A5: Volume: 460ul

  21. Evaluation of Visual Function of A6, 3mpi and 6mpi

  22. Evaluation of Visual Function of A1, 11mpi, ON2

  23. Conclusion • RPE65-deficient dogs treated with Dox-regulatable AAV vectors displayed retinal function recovery, but not rescue of vision. • Injection of constitutive vectors in RPE65-deficient dogs showed improvement of vision when the amplitude of the dark-adapted b-wave was above 60V.

  24. Questions • What is gene therapy? • What is Dox-regulatable system? • What is the difference between TetON and TetOFF? • What is the role of RPE65? • What is electroretinography (ERG)?

  25. How might one resolve the problem these authors encountered in improving vision in RPE65-deficient dogs?

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