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

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

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

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

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

14. AAV2/4TetOff.rpe65 (A2) CT6mpi AAV2/4TetOn.rpe65 (A4) CT 6mpi AAV2/4CMV.rpe65 (A6) CT6mpi Zone of the bleb OCT scanning path Retinal vessel Fundus photography Optical coherence tomography (OCT) image

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

16. 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).

17. Sibling of RP patientRP patient

19. Left eyeRight eye

20. ERG Recording of TetOff Vector Treated Dogs

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

22. ERG Recording of TetOn Vector Treated Dogs

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

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

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

27. 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.

28. 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)?

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