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

• 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.

Virus Vector

•Adenovirus•Adeno-associated virus•Herpes simplex virus type 1•retrovirus

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

Tet-OFF Regulatable Switch

tTA (Tet-off) or rtTA (Tet-on) : Tet-controlled transcriptional activator

TRE: tetracycline response element

Tet-ON Regulatable Switch

tTA (Tet-off) or rtTA (Tet-on) : Tet-controlled transcriptional activator

TRE: tetracycline response element

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.

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

Results and Methods

cDNApromotertransactivator

cDNAtransactivator

promoter

promoter promoter

A1 to A3

A4 and A5

A6

Subretinal Injection

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

AAV2/4TetOff.rpe65 (A2)

CT 6mpi

AAV2/4TetOn.rpe65 (A4)

CT 6mpi

AAV2/4CMV.rpe65 (A6)

CT 6mpi

Zone of the bleb

OCT scanning path

Retinal vesselFundus photography

Optical coherence tomography (OCT) image

immunogenicity

AAV2/4TetOff.rpe65 (A2)

AAV2/4TetOn.rpe65 (A4)

AAV2/4CMV.rpe65 (A6)

Mac: macaque, positive control

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).

Sibling of RP patient RP patient

Left eyeRight eye

ERG Recording of TetOff Vector Treated Dogs

AAV2/4TetOff.rpe65 in RPE65-deficient dogs

A1:Volume: 600ul

A2:Volume: 350ul

A3:Volume: 590ul

untreated eye

treated eye

ERG Recording of TetOn Vector Treated Dogs

AAV2/4TetOn.rpe65 in RPE65-deficient dogs

A4:Volume: 540ul

A5:Volume: 460ul

untreated eye

treated eye

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

Evaluation of Visual Function of A1, 11mpi, ON2

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 60V.

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)?

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