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.
tTA (Tet-off) or rtTA (Tet-on) :
Tet-controlled transcriptional activator
TRE: tetracycline response element
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
promoter
cDNA
transactivator
promoter
A1 to A3
transactivator
promoter
promoter
cDNA
A4 and A5
A6
Subretinal Injection
• http://www.jove.com/video/4286/subretinal
-injection-gene-therapy-vectors-stem-cellsperinatal-mouse
AAV2/4TetOff.rpe65 (A2)
AAV2/4TetOn.rpe65 (A4)
CT
CT
6mpi
6mpi
AAV2/4CMV.rpe65 (A6)
Zone of the bleb
CT
OCT scanning path
6mpi
Retinal vessel
Fundus 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
Right eye
Left eye
ERG Recording of TetOff Vector Treated Dogs
AAV2/4TetOff.rpe65 in RPE65-deficient dogs
treated eye
untreated eye
A1:
Volume: 600ul
A2:
Volume: 350ul
A3:
Volume: 590ul
ERG Recording of TetOn Vector Treated Dogs
AAV2/4TetOn.rpe65 in RPE65-deficient dogs
A4:
Volume: 540ul
treated eye
untreated eye
A5:
Volume: 460ul
Evaluation of Visual Function of A6, 3mpi and 6mpi
Evaluation of Visual Function of A1, 11mpi, ON2
Conclusion
• RPE65-deficient dogs treated with Doxregulatable AAV vectors displayed retinal
function recovery, but not rescue of vision.
• Injection of constitutive vectors in RPE65deficient dogs showed improvement of
vision when the amplitude of the darkadapted b-wave was above 60V.
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?