GENE THERAPY 101

Shannon Boye, Ph.D.Assistant ProfessorDepartment of OphthalmologyAchromacorp Conference 6/2014

Let’s start from the beginning…..

Achromatopsia is caused by defects in genes that encode proteinsexpressed in cone photoreceptors

Vitreous

humorLens

Optic

Nerve

Fovea

Pupil

Iris

Cornea

Ciliary body

Anterior

chamber

Sclera

Choroid

GCL

INL

ONL

IS

OSRPE

ILMShannon Boye

Retina

photoreceptors

RODS CONES

Photoreceptors are where vision begins

Spatial distribution of rods and cones

macula/fovea

CNGA3

CNGB3

GNAT2

PDE6C

Achromatopsia

cytoplasm IPMphotoreceptor disk membrane

NCKX

K+,Ca

++

Phototransduction

cascade

GTP

CNGopen

cations

CaM

CaM

GCAP

GC

GCAP

-Ca2+

GC*Arr

RP

REC

Rhk

Rhk*

-Ca2+

Ca++

Ca++ R*

PDE

g g

cGMP

GMP

closed

CNG

Ta*

GTP

PDE*

Tbg

T

Ta*

GTP

rgs9

Gb5

Ta

GDP

CaM -Ca2+

R

hn

ret

ret

GDP

GNAT2

PDE6C

CNGA3/CNGB3

DNA encoding ‘normal’ gene

Virus carrying ‘normal’ gene

Proteins producedby normal gene

How does gene therapy work?

What if we could put a normal copy of CNGB3 (or CNGA3or GNAT2 or PDE6C) back into cones of ACHM patients?

Adeno-associated virus (AAV)

• non pathogenic

• non immunogenic

• drives efficient, sustained expression of genes

• can infect cone photoreceptors

CLINICALLY RELEVANT

a-1-Antitrypsin deficiency (AAT)Alzheimer's

Rheumatoid arthritisCystic Fibrosis

Duchenne muscular dystrophyEpilepsy

HemophiliaParkinson’s

Batten’s Leber congential amaurosis 2 (LCA2)

wet AMDchoroideremia

Ocular gene therapies

We remove the native (wild type) genes of the virus and

replace them with our gene of interest

An example:

green fluorescent protein (GFP)

Green fluorescent protein

Testing vectorin cells in a dish

RPEChoroidSclera

Subretinal

injection

How to get vector into cone photoreceptors of a living organism?

Testing vector in mouse retina

In order to test whether a gene therapy will be effective,

you need a good animal model in which to test it.

A good animal model = one that faithfully mimics the human condition

• Displays a loss of cone photoreceptor function

• Some degree of cone degeneration

After injecting animal model with gene therapy vector, the following tests are performed:

• Electroretinogram (ERG)- a measure of retinal function

• Optical Coherence Tomography (OCT)- a measure of retinal structure

• Behavior testing

• Microscopic analysis of the retinal tissue from treated mice

Gene replacement therapy is dependent on preservation of target cells and knowledge of patient’s gene mutation

Alternatives to gene replacement therapy:• Optogenetics• Stem cells• Retinal prosthesis

Signals originating in photoreceptors are transmittedthrough ganglion cells to the optic nerve and thento the brain where they are translated as “vision”

Optic nervehead

Optogenetics• Deliver a gene that encodes a light-sensitive protein

• Forces the surviving retinal cells to become light sensitive

• If the retina is light sensitive, it can send signals to the brain which can be processed as vision.

Signals coming from light sensitive middle retinaare sent to the brain and processed as vision

Stem Cells

Treat photoreceptors derived from stem cells with gene therapy vector then

deliver treated cells to the patient’s retina

Injection of stem-cell derived photoreceptors Integration of functioning, transplanted photoreceptors