Week 3 Genetic therapy for retinal disease
Targeting DNA/RNA
LE Genetic Therapy for inherited Retinal Disease
Central dogma in Molecular Genetics = relationship between DNA, RNA and proteins
In the retina there are the cells (cones and rods) that converts light into an electric signal. Retinol
pigment epithelium (RPE) on top of the cones and rods, is to protect the cells.
Inherited retinal diseases (IRDs) are clinically and genetically heterogeneous.
- Clinically heterogeneity = umbrella term for different subtypes of retinol disease based on
different criteria (age of developing the disease, effected cells)
- Genetic heterogeneity =depending on the genes first the rods and then cones can be
effected or the other way around. Depending on the gene of interest you can have a
different subtype of retinal disease.
The retina/eye has a local immune system (immune-privileged) so drugs in the eye don’t end up in
the rest of the body (= contained). So it is also safer to try drugs in the eye (non-invasive read-outs).
You have two eyes so the other eye can be a control. It is easily accessible (compared to the liver
e.g.).
Diagnosis
- Letter chards say something about cone function (recognizing faces and details)
- Perimetry assay to test rod function in large visual field (chin on device, look in the globe,
dots coming from everywhere, every time you see a dot press the button). A defect in rod
cells limits the visual field.
- Electro retina gram, stimulates the eye with light flashes, electro response (a/b waves) of
retinal cells is measured.
- Imaging tools to look at the retina
- DNA sequencing
Disease stages: (1st) dysfunction of cells, they are there and not dead. (2nd) Degeneration is where
cells die if they do not function for a while. Retinal cells lost over time. Early and late degeneration.
, Possible therapeutic interventions.
- Cell therapy = not fixing cells but adding cells. Making cells in vitro and then transplant them.
In early degeneration stage to manage the degeneration.
- Electronic implants = put in device behind the retina (artificial retina). Glasses film and signal
to the device which sends signal to the brain. In late degeneration stage because patients
hardly see.
- Genetic therapy = interfering with mistakes in the DNA, to try correct the defects in DNA or
RNA in a way that the ineffective protein starts to function normally again. In dysfunction
stage because if the cells are not there to take it up there is nothing to restore.
Different types/strategies of genetic therapy
- Adding DNA (also called gene augmentation therapy = e.g. RPE65 gene mutated in retinal
disease expressed in RPE cells. Try to get new RPE65 protein in the right cells by e.g. a virus
as a transporter molecule (promotor and cDNA).
- Fixing DNA = take out wrong nucleotide and put right nucleotide back
- Fixing RNA = correcting RNA
- Degrading RNA = degrade the second allele with mutation so only the correct allele stays.
Usually only possible with autosomal dominant because with recessive both alleles are
mutated and there is no protein anymore.
Knowledge needed for treatment: patient diagnosis (stage), genetic defect, and molecular
mechanism (type and effect of mutation (missense and LOF/GOF e.g.)
Tools needed for treatment: therapeutic strategy, cellular (stem cells)/animal (mouse, dogs,
zebrafish) models, and delivery vector (viral (DNA (AAV) and RNA virus) and non-viral vectors (Naked
DNA, liposomes, polymer, DNA complex)
LE Gene Augmentation Therapy
= Adding DNA in a patient in which the gene is missing or mutated
Building blocks of a therapeutic construct: promotor (drives expression), cDNA, and poly A tail
(needed for protein synthesis, transporting to ribosomes).
RPE65 is an enzyme that plays a role in the visual cycle. When RPE65 gene is mutated, the RPE65
enzyme doesn’t work properly and the visual cycle cannot properly continue. RPE65 mutation is one
of the most frequent causes of leber congenital amaurosis = childhood blindness (LCA). LCA has
severe and early (<1 year) visual loss. In all exons of RPE65 gene there can be many mutations found
(missense, nonsense etc). These loss-of-function mutations are inherited in autosomal recessive way.
As a treatment, they used a therapeutic construct of recombinant adeno-associated viruses (rAAVs).
Took out the replication gene and capsid gene of the virus (normally in the AAV). And put in the
promotor and REP65 cDNA in the viral vector. Different viruses go to different cell types.
For the clinical drug trials you don’t want healthy individuals because they already have RPE65. Too
much enzyme can also be harmful. And subretinal injection is a complicated surgery so its unneeded
risk. Phase 1 looks at safety and phase 2 at efficacy, Phase 1 and 2 were combined for the drug since
only patients are used. Signs of efficacy were a bit low, younger subjects were tested with the
hypothesis that the success would be higher due to the lower amount of degenerated RPE cells.
Long-term effects were tested with imaging (response to visual input). RPE cells were still
degenerating long-term after treatment. Phase 3 drug safe and effective, led to approval of the drug.
Targeting DNA/RNA
LE Genetic Therapy for inherited Retinal Disease
Central dogma in Molecular Genetics = relationship between DNA, RNA and proteins
In the retina there are the cells (cones and rods) that converts light into an electric signal. Retinol
pigment epithelium (RPE) on top of the cones and rods, is to protect the cells.
Inherited retinal diseases (IRDs) are clinically and genetically heterogeneous.
- Clinically heterogeneity = umbrella term for different subtypes of retinol disease based on
different criteria (age of developing the disease, effected cells)
- Genetic heterogeneity =depending on the genes first the rods and then cones can be
effected or the other way around. Depending on the gene of interest you can have a
different subtype of retinal disease.
The retina/eye has a local immune system (immune-privileged) so drugs in the eye don’t end up in
the rest of the body (= contained). So it is also safer to try drugs in the eye (non-invasive read-outs).
You have two eyes so the other eye can be a control. It is easily accessible (compared to the liver
e.g.).
Diagnosis
- Letter chards say something about cone function (recognizing faces and details)
- Perimetry assay to test rod function in large visual field (chin on device, look in the globe,
dots coming from everywhere, every time you see a dot press the button). A defect in rod
cells limits the visual field.
- Electro retina gram, stimulates the eye with light flashes, electro response (a/b waves) of
retinal cells is measured.
- Imaging tools to look at the retina
- DNA sequencing
Disease stages: (1st) dysfunction of cells, they are there and not dead. (2nd) Degeneration is where
cells die if they do not function for a while. Retinal cells lost over time. Early and late degeneration.
, Possible therapeutic interventions.
- Cell therapy = not fixing cells but adding cells. Making cells in vitro and then transplant them.
In early degeneration stage to manage the degeneration.
- Electronic implants = put in device behind the retina (artificial retina). Glasses film and signal
to the device which sends signal to the brain. In late degeneration stage because patients
hardly see.
- Genetic therapy = interfering with mistakes in the DNA, to try correct the defects in DNA or
RNA in a way that the ineffective protein starts to function normally again. In dysfunction
stage because if the cells are not there to take it up there is nothing to restore.
Different types/strategies of genetic therapy
- Adding DNA (also called gene augmentation therapy = e.g. RPE65 gene mutated in retinal
disease expressed in RPE cells. Try to get new RPE65 protein in the right cells by e.g. a virus
as a transporter molecule (promotor and cDNA).
- Fixing DNA = take out wrong nucleotide and put right nucleotide back
- Fixing RNA = correcting RNA
- Degrading RNA = degrade the second allele with mutation so only the correct allele stays.
Usually only possible with autosomal dominant because with recessive both alleles are
mutated and there is no protein anymore.
Knowledge needed for treatment: patient diagnosis (stage), genetic defect, and molecular
mechanism (type and effect of mutation (missense and LOF/GOF e.g.)
Tools needed for treatment: therapeutic strategy, cellular (stem cells)/animal (mouse, dogs,
zebrafish) models, and delivery vector (viral (DNA (AAV) and RNA virus) and non-viral vectors (Naked
DNA, liposomes, polymer, DNA complex)
LE Gene Augmentation Therapy
= Adding DNA in a patient in which the gene is missing or mutated
Building blocks of a therapeutic construct: promotor (drives expression), cDNA, and poly A tail
(needed for protein synthesis, transporting to ribosomes).
RPE65 is an enzyme that plays a role in the visual cycle. When RPE65 gene is mutated, the RPE65
enzyme doesn’t work properly and the visual cycle cannot properly continue. RPE65 mutation is one
of the most frequent causes of leber congenital amaurosis = childhood blindness (LCA). LCA has
severe and early (<1 year) visual loss. In all exons of RPE65 gene there can be many mutations found
(missense, nonsense etc). These loss-of-function mutations are inherited in autosomal recessive way.
As a treatment, they used a therapeutic construct of recombinant adeno-associated viruses (rAAVs).
Took out the replication gene and capsid gene of the virus (normally in the AAV). And put in the
promotor and REP65 cDNA in the viral vector. Different viruses go to different cell types.
For the clinical drug trials you don’t want healthy individuals because they already have RPE65. Too
much enzyme can also be harmful. And subretinal injection is a complicated surgery so its unneeded
risk. Phase 1 looks at safety and phase 2 at efficacy, Phase 1 and 2 were combined for the drug since
only patients are used. Signs of efficacy were a bit low, younger subjects were tested with the
hypothesis that the success would be higher due to the lower amount of degenerated RPE cells.
Long-term effects were tested with imaging (response to visual input). RPE cells were still
degenerating long-term after treatment. Phase 3 drug safe and effective, led to approval of the drug.
