Transcript Slide 1

Class 10
Dr. Pittler
The Inherited Retinal Degenerations fall into 2
broad categories:
1)
2)
The Retinitis Pigmentosa Family in which
rod photoreceptors are first affected. Thus,
peripheral vision and low light vision are lost
first. Vision loss can be very early or
somewhat later in life.
The Macular Degeneration Family in which
cone photoreceptor are first affected. Thus,
central, sharp vision and color vision are
affected first. They can strike in early
childhood (Stargardt Disease) or much later
(AMD).
Prevalence: 100-200,000 affected in USA;
general prevalence of about 1:3,400
Genetics: dominant, recessive and X-linked
genetic forms (also mitochondrial and
syndromic).
Over 130 gene mutations identified yielding
many phenotypes (physical characteristics),
e.g.
Usher Syndrome – early onset hearing loss (some
have balance problems) along with RP vision loss.
25 K affected; 11 genes identified.
 Leber Congenital Amaurosis – very early, severe
vision loss (often congenital); nystagmus. 10-15 K
affected; 9 genes identified.
 Bardet-Beidl Syndrome – vision loss with many
other (e.g., mental) problems. 10 K affected. 12
genes identified.
 Choroideremia – choroid involved; 10 K affected.
Gene defect known
 Retinoschisis – retina “splitting”. 6 K affected.
Gene defect known.
Many others………
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Clinical – what the professional sees:
- thinning of the retina, mainly rod
photoreceptor loss
- attenuated blood vessels
- abnormal pigment clumping (bone spicule)
Visual – what the patient sees:
- loss of peripheral vision (tunnel vision)
- loss of night vision
Progression is variable sometimes very fast,
sometimes slower. Inexorable constriction of
visual field in most cases. Loss of functional
vision or total blindness are often the end
results.
SEE THE LIGHT
What a patient with retinitis pigmentosa sees.
Our Goal:
To rapidly move from research at the laboratory bench to the clinic such that we can deliver
preventions, treatments and cures to all patients with inherited retinal degenerations (RDs).
This includes RP, AMD and all the rare RDs such as Stargardt, Usher, Leber, choroideremia,
etc.
How do we reach the Goal?
The path starts with scientific Proof of Principle. Basic scientists have done a thorough job
providing much information on the RDs including potential treatments and cures. We must
now move to Clinical Trials that lead to effective treatments.
1) Basic Science – start with progress in genetics, cell
biology, etc.
2) Proof of Principle - establish animal models,
understand disease mechanisms and design modes of
treatment. This is called “Proof of Principle” – it works!
3) PreClinial Trials – work with companies; do efficacy
and safety trials in animals; drug delivery studies, etc.
Much money is needed.
4) Clinical Trials – get government approval and move
to and through the human Clinical Trial to a successful
conclusion. This usually occurs collaboratively with a
biotech or pharmaceutical company.
Phase I:
 Phase I is designed to test safety and to
determine the best and safest dose of treatment.
The main question here is” “How well is the new
treatment tolerated in a small number of
patients? Are there bad side effects?”
 Thus, a Phase 1 trial is not designed to see if
the treatment works. Rather, only to see if it is
safe in a small number of patients.
Phase II is designed to determine whether the
treatment has any positive effect in the human.
Hopefully this “efficacy” phase shows a useful
effect of the treatment in man as it did in animals.
It usually uses a larger number of patients than in
Phase I.
Another aim is to provide more information on
safety and any side effects.
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Phase III is designed to fully evaluate the
effectiveness of a treatment.
Usually, it involves a larger number of patients than in
Phase II. One of the main problems we will encounter
in a Phase III trial for RP patients is the very small
patient population. This makes it difficult to recruit
enough patients such that enough data are collected
to make the results statistically significant.
The whole Clinical Trial process usually takes 2-5
years and costs a lot of money. Therefore, working
with pharmaceutical companies is essential.
In 1990, the first gene mutation was found in the
rhodopsin gene (Humphries; Dryja et al.).
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A defective gene can be replaced with a new
functional one. This is the simplest form of Gene
Therapy. However, you must know the gene
mutation first!
http://www.sph.uth.tmc.edu/Retnet/sum-dis.htm
RETNET RETINAL DISEASE DATA
Retinitis pigmentosa,
autosomal dominant
Retinitis pigmentosa,
autosomal recessive
Retinitis pigmentosa, Xlinked
16
15
19
15
6
2
Macular degeneration,
autosomal dominant
12
6
Macular degeneration,
autosomal recessive
2
2
9
4
13
11
6
6
9
7
192
144
mapped
cloned
Other retinopathy,
autosomal dominant
Other retinopathy,
autosomal recessive
Other retinopathy,
mitochondrial
Other retinopathy, Xlinked
TOTALS
197 (2009)
154 (2009)
RETNET RETINAL DISEASE DATA
http://www.sph.uth.tmc.edu/Retnet/sum-dis.htm
is the introduction of genetic material into an organism to
slow, prevent or reverse the progression of disease. It
involves replacement of defective or absent proteins in the
case of loss of function defects or the inhibition of new
function in gain of function defects.
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No Longer Science Fiction
◦ Significant advances have been made in research on
neurodegeneration and treatment across a range of
eye diseases (retinitis pigmentosa, macular
degeneration and others)
 One
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of several potential interventions
Pharmacological (drug intervention)
Surgical
Prosthetics
Cell replacement (or tissue/organ transplantation)
Challenges facing gene therapy
Controlling gene expression. For some diseases, the correct amount of protein has to be made for the
right amount of time. Genetic diseases like cystic fibrosis need a continual supply of the therapeutic protein
throughout a patient’s lifetime to keep the disease in check. Other diseases don’t require such tight control.
For different reasons, gene expression also sometimes works poorly or shuts off altogether shortly after it
has been introduced. Scientists are not yet able to control gene activity after it’s been introduced into the
body.
Getting genes to their proper targets. One big problem is getting the corrected gene into the right cells
and functioning at the desired site. Often, cells other than the intended targets take up the gene as well.
Preventing destruction of the introduced gene. Some enzymes will chew up DNA that is not protected.
In other cases, the immune system will recognize a viral vector and destroy both it and the inserted gene.
Delivery methods. Another challenge is how to most effectively administer the gene so that it ends up
where you want it. In some cases, it’s possible to take the desired cells out of the body and insert the
gene. Others inject the gene directly into specific sites, such as heart muscle.
Condition of the host. For some genetic diseases, irreparable damage occurs early in life. In cystic
fibrosis, for example, the lungs are damaged during childhood. Treating some of these diseases will mean
having to intervene before permanent damage has already occurred.
Host immune response. Another concern is how the person’s immune system will react to a foreign
protein for the first time. Alternatively, the immune system may not react adversely the first time the vector
or gene product (the protein) is encountered but can mount a severe response on subsequent exposures.
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Targets the actual cause of the disease rather than
symptoms
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Expression of the desired gene may exceed the duration
of action of currently available drugs
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Gene expression may be targeted to a specific cell type
(using a specific promoter to achieve spatial regulation)
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Gene expression may be regulated, i.e. turning the gene
on and off (using a regulatory cassette to achieve
temporal regulation)
 The
advantage of ocular tissue for
gene therapy
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Small size
Easily accessible
Immune-privileged
Tissue boundaries that prevent leakage of the
therapeutic material to other sites and separation
from the systemic circulation
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Where – mode of delivery
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What – knowledge on the genetic cause of
the blinding disease (for specific treatment)
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How – expression in the right place
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Functional copy of the gene
◦ Gene replacement for recessive mutations
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Ribozymes, antisense oligonucleotides,
siRNA
◦ Gene silencing for dominant mutations (new -small
interference RNA)
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Genes encoding cytokines, growth factors,
neuroprotecting agents, and anti-apoptotic
products
◦ Slows or prevents degeneration but does not cure
or restore
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rd1 mouse
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rds mouse
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RPE65 mutation
◦ Premature stop codon mutation in the gene encoding the rod
photoreceptor cGMP phosphodiesterase β subunit
◦ Deliver functional PDEβ gene through Ad, lentivirus, AAV rescued
photoreceptors in the rd1 mouse
◦ Lacking a functional gene for peripherin-2
◦ Leber congenital amaurosis
◦ Canine model
 RPE65-deficiency, a 4 bp deletion resulting in a premature stop and truncation
of the protein product. The dogs are vision-impaired
 AAV-mediated delivery of the normal RPE65 gene restored vision.
Ribozyme therapy
Antisense therapy
SiRNA
Gene replacement
therapy
Growth factor
therapy
Suicide gene
therapy
Ribozyme therapy: Ribozymes hybridize
with mRNA from a mutated gene. The
ribozyme enzymatically cleaves the mRNA
and functionally silences the gene by
preventing synthesis of the abnormal
protein from the transcript
Antisense therapy: Complementary DNA
molecules hybridize with the mRNA from
the mutated gene. The ribosomes cannot
bind to the double
stranded heteroduplex, preventing
synthesis of the abnormal protein
Gene replacement therapy: Mutated genes
causing autosomal recessive retinal
degenerations can be replaced using viral or
non-vial methods
Growth factor therapy: Growth factors are
synthesized and secreted from cells
expressing growth factor transgenes. The
growth factors have positive neurotrophic
effects on surrounding retinal cells and also
on the secreting cells via specific receptors
Suicide gene therapy: Gancyclovir is
converted to a cytotoxic nucleotide by a
transfected viral thymidine kinase. The drug
inhibits DNA synthesis, leading to cell
death. Dying cells are also cytotoxic to
nearby untransduced cells, causing the
“bystander effect”
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Contain highly conserved sequences that
direct catalytic hydrolysis of the mRNA at
specific tri-nucleotide motifs
Contain variable sequences that determine
the specificity of the ribozyme for its target
Each can catalyze the hydrolysis of many
transcripts within the cell
Two commonly used ribozymes
◦ Hammerhead and hairpin ribozyme
◦ Hammerhead ribozymes have been used more
commonly, because the target site has few
limitations
Secondary structures of some naturally occurring ribozymes
green-ribozyme or intron region
black-substrate or exon region
Takagi, Y. et al. Nucl. Acids Res.
29, 1815-1834 (2001)
Inhibition of ftz synthesis by a ftz hammerhead ribozyme
5’-m7GpppG
3’-nAAA
5’-m7GpppG
cleavage site
U
U
C
A
A
U
U
G
G
U
C
GGAGCUACACG
CCUCGAUGUGC
C
U
red-ftz mRNA
green-ribozyme
AAAn-3’
A
A
G
U
U
A
A
C
C
A
wildtype
A
GAU
A
A
GCAGG G
GUCC A
G
A
G
+ ftz ribozyme
+ ftz ribozyme
Zhao, J. J. and Pick, L. Nature 365, 448-451 (1993)
Uninjected eye from a rat at P130.
Retina from the opposite eye from the same rat as
in A, which was injected subretinally with Hh13
ribozyme at day p15 now at P130.
Ribozyme
Therapy
Uninjected eye from a rat at P240.
Retina from the opposite eye of the
same rat in C, which was injected with
Hh13 ribozyme at P15 now at P240.
P23H transgenic rat retinas taken at postnatal day P130
Growth Factor Therapy
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The majority of vectors available today to
deliver genes to cells are of viral origin
◦ Viruses, by means of their own nature, infect cells
very efficiently
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Adenovirus
◦ Non-enveloped, double-stranded DNA virus
◦ Infects a broad range of human and non-human cell types by
binding to specific cell-surface receptors
◦ The virus in not incorporated into the genome (remains as an
episome within the nucleus)
◦ The episome is eventually degraded or lost – transient nature of
gene expression (peak expression – 2 d to 2 wks)
◦ Infectious nature
◦ Tissue inflammation
◦ need to use replication-defective or helper-dependent Ad
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Adeno-associated virus
◦ Non-enveloped, single-stranded DNA parvovirus
◦ efficiently infects dividing and non-dividing cells
◦ Inserts into the host genome at specific locus (stable
expression)
◦ Not associated with any known human infectious disease
◦ Less tissue inflammation than Ad
◦ distinct differences among the cell-targeting specificities of the
8 serotypes
 For RPE, AAV1, AAV2 or AAV5
 For photoreceptors, AAV2 or AAV5
◦ AAV capsid proteins affect both cellular tropism and the
speed of onset and intensity of gene expression
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RPE65 is a pigment epithelium protein that is required for regeneration of 11-cis
retinal in the visual cycle
RPE65 is the retinal isomerase that converts all-trans retinol to 11-cis retinol
In humans defects in the gene cause Leber congenital amaurosis, the congenital
form of retinitis pigmentosa
Defects in the corresponding gene in Briard dogs have also been found.
Researchers at the University of Pennsylvania created an adeno-associated gene
transfer vector to replace the defective Briard dog RPE65 gene.
photoreceptor
promoter
RPE65 protein coding region
AAV genome, selectable markers, and
replication origin
Functional recovery in RPE65 mice
An exciting new area of transplantation study is that of
Stem Cell research. Stem cells are primitive,
undifferentiated cells that have the ability to differentiate
into many types of mature, adult cell types – liver, skin,
photoreceptor cells.
Where are stem cells found? Stem cells are, of course,
present in embryonic tissue. However, small numbers
have recently been found in many adult tissues. For
example, Van der Kooy et al. was the first to find stem
cells at the edge of the retina in the eye of the adult
mouse.
The Needs and Challenges:
Source: We need good sources of stem cells. We already
have evidence that stem cells from different sources
such as the brain can be transplanted into the retina.
Development: We need to determine the factors that will
push the stem cells to develop into mature, functioning
cells such as photoreceptor cells.
Thus, stem cell research has a long way to go before it
fulfills its promise.
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Issues with Stem Cells
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Adult stem cells are plastic to some extent, but
how useful remains to be determined.
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Again, we do not know how to guide adult stem
cells to differentiate into a particular type of
cells.
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The use of nutritional supplements as a therapy in RD has
been controversial but now must be taken seriously in
prevention or at least slowing down the RDs, especially
AMD.
In 1983, Converse et al. found that the concentration of a
fatty acid called DHA was abnormal in the blood of some
RP patients. DHA is heavily concentrated in photoreceptor
outer segments and is thought to be vital for their
functioning. Two human Clinical Trials have been
conducted to examine the effects of DHA supplementation
on RP patients (no results yet).
In 1993, Berson et al. found that vitamin A supplementation
slows RP to a small extent in some RP patients. This was
the first agent shown to be effective in treating RP.
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The AREDS Clinical Trial of the NEI has ended after
many years of study. It found that some nutritional
supplements helped in delaying the progression of
AMD. The nutrients studied were zinc, B-carotene
and vitamins C and E.
Lutein is a nutrient that is highly concentrated in the
macula. It is thought to be an antioxidant and thus
may protect photoreceptor cells in the macular
region. It is currently being tested as to its effects in
both AMD and RP.
These fall into 2 categories:
1) Brain (cortical) electronic implants
2) Retinal implants – in front or behind
the retina
For the retinal implants, there are many
different designs and surgical
approaches from groups around the
world.
Cortical Implant
The Active Implantable Device:
◦ Uses electrical stimulation to bypass
defective or dead photoreceptors and
stimulate remaining viable, nonphotoreceptor cells of the retina.
◦ Image data from an external camera is
wirelessly transmitted to the implant
which stimulates electrodes in an array on
the retina to produce formed vision.
1) A small video camera and a transmitter are
hidden behind glasses worn by the patient.
The camera captures the visual images.
2) The images are relayed to a small computer.
The computer processes the signals and
sends them to the electrode array implanted
on the retina.
3) The implant electronically stimulates the
retina, mimicking the original visual image.
4) The inner retinal neurons biologically
process the signal and pass it down the optic
nerve for final processing in the brain.
Visual Prosthetic Devices
The Retinal Chip
The Retinal Chip Electrode
Chronic studies on human implants have been
done at Doheny/USC on the Argus 16
electrode device -- from 2/02 and yet
continuing with functional testing.
 Six patients were implanted. NO device
failures.
 All subjects saw discrete visual images
(phosphenes) and could perform visual
spatial and motion tasks.
 Mobility (walking and navigation) has been
improved
 One device had to be removed for unrelated
health reasons.
 The remaining 5 patients use the device at
home.
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The new device has 60 electrodes – not just
16. This does allow for much finer detail in
the visual image.
The Argus II is much smaller than the first
generation device. It is designed to last a
lifetime.
An Argus III 200 electrode array device is
now in development.
Progress of the Artificial Retina
Vision
1000
electrodes
Face Recognition
60
electrodes
240
electrodes
Finger Count
Hand Motion
16
electrodes
2002
2006
Years
2010
2014
People to be Helped in the US
20/20
2018
12,000,000
1,000,000
100,000