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PRESENTATION
DR PRATEEK BUCH
RESEARCH FELLOW, DEPARTMENT OF GENETICS, INSTITUTE OF
OPHTHALMICS , UNIVERSITY COLLEGE LONDON
I’d like to share with you the timeline or story of how we have gone
from understanding retinal disease to commencing gene therapy in
the clinic, illustrating important work along the way to demonstrate
how research is translated from the laboratory to the clinic. I intend to
cover the following areas: why it is we feel the eye has particular
advantages as a target organ for gene and cell therapy; how viruses
can be engineered to achieve gene delivery to the cells we’re
interested in; how under the right conditions stem cells can be used
to replace degenerating cells in the eye; how proof-of-principle for
gene therapy was established first in rodent models of disease, then
in pre-clinical studies; and finally how our group is amongst the first to
conduct a clinical trial of gene therapy in the eye and how these
studies are proceeding.
In a sense for this audience I needn’t cover quite why the eye is an
attractive site for gene and cell therapy – but just give you an idea of
the advantages of working in the eye, I’d say that with the eye being
transparent and on the outside of the body it’s easy to monitor after
treatment, using routine clinical assessment. In addition the cells
encasing the eye form a sort of blood-retina barrier, meaning there is
minimal immune response to whatever we put into it, and importantly
there is very little spread of therapeutic agents outside of the eye.
This may seem obvious but compared to those treating liver disease
or systemic conditions we ocular types do have considerable
advantages.
Going back to that timeline, before we can even think about treating
diseases we need to understand the mechanisms that underlie them,
why and how they arise in the first place - hence one very important
advance in understanding retinal disease is the continual discovery of
genes that, when mutated, cause the different types of blinding
disorders. Since the mid-1980s hundreds of mutations in dozens of
genes have been shown to cause inherited retinal diseases, and the
discovery of each new disease gene allows us to better understand
how vision is lost.
Once we know this, the simplest form of gene therapy is to deliver to
cells in the retina the genes that are missing or damaged to as to
restore structure and function. To achieve this, our lab and many
others use modified viruses to deliver genes to the light-sensitive
photoreceptors and other cells of the retina. We use a close relative
of the adenovirus that causes the common cold, AAV, prepared in the
lab, and most experiments use a modified version of the most
common type of AAV, 2. Later in the talk I’ll refer to more effective
forms of AAV that are currently being tested in the lab. Early proof-of
concept studies indicated efficient delivery of genes to retinal cells as
demonstrated by the delivery of the gene for a green fluorescent
protein. From delivering genes to the odd cell in the eye in the early
1990s, the efficiency of gene delivery using new vectors is close to
100%, but the point is not to turn the back of the eye green, fun
though that is – using this technology the idea is to replace genes
damaged in disease.
Amongst the various clinical disorders of the eye we can make two
rough categories, one being clearly inherited diseases which are
often caused by single mutations, and the other being diseases which
may have an inherited component but which also depend upon age,
health status and environmental factors. Of course once it’s
established that you can deliver genes to the eye we needn’t focus on
replacing missing or damaged genes, we can use gene delivery
much in the same was as conventional drug therapy to deliver genes
that may be beneficial in more complicated diseases such as those
that involve aberrant blood vessel growth or inflammation.
Nonetheless the story of retinal gene therapy really begins with the
inherited conditions, and in particular retinitis pigmentosa. A family of
diseases that ranges in severity and age of onset, RP is the biggest
cause of inherited vision loss and is caused by hundreds of mutations
in any one of dozens of genes involved in the signalling pathway that
leads to light perception by the eye. One such form of RP is caused
by mutations in a protein that forms a scaffold in the rod and cone
photoreceptors of the retina, a scaffold that houses the light-sensitive
pigments and other proteins of the signalling cascade in the cells’
outer segments. When this protein, RDS, is missing, rods and cones
can’t form these outer segments and there’s a consequent loss of
structure and function. Using AAV to deliver the RDS gene to the eye
allows the formation of these scaffolds and a restoration of function,
showing that gene replacement can have therapeutic benefit. So, job
done, pass go, collect £200…
As with most things in life it’s never that straightforward. The
improvement in function is often transient – after some weeks
treatment benefit wears out. This is because in most cases the
restoration of function does not prevent the cells themselves from
dying. Tackling this takes two forms – the first is yet more gene
delivery, this time delivering genes that convince cells not to commit
suicide despite carrying mutations – literally. There are many genes
that prevent cell death in this way, and although I won’t go into too
much detail here we do need to exercise caution as some genes are
potent at preventing cell death but depress function – work from our
lab suggested that there are genes that avoid these side effects, but
one protein that does appear to damage function in some contexts is
already in clinical trials and so care needs to be taken.
Neuroprotection
is
not
always
required,
however,
as
gene
replacement can in some cases restore function in the long term.
We’ll get back to that later but now I’d like you to imagine a case
where somebody presents with late-stage disease – where a
mutation has caused the rods and cones to die and to a large extent
there is nothing left for a gene therapy vector to deliver genes to –
what next? This is where stem cells hold great promise. True stem
cells have to potential to form any cell in the body, but past
experiments using embryonic stem cells showed that although they
survive after injection, they don’t develop into mature rods and cones,
don’t mesh with the host retina or make the connections with other
neurons in the eye that are required for a visual signal to be
transmitted to the brain. It is thought that many organs in the adult
have precursor cells that have the potential to form many of the cell
types in that organ. As the eye is formed during foetal development,
one set of these precursors in the retina stops dividing and forms
mature rod cells – using a mouse that expresses green fluorescent
protein in all its cells, Rachael Pearson and Robert MacLaren took
retinal cells from this point in a mouse eye’s development and
transplanted them into a normal eye. They saw integrated cells
looking like normal rods (only green!), showing for the first time that
cells from the right developmental stage could form rods. By injecting
them into a degenerating retina they also proved they could replace
rods lost as a consequence of genetic mutations. This was clearly an
exciting finding, but the real question was whether replacing lost cells
improved the function of the eye. Although there were on average a
few thousand cells integrating into the recipient retina, these must
have correctly wire up with the partner neurons in the eye because
the eyes which received cell transplants had their pupil reflex restored
– their pupils responded to light exposure by dilating, much as mine
did when I woke up at 5a.m to catch the train to Newport! To
determine which precise cells were the ones doing the replacement,
again Rachael and Robert used GFP – this time in mice where only
those cells which are about to become rods are green. Turns out it’s
only these cells that can successfully be transplanted – showing that
the potential to integrate is restricted to cells already committed to
becoming rods. Which is fair enough in mice as these cells commit to
a rod fate in the first three to five days after birth – in man the eyes
develop much earlier and the equivalent cell type is found in the
second trimester. So one challenge that remains is to generate
precursor cells – those which can turn into rods and cones – from
either embryonic stem cells of ideally from adult cells. The other
parallel challenge is to improve the rate at which donor cells integrate
into the host retina. To date we and others have not managed to
restore the mouse electroretinogram (ERG), a clinically relevant
measure of visual function. As to the generation of useful precursor
cells from embryonic tissue, a Japanese group reported last year that
they had taken cells from a very early embryo, cultured them in a dish
along with a very specific cocktail of growth factors and molecules,
and managed to pick out a set of cells that produce many of the
proteins you find in rods and cones. In a key development researches
have also created cells from skin fibroblasts – adult cells that were
reprogrammed to become capable of developing, potentially, into
many different cell types including retinal cells. I won’t discuss in too
much detail how we plan to increase the numbers of cells integrating
into the retina, save to say that there are thought to be several
physical barriers to cells gaining access to the right environment
where they can connect up, and manipulating these barriers results in
significantly improved integration. Combining these approaches could
yield a viable treatment for many disorders where cells die at a fast
rate.
Let’s return to gene therapy now and think about how the advances in
gene delivery could be applied to the clinic. The first task in
translating research technology into the clinic is to choose an
appropriate form of a disease to target – in the first instance a form of
degeneration caused by a single gene defect, preferably with a
window of opportunity between presentation of symptoms and endstage disease, and a form where there is the chance not only for
patients to retain their current level of vision but for vision to actually
be improved. A number of forms of disease fit these criteria, one of
these being a severe early-onset form of retinal degeneration called
Leber’s congenital amaurosis (LCA). First described by Theodore
Leber in 1869, this form of retinal degeneration is now known to be
caused by mutations in several genes, one of which is known as
AIPL1. There are mice which lack AIPL1. This gene codes for a
protein that transports and assembles other vital protein in
photoreceptors, and our group recently showed that using AAV we
can restore both the localisation of these client proteins, and retinal
function, in these mice. Interestingly because of the speedy progress
of this disease it was necessary to use an alternative form of AAV –
one where DNA based on the common form, AAV2, is packaged
inside
virus
particles
originating from
AAV8.
This
so-called
pseudotyping allows AAV to infect cells faster and more efficiently,
and may be useful in future clinical trials of more severe disease.
Another form of LCA is cased by defects in a gene found in RPE cells
– a single layer of cells which underlie the retina and provide nutrients
to rods and cones, and that replenish the visual pigment by recycling
rhodopsin once it’s been activated by light. It’s long been thought that
RPE defects that lead to retinal degeneration might be more
amenable to therapy, as not only does AAV infect these cells more
readily the majority of such diseases leave the photoreceptors
themselves intact and healthy, so that addressing the RPE defect
should allow these inherently healthy rods and cones to function
normally. Indeed mice lacking RPE65, a protein that is involved in the
recycling of visual pigment, have their vision restored by AAV gene
delivery. In 2001 an American group showed that dogs which have a
very similar RPE65 defect also benefit from AAV gene delivery, and
we and others have shown that this improvement can last for a long
time – the latest is at least five years post-therapy. Not only that, as
this video shows, this therapy leads to improved visual behaviour.
So based on this pre-clinical data we decided to initiate a clinical trial
of AAV-mediated RPE65 gene delivery in patients. Designed as a
Phase I/II safety and efficacy trial, and beginning with patients with
late-stage disease, we delivered AAV encoding RPE65 to one eye in
each patient and carefully monitored how well the vector was
tolerated and if there were any improvements in vision.
Importantly, the surgery itself lead to no complications or damage,
and there were no severe immune reactions noted. This is important
as it goes back to why we think viral gene therapy has such promise
in the eye – being a virtually sealed compartment, no vector leaks out
into the blood and there is very little immune reaction to the vector or
gene product.
We looked at visual acuity in the patients we treated and whilst we
saw some modest improvement in one or two eyes we were not
confident that this was significant. What did appear significant,
however, was the improvement in more objective measures of retinal
function such as microperimetry – where the patient is shown a light
focussed on a very specific point in the retina and asked to press a
button if they see it, which assesses sensitivity to light. One patient in
particular improved considerably by this measure, and we were able
to test his vision-guided movement in a special facility at UCL. This is
shown in a video where the patient before therapy struggles to
navigate a maze in very dim light, hesitating at many points and
bumping into objects and walls placed in his path. He eventually
navigates the maze after some 77 seconds and having made 8
navigation errors. Six months after treatment the patient enters the
maze confidently and walks straight through the fastest route
bumping into no walls, in under 14 seconds – clear evidence that
there is improvement in his vision.
So whilst we’ve come a long way since the early days of gene and
cell therapy for retinal degeneration, there is still a great deal left to
achieve. The current trial continues with the dose escalation phase
and we hope to treat younger patients as a part of that. There are a
raft of other forms of disease which may benefit from similar gene
replacement therapies, and there’s considerable progress been made
in treating more complex conditions such as those which are
dominantly inherited, which would require not replacing a gene but
interfering with a toxic gene product, and those conditions affecting
primarily cone vision. And of course there is a great deal of promise
in the use of stem cells but much remains to be done before we see
this technology in the clinic.