Restoring the retina

Advances in cellular biology that have taken place over the last half century are making a slow advance into the clinic in the treatment of retinal diseases. 
The research has revealed in increasing detail the complex biomolecular interplay involved in maintaining the function and metabolism of the cells of the retina. Moreover, by combining that understanding with genome mapping, investigators have pinpointed the underlying molecular basis and genetic origins of several hereditary retinal dystrophies. 
Recombinant DNA techniques first developed in the 1970s have since enabled the development of in situ gene therapy techniques that can restore the function of diseased retinal cells. The same technology has lead to the creation of genetically modified animal models of the diseases for testing new therapeutic strategies. Also important has been the mapping out of the evolution of stem cells, from the zygote to the retina.
Based on this research, there are already in routine clinical use a range of biomolecular agents designed to inhibit angiogenesis in eyes with exudative age-related macular degeneration (AMD), and trials are now under way with the use of a monoclonal antibody in the treatment of geographic atrophy. 
Gene therapy for hereditary retinal diseases and the use of stem cells to regenerate damaged or absent retinal tissues are two products of the scientific advances that are taking further steps towards clinical use. And while progress in the clinic has been slow, it has nonetheless in several instances yielded confirmation of the safety and potential efficacy of the new therapeutic techniques.

GENE THERAPY
Of the two approaches, gene therapy has the most advanced pedigree in terms of clinical trials, dating back to 2008 with the publication of three separate gene therapy trials, conducted in the UK, Pennsylvania and Florida, and involving patients with Leber congenital amaurosis type 2 (LCA2). 
In the UK study, one patient showed significant improvement in dark adapted perimetry and subjective testing of visual mobility. In the Pennsylvania trial, patients had improvements in visual acuity, light sensitivity and mobility. In the Florida study, patients had a functional improvement in the areas of the visual field corresponding to areas of the retinal pigment epithelium (RPE) injected with modified adeno-associated virus (AAV) carrying the gene for the RPE65 enzyme.
These early gene therapy studies established that it is possible to safely deliver billions of AAV particles into the subretinal space. Subsequent studies from the Philadelphia team also showed that a second injection into the fellow eye did not provoke an immune reaction. However, the long-term benefit has yet to be established with certainty. Follow-up reports from the Florida study indicate that there is improved visual function in the short-term but that it declines after a few years. 
Robert MacLaren DPhil, FRCOphth, from the University of Oxford, UK, noted that the seeming loss of effect in RPE65 was more a result of a lower than optimum gene transduction in eyes with disease that was too advanced. 
“The most important factor in the decline in vision is that it is unlikely that all of the RPE cells will be transduced. If you look at some of the data from the Florida study you will see that there is a boost initially in visual function but then it diminishes. But it reaches a plateau which is still much higher than it was at baseline,” said Dr MacLaren. 
“That is because, when only 50 per cent of cells have been transduced, you will suddenly get a boost from the improved function of those cells and you still have some residual output from the remaining cells which have not been transduced. Over a period of time, the 50 per cent of cells which have not received the virus will die off, so following that initial peak you will have a decline,” he explained. 
However, another hurdle gene therapy techniques have to overcome is their regulatory approval for use in trials involving patients at an early stage of their disease. By necessity, early trials involve patients with advanced disease with less to lose but also less tissue to salvage.
“Gene therapy is always going to be much more efficacious when the treatment is applied before the onset of retinal degeneration,” Dr MacLaren said. 
Meanwhile, recently published results involving six patients undergoing choroideremia have been involving gene therapy for choroideremia (MacLaren et al, The Lancet 2014, 29;383(9923):1129-37), showed that patients gained a mean of 3.8 letters of visual acuity, and that the two more severely affected individuals gained 21 letters and 11 letter letters, respectively. 
In addition, regulatory approval has been granted in Germany for a gene therapy trial involving achromatopsia, a rare condition due to mutations in the CNGA3 gene that causes complete loss of function to the cone cells. Further AAV gene therapy studies for X-linked retinoschisis (XLRS), Leber hereditary optic neuropathy (LHON) and retinitis pigmentosa due to mutations in the MerTK gene are also ongoing.
“I think we’ll see gene therapy used in a number of retinal diseases and it will simply be taken for granted as a treatment. We need to do more work on the surgery so that we can apply the virus safely in the early stages of disease and we need to work out a way of delivering larger genes. Apart from that, we’re en route,” Dr MacLaren added.

STEM CELL TRANSPLANTS
Stem cell transplants for retinal diseases have only very recently been used in clinical trials in small numbers of patients. Although it is too early to provide an assessment of their therapeutic value, in-vitro and animal studies indicate that the approaches have great potential, showing that it is possible using three-dimensional stem cell culture techniques. It is possible to generate stratified layers of retinal cells and that multilayered retinal stem cell implants can provide vision to genetically blind mice.
The two competing stem cell technologies for clinical use are those using embryonic stem cell techniques and those using autologous induced pluripotent stem (iPS) cells. Embryonic stem cells have the advantage of being producible on a grand scale at a fairly modest expense using closely monitored cell lines of proven safety and efficacy. In contrast, autologous iPS cell grafts take a year to create at an expense of around $400,000 to $800,000 per patient. 
Both approaches have been used in phase1/2 trials to generate retinal pigment epithelial cells for implantation in the subretinal space of eyes with AMD. The RPE plays a pivotal role in the pathophysiology of AMD. 
The first application of stem cell therapy for dry AMD in humans was reported by Steven D Schwartz MD, Jules Stein Eye Institute, UCLA, USA. The researchers used cell cultures derived from blastocysts and induced them to differentiate into RPE cells. They then injected the cells in a suspension subretinally in nine patients with dry AMD and nine patients with Stargardt’s disease. The patients received strong immunosuppressant therapy. (Schwartz et al, Lancet 2015 ;385 : 509-516) 
After the injection, clumps of hyperpigmented stem cells were visible. Furthermore, there was no evidence of inflammation or rejection. And although the trial was mainly focussed on the safety of the technique, the researchers reported improvement in several different functional measures. However, there was no clear topographic correspondence between retinal regions with improved function and regions where there was hyperpigmentation.
“Emphasis needs to be placed on the fact that this is the first use of ESC-engrafting of any kind and that it established proof of principle, that the transplanted cells could survive without the feared adverse side effects like teratoma or immune reaction. Much to our surprise, some patients had an improvement in their vision,” Dr Schwartz told EuroTimes in an interview.
He added that while iPS cell transplants avoid any ethical, religious or regulatory concerns regarding the use of embryonic stem cells, the stem cell line he used in his study were derived not from abortions but from unused zygotes obtained through fertility treatment. He added that, compared to iPS cell lines human which hold great promise and may eventually be safe and scalable, embryonic cell lines seem at present to be less likely to be tumorigenic. Induced stem cells have a tendency to revert to their original cell type and are more prone to mutation, he said. 
Still open to question is whether embryonic stem cell therapy will be more immunogenic than techniques using autologous iPS cells, or whether the retina’s immune privilege will make any difference irrelevant, he said. 

iPS CELL APPROACH
Masayo Takahashi MD, PhD and her associates at the Riken Centre for Developmental Biology in Kobe, Japan, have described the subretinal implantation of sheets of autologous iPS-cell derived RPE cells (Kamao et al, Stem Cell Reports 2014; 2:205-208). They have so far implanted the autologous RPE-sheet in one patient with neovascular AMD, observed no immune rejection without immunosuppression, but have had some setbacks. 
The first trial they initiated with the treatment was halted because of a debate induced by mutation detected in one of the autologous iPS cell lines. In addition, subsequent changes in legislation meant that clinical research in Japan can no longer be led by research facilities, only by hospitals.
However, Dr Takahashi told EuroTimes in an interview that although the first patient in the trial has not had any noticeable changes in vision, she has been able to cease her anti-VEGF injections. She added that the type of mutation they have seen has been proved not tumorigenetic in the animal test. She also noted that she and her team are now planning clinical research involving the use of stem cells derived from a iPS cell bank derived from human blood cells. 
“We confirmed with the animal experiments that no immune rejection occurred if the HLA is matched. For the HLA-matched patients we may not use immunosuppression. For HLA-mismatched patients, of course we will use it.”
Regarding the future use of gene therapy and stem cell transplantation, Dr MacLaren said it is likely that, as experience grows with the techniques and research continues to expand in the area, possibilities will emerge that stretch the imagination.
“There are lots of different approaches. We may be able to switch on the repair process in the stem cells already present for the retina so we need to stay open-minded, but what we also need to do is more clinical trials,” he added.

Robert MacLaren: enquiries@eye.ox.ac.uk
Masayo Takahashi: mretina@cdb.riken.jp
Steven D Schwartz,
Contact – Stephanie Wynbrandt, Jules Stein Eye Institute, UCLA: 
wynbrandt@jsei.ucla.edu 

Tags: gene therapy, retina