Face-to-face time: the retina "from a test tube" will help to save people from blindness

A team of researchers from MIPT has developed a method for cultivating human retina fragments in the laboratory. The technology will allow evaluating the effectiveness of viral vectors for gene therapy even before the start of expensive animal trials, which will accelerate and make safer the development of treatments for blindness caused by photoreceptor degeneration. Retinal fragments are obtained postmortem from donors, then they are cultured. Such models can be used to test new delivery viruses and platforms that are considered good candidates for gene therapy. At the same time, scientists will have to solve a number of tasks related to the features of such models in order to accelerate the introduction of new methods of treating blindness into clinical practice, experts told Izvestia.

Retina "from a test tube"

Scientists at the MIPT Center for Living Systems have proposed a promising model for gene therapy of blindness — fragments of donor retina cultured in the laboratory. As the experts explained, optogenetic gene therapy is at the heart of many modern approaches to the treatment of hereditary diseases: with the help of a harmless virus, genes encoding photosensitive proteins are delivered to retinal cells. These proteins, embedded in the remaining neurons, restore their ability to respond to light, partially compensating for the lost photoreceptors.

The main problem is that the preclinical evaluation of new viral vectors traditionally relies on testing in animal models and cell cultures. But the results obtained in mice, rats, or even primates are often poorly reproduced in the human eye. As a result, a promising vector that has shown excellent tropism (affinity for certain cell types) in animals may actually be ineffective or even toxic to humans. In addition, such experiments require a lot of time, money and raise ethical issues.

"Our approach, based on culturing explants from a donor retina, allows us to directly study the interaction of viral vectors with human tissue, while maintaining a complex architecture and cell types. But this method involves several complex key steps," said Alsallum Almakdad, head of the retinopathy gene Therapy group, researcher at the Laboratory of Genomic Engineering at the Moscow Institute of Physics and Technology.

Within six hours after the death of the donor, the retina must be removed and separated from the pigment epithelium and vitreous body. Then, using a special round punch with a diameter of 5 or 8 mm, cut out identical fragments from different areas. These fragments are placed on membrane inserts with 0.4 micron pores in a special nutrient solution. The retina is not completely immersed in the solution, but is located at the interface of the media: nutrients enter from below through the pores, air access is provided from above. This approach allows you to recreate conditions close to natural, so that the tissue remains viable for up to two weeks.

— By isolating the retina and then cultivating it, we are able to preserve its viability as much as possible by creating a special environment. This makes it possible to test new associated viruses or lentiviruses on a specific organ, or new platforms that are considered good candidates for gene therapy. It is enough to literally drip these candidates onto the retina on the first or second day after isolation and wait 7-14 days until they enter specific cells. Then send the samples for histology or using fluorescence to determine where your viruses are located, which cells they eventually targeted," commented Alsallum Almakdad.

In other words, this is a fairly economical and effective method of screening new therapies, the specialist concluded. The technology can already be used in laboratory practice. The method has its limitations, but, in fact, the researchers have created a biological "test bench" that is much closer to reality than standard cell cultures. This will accelerate the development of therapies for diseases such as retinitis pigmentosa, Stargardt's disease and age-related macular degeneration, which affect millions of people worldwide.

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Prospects for the creation of new methods of gene therapy for blindness

In ophthalmology, there are many serious diseases of the retina that lead to disability. This is a huge social problem all over the world, and it still remains unresolved, Andrey Demchinsky, Director of Science at Elvis Neuroimplants and president of the Aspectum Association, told Izvestia. For decades, scientists have been looking for approaches that could help such patients. There are drugs, stem cells, visual prostheses, and optogenetics. The latter could potentially help thousands of totally blind people regain some of their visual functions. However, there are no reliable approaches that can be effectively and widely implemented in clinical practice.

— Therefore, any tool that will speed up experiments is highly valuable. If scientists have the opportunity to increase the frequency of iterations in experiments, as well as use more objective methods of monitoring the result, this should bring science closer to helping many blind people," the expert noted.

The method of culturing retinal cells and growing three—dimensional organoids from them is one of the most significant achievements in ophthalmology. It allows us to better understand the processes occurring in the retina and find approaches to the treatment of diseases that were considered incurable, ophthalmologist, PhD, General Director of Vista Ophthalmological Clinic Anton Kazantsev told Izvestia. Previously, two-dimensional structures in a Petri dish were used, but now they have realized that three-dimensional structures are more viable, self-organize, exchange information, and increasingly resemble the complex organization of a real retina.

— Until now, new pharmacological substances could be tested mainly on animals, but the structure of their eyes is significantly different from that of humans, which created a barrier. It is impossible to conduct such studies on living people. Volumetric structures allow us to study the mechanisms of diseases and test various effects — viral vectors, drugs, new molecules. This is the near future, the methods of which are already widely used," said the specialist.

However, according to him, there are difficulties: the process takes at least six months, there are no vessels and immune cells in the organoid, the structure of the retina is not fully recreated, since the macula, the central zone and the periphery differ from each other. Therefore, scientists face many ambitious tasks in this direction. And it may still take years to introduce new methods of gene therapy for blindness into clinical practice.

The study is published in Springer Nature Link as part of the book Retinal Gene Therapy: Methods and Protocols.