Retinal dystrophies: gene-agnostic therapy based on miRNAs

New miR-based gene-agnostic strategies target shared mechanisms in retinal dystrophies, opening new therapeutic options beyond single-gene therapy. 

February marked Retinitis Pigmentosa Awareness Month, highlighting a rare, inherited and degenerative condition affecting retinal photoreceptors and leading to progressively worsening visual impairment, which may ultimately result in severe vision loss or blindness. Retinitis pigmentosa belongs to the broader group of inherited retinal diseases, which also includes Stargardt disease, Best vitelliform macular dystrophy, and Leber congenital amaurosis. 

These disorders are caused by mutations in hundreds of different genes, making the use of conventional gene therapy particularly challenging. Traditional gene replacement strategies are based on the paradigm of “one gene, one mutation”, in which the defective gene is replaced with a functional copy. However, such an approach becomes difficult to apply in diseases characterised by extreme genetic heterogeneity. 

As with many rare and ultra-rare conditions, additional barriers complicate therapeutic development, including high development costs, long timelines, and small patient populations, which affect both clinical trial feasibility and long-term economic sustainability. When analysed at the mutation level rather than by clinical phenotype, some variants may even be unique to a single patient worldwide. 

For these reasons, researchers have increasingly turned towards a mutation-independent, or gene-agnostic, therapeutic strategy. 

Within this framework, scientists at TIGEM (Telethon Institute of Genetics and Medicine) are developing miR-based gene therapies designed to target shared pathogenic mechanisms, rather than individual genetic defects. The goal is to create therapeutic approaches applicable to all patients who share the same clinical manifestation of disease, regardless of the underlying mutation. 

The most widely recognised concept of gene therapy is based on identifying a mutated gene whose defective product causes a specific disease. In this framework, the therapeutic intervention aims to correct or replace that gene with a functional version. 

A mutation-independent (or gene-agnostic) approach follows a different rationale. Rather than targeting a specific gene and its multiple variants, this strategy focuses on pathogenic mechanisms shared across different forms of the same disease. The intervention does not address the primary genetic defect itself, but the downstream biological processes that drive disease progression. 

A well-known example of classical gene therapy is the treatment for Leber congenital amaurosis, a rare inherited retinal dystrophy and a frequent cause of severe visual impairment or blindness in early infancy. This therapy is based on gene replacement: patients carrying mutations in the RPE65 gene receive a functional copy of the same gene, directly correcting the underlying genetic defect. 

The gene-agnostic approach differs fundamentally. Instead of acting on an individual mutated gene, it targets shared pathological pathways activated downstream of diverse mutations, with the aim of slowing disease progression and reducing severity regardless of the specific genetic cause. Such strategies are particularly relevant in conditions characterised by high genetic heterogeneity, where numerous genes may lead to a similar clinical phenotype. This is the case for inherited retinal diseases as well as many mitochondrial disorders. Overall, inherited retinal dystrophies alone may result from mutations in 300–400 different genes. 

This approach may also prove advantageous in diseases where the pathogenic mechanism requires gene silencing rather than gene replacement, for example when pathology arises from gene overexpression or the acquisition of toxic gain-of-function properties. In these scenarios, conventional gene replacement therapy becomes significantly more difficult to implement. 

“Developing a specific therapy for each individual gene takes time. This is why, to date, the only gene therapy approved and available in clinical practice for retinal dystrophies is the one for Leber congenital amaurosis, whose development — from initial experimental evidence to regulatory approval — required more than fifteen years” explains Sandro Banfi, researcher at TIGEM and Professor of Medical Genetics at the University of Campania “Luigi Vanvitelli”. 

Gene-specific therapies also require substantial financial investment. When mutations are relatively common and affect larger patient populations, developing a targeted therapy may remain economically viable. 

“However” notes Alessia Indrieri, TIGEM and CNR researcher specialising in mitochondrial medicine, “there are also rare and ultra-rare cases. Given the cost of developing these therapies, it is very likely that — without a major technological leap in the coming years — many of these patients will remain without treatment. The real challenge is the economic sustainability of developing therapies for only ten or fifteen patients”. 

The key advantage of gene-agnostic or mutation-independent gene therapies lies precisely in their broader applicability: they can potentially be used in all patients sharing the same clinical phenotype or common pathogenic mechanism. This increases the likelihood of delivering treatments more rapidly and at more sustainable costs, even for rare and ultra-rare conditions. 

As discussed, the mutation-independent approach begins with a shift in perspective. Rather than intervening directly on the gene responsible for the disease, the strategy focuses on pathological mechanisms activated downstream of the mutation. In disorders such as retinitis pigmentosa, the shared outcome is a progressive degeneration of specific retinal cell populations, regardless of the original genetic defect. 

Gene-agnostic therapies aim to slow disease progression by targeting mechanisms common to different forms of the disease. The goal is to preserve retinal tissue functionality for as long as possible, maintaining the eye’s potential responsiveness to future gene-specific therapies, should these become available for individual mutations. 

Identifying a shared therapeutic target relies on detailed investigation of disease pathogenesis, made possible through the cumulative work of an entire scientific community. 

“If we look, for example, at neurodegenerative disorders — which include both retinal diseases and conditions affecting the brain — we see that they share several altered biological processes” explains Alessia Indrieri. 

From this observation arises the hypothesis of a common therapeutic target. Experimental validation then becomes essential: specific assays can determine whether modulating a given mechanism produces measurable therapeutic benefit. Once confirmed, the same strategy can be extended to different forms of a disease that share a clinical phenotype, even when driven by distinct genetic mutations. 

“We also know that several molecular pathways are consistently involved in neurodegenerative diseases. One example is the reduced capacity of cells to produce energy” adds Sandro Banfi. 

Within this pathway, cellular attempts to compensate for energy deficiency may themselves activate toxic processes that further damage the cell. 

Another key mechanism is the inflammatory response. As cells begin to degenerate, neighbouring cells trigger inflammation in an attempt to eliminate damaged tissue. However, what initially represents a protective response can become a double-edged sword: chronic inflammation ultimately harms healthy cells and accelerates disease progression. 

These shared processes — including impaired energy metabolism and chronic inflammation — therefore emerge as promising therapeutic targets. Building on consolidated biological knowledge, research can then investigate disease-specific mechanisms in greater depth. A precise understanding of pathogenesis allows scientists to identify critical intervention nodes and design strategies capable of counterbalancing degenerative processes. 

MicroRNAs (miRNAs) are small regulatory molecules whose function is to modulate cellular activity. Rather than acting as simple on–off switches, they fine-tune gene expression. A single microRNA can regulate not only one, but multiple cellular functions simultaneously. This represents their key advantage: they are endogenous molecules, naturally used by the organism to precisely control complex biological processes. 

“MicroRNAs are like orchestra conductors: they do not switch an instrument off, but adjust its volume, asking it to play more softly or more intensely while maintaining overall harmony” explains Alessia Indrieri. 

This means that increasing microRNA expression by delivering additional molecules to the cell — or reducing their activity — does not produce disruptive effects, but rather a coordinated and finely tuned modulation of the targeted function. A single miRNA can simultaneously influence, for example, both inflammatory responses and cellular energy production. 

“Of course, safety and potential side effects must be carefully evaluated, but the fundamental idea behind microRNA-based approaches is precisely this: to modulate rather than overturn biological systems” adds Sandro Banfi. 

Another important advantage is that miRNAs represent regulatory tools already selected through evolution to control the very biological pathways involved in disease pathogenesis. The strategy does not rely on introducing entirely artificial mechanisms, but on harnessing regulatory systems that are intrinsically present within cells. 

Research on miR-204 originated from a systematic project aimed at identifying the most abundant microRNAs in the retina. Among them, miR-204 emerged as particularly highly expressed. To investigate the consequences of altering its expression levels, researchers used an animal model — a small fish closely related to zebrafish — observing that both increased and reduced expression of miR-204 impaired retinal development. 

Definitive evidence of the involvement of miR-204 in retinal function came through the identification, in collaboration with an international consortium of geneticists, of a family affected by retinitis pigmentosa carrying a mutation in miR-204. This represented the first documented case of a retinal disease caused by a microRNA. 

“At that point, the question became therapeutic: if a microRNA is involved in the disease, could it also be used to counteract it?” explains Sandro Banfi. 

In murine models of retinitis pigmentosa, the research team delivered miR-204 using viral vectors similar to those employed in gene therapy. The results were encouraging: the treated eye showed slower retinal degeneration compared with the untreated eye. 

“This was a first indication of a protective effect, later confirmed across several experimental models” Banfi concludes. 

The development of miR-181 arose from the convergence of two research areas: retinal microRNA biology and mitochondrial disease research. Mitochondrial disorders — an extremely heterogeneous group of conditions caused by mutations affecting either nuclear DNA or mitochondrial DNA — share a central pathogenic feature: impaired cellular energy metabolism. 

Researchers identified miR-181 as a potential regulator of cellular energy homeostasis and mitochondrial activity. In this case, however, therapeutic benefit was not expected from activation but from inhibition of the microRNA. Studies in models of mitochondrial disease — including severe systemic conditions such as Leigh syndrome, a paediatric neurodegenerative disorder — showed that suppressing miR-181 produced beneficial effects. 

These findings led to the development of a therapeutic strategy based on a miR-181 inhibitor, delivered to target tissues — retina or brain — through gene therapy approaches. The programme is currently in an advanced preclinical stage. 

“Because mitochondrial dysfunction represents a shared mechanism across many neurodegenerative diseases, research has expanded to additional conditions such as retinitis pigmentosa and glaucoma. The ambition is to develop a single therapeutic molecule capable of acting across multiple diseases linked by the same biological mechanism, regardless of the mutated gene” says Alessia Indrieri. 

The therapeutic potential of miR-181 does not stop at the retina. Researchers have extended their investigations to Parkinson’s disease, a condition that includes both well-defined genetic forms and more complex idiopathic variants. The link between impaired cellular energy production and Parkinson’s disease is also supported by environmental evidence. Exposure to rotenone, a pesticide that inhibits mitochondrial respiratory chain complex I, has been associated with increased disease risk in exposed populations. 

On this basis, researchers tested miR-181 inhibition in several murine models of Parkinson’s disease. Although the results remain preliminary, they have been encouraging. Translation to clinical application, however, is more challenging than in ocular diseases, which benefit from an accessible target tissue and well-established delivery technologies. 

“In the case of the brain, the main challenge is how the therapy reaches the target tissue. However, viral vectors already used in clinical settings are capable of crossing the blood–brain barrier and reaching the brain following systemic administration. This makes the approach technically plausible,” explains Alessia Indrieri. 

Delivering microRNAs — or their inhibitors — into target cells first requires identifying the most effective and safest delivery platform. For retinal diseases, the TIGEM group has relied on adeno-associated viral (AAV) vectors, a technology already validated in clinical practice. 

Both miR-204 and miR-181 therapeutic strategies are based on this platform: AAV vectors are used to deliver either the microRNA itself or a specific inhibitor directly into the retina. Alongside this approach, researchers are exploring more flexible alternatives, including RNA molecules encapsulated in lipid nanoparticles, similar to those employed in mRNA vaccines. 

“The COVID-19 pandemic dramatically accelerated the development of these platforms, making them more mature and technologically advanced. This is not only a scientific issue but also an economic one. Producing a clinical-grade viral vector is complex and costly — gene therapies can reach several million euros per dose. Lipid nanoparticles, by contrast, could prove less expensive and more versatile” notes Alessia Indrieri. 

Logistics also play a crucial role. 

“In retinal diseases, gene therapy currently requires subretinal injection — a delicate surgical procedure that can be performed only by highly specialised clinicians in a limited number of centres. A less invasive delivery route, such as an outpatient or systemic administration, would make treatment more accessible and potentially more widespread” adds Sandro Banfi. 

The ultimate goal is therefore not only to demonstrate the biological efficacy of microRNAs as therapeutic targets, but also to identify the most sustainable, safe and clinically feasible platform capable of translating these therapies from laboratory research into real-world medical practice. 

The decision to begin with ocular diseases was not accidental. The retina is a relatively accessible tissue, allowing faster translation from experimental research to patients. From a therapeutic development perspective, the most advanced programme currently focuses on miR-181 inhibition in mitochondrial optic neuropathies. In these models, the preclinical phase is essentially complete: collected data show that silencing miR-181 can improve pathological features and slow neurodegeneration. The next step will be the initiation of a clinical trial, which will require dedicated funding. 

For miR-204, as previously described, the research team has demonstrated a protective effect following modulation of its expression in animal models. However, additional validation studies are still required before clinical translation. 

Alongside these two main research lines, a broader and more systematic effort is underway. The creation of a retinal microRNA expression atlas has enabled researchers to identify the most abundant miRs in the human retina. 

“We then asked whether, among the hundreds of known microRNAs, there might be others with unexplored therapeutic potential” explains Sandro Banfi. 

This question led to the launch of the RetMIR project, designed to systematically test the modulation of multiple microRNAs in models of retinitis pigmentosa. By both increasing and reducing miR expression levels, researchers aim to identify protective effects and uncover new therapeutic candidates. 

“This represents a change of scale: no longer studying a single microRNA driven by an initial hypothesis, but performing systematic screening that could significantly expand the number of available therapeutic targets” Banfi continues. “The work is carried out together with Alberto Auricchio, Scientific Director of TIGEM and Professor of Medical Genetics at the University of Naples Federico II, and relies on a dedicated library of adeno-associated viral vectors developed specifically for screening purposes. We have already identified a couple of new candidates that are now undergoing deeper preclinical investigation”. 

Beyond economic constraints, regulatory caution represents another important challenge. Because microRNAs simultaneously modulate multiple genes, concerns remain about potential off-target effects. However, these molecules do not act randomly; they regulate coherent biological networks that have been conserved through evolution. In ocular models, no significant toxicity signals have emerged so far, although safety remains a central issue, particularly when extending the approach to more complex tissues such as the brain. 

MicroRNA modulation is not a new concept. Similar strategies are already under investigation in oncology and cardiovascular diseases, with several advanced clinical trials underway and at least one Phase III study potentially leading to the first regulatory approval of a microRNA-based therapy. The scientific community working on microRNAs as therapeutic targets is rapidly expanding, and numerous clinical studies are currently in progress. 

Such approaches appear especially promising for complex diseases, where coordinated intervention across multiple biological pathways is required. 

According to the TIGEM research teams, future development will follow three main directions: 

“The future moves along three lines: bringing the most advanced candidates into clinical trials, continuing to identify new microRNAs with therapeutic potential, and developing new delivery platforms and administration strategies to make therapy simpler and less costly” says Sandro Banfi. 
“Moreover, the screening framework developed for the retina can be extended to other tissues, potentially adapting the type of vector used”. 

Ultimately, the objective remains unchanged: to reach the biological target in the most effective, safe and sustainable way possible, translating molecular insight into therapies capable of addressing unmet medical needs in retinal and neurodegenerative diseases.