Organoids: advancing towards new personalized gene therapies



Assistant Professor at Radboud University Medical Center Nijmegen, Holanda)

Specialist in molecular therapies for inherited retinal diseases and neurometabolic disorders

Dr. Alex Garanto carried out his doctoral thesis at the University of Barcelona (2011) under the supervision of Drs. R. González-Duarte and G. Marfany, studying the function of CERKL, a gene associated with Retinitis Pigmentosa and Cone-Rod Dystrophy. After his PhD he was granted a postdoc position and integrated in the Blindness Genetic Therapy group, at the Department of Human Genetics at Radboud University, Nijmegen (The Netherlands). Under the direction of Dr. Rob Collin, he generated in vitro and in vivo models that opened new perspectives and set the bases for the development of novel therapies for retinal disorders. He is currently (2022) Assistant Professor at the Departments of Pediatrics and Human Genetics of the Radboud university medical center, where he leads a research group focused on molecular therapies for the treatment of inherited neurometabolic and retinal diseases.


In your professional career, there is a transition from studying the function of CERKL and the generation of animal models to the development of cellular models (organoids). Which were the key steps and aims of this process?

Animal models have contributed significantly to identify gene function and to understand many functional processes. However, the retina is a very specialized tissue and not always do the observations in animal models recapitulate the phenotype in humans. In my experience, both during my PhD and my postdoc, I generated and characterized two animal models (one for CERKL and another for CEP290) and both of them did not recapitulate the human phenotype. This has also been shown for other models in literature, such as ABCA4 mouse models.

Furthermore, within my research group, we develop genetic therapies mainly based on antisense oligonucleotides (AONs) and genome editing techniques. These approaches are sequence-dependent, and therefore the same molecule designed for humans is not compatible with, for example, the mouse sequence. For all these reasons, we started to develop human-based models based on stem cell technology. The advantages of these models rely on the fact that from blood cells of the patient, we can produce pluripotent stem cells that can be subsequently differentiated to retina-like cells that allow the study of the disease mechanisms, therapy screening and functional readouts.


Innovative methodological approaches for the generation of retinal organoids are one of the key features of your research. Could the use of retina cell derived 3D organ-like structures to improve the knowledge of disease mechanisms and the development of new treatments? Could you explain your differential contribution in relation with previous approaches?

The generation of induced pluripotent stem cells has revolutionized the development of cellular models. In the past, culturing neurons was difficult as they do not divide, but nowadays in weeks or months we can have different types of cells starting from the same origin. These models are usually human-based, recapitulate certain features of the diseases, and allow the screening of known or new therapies. In the past, all these processes were tested in animals, and there have been examples in which something that seemed to not be toxic in mice, was in humans and vice versa. Another advantage of these models is that we can generate 2D cultures or also 3D organoids. The latter are structures that resemble relatively well the retina or the brain for example. In the 2D cultures, usually we have a pure population of cells or a combination of, but they are not in the proper three-dimensional spatial structure. In fact, in the Netherlands, there is a strong focus on the organ-on-chip technology, to develop human-based cellular models that can mimic the human situation and allow the assessment of therapies, therefore reducing the number of experimental animals.

In particular, when talking about the eye, these 3D organoids are still not complete. Photoreceptors lack the outer segment, and there is no RPE (retinal pigmented epithelium) surrounding the photoreceptors. In addition, the differentiation process is costly and lengthy (minimum 150-180 days, and nowadays there are articles suggesting 250-280 days). However, the overall retinal structure is there, and this has allowed to study localization of proteins or the effect of mutations. One example on how this technology was employed to test therapeutics can be found in this article . Using organoids, it was possible to determine the efficacy of an antisense oligonucleotide molecule at RNA and protein level. In addition, it was possible to evaluate toxic effects caused by the binding of the molecule to the wrong place (off-target effect) and potential toxicity of the chemical modifications that are present in the molecule. With this information, and minimal toxicology (not efficacy) in rabbits and non-human primates, the molecule was approved for a phase 1/2 clinical trial.


Based on your experience in the use of antisense oligonucleotides (AONs) for therapeutic purposes in animal models, how do you assess their present and future applications in relation to other therapies?

Antisense oligonucleotides are small acid nucleic molecules that can have multiple functions. We mainly use them to module splicing. In particular, we use AONs to remove pseudoexons that are included in the final mRNA due to deep-intronic variants. On the positive side, AONs are relatively small molecules that can be delivered to the eye by an intraocular injection and penetrate into the cells of the retina. Because the eye is a closed environment with non-dividing cells, the effect of these AONs can last longer than when delivered systemically to other tissues. On the negative side, they have a half-life of 3-6 months in the eye, and this means that injections will be required throughout the entire life of the individual. This is at the same time something positive when compared to other therapies like gene augmentation. In the latter, a correct copy of the gene is delivered in a virus. This is usually one single injection with an effect for, in principle, the entire life. So, if there is a potential toxic effect, in the case of gene augmentation, the virus is already in the eye and cannot be removed. However, if the AONs are not well tolerated, by stopping the treatment the toxic effects should revert.

The challenge nowadays for AONs for retinal therapies is to assess efficacy in relevant models. In contrast with other diseases, delivery to the eye is easy. However, when other organs or tissues need to be targeted, the delivery is still a big challenge. For that, nanoparticles, conjugating peptides, new chemical modifications, etc. are being tested to increase the uptake by those organs in which local delivery is not an option and are difficult to target by systemic delivery.

In vivo, we usually have the issue when assessing efficacy, that these AONs are directed to a human sequence. Therefore, in the ideal case, a humanized model would be the ideal candidate. However, this is not always possible and as I explained in the previous questions, those animals do not always recapitulate the human phenotype. Nowadays, most of the efficacy data is usually done in vitro, but often measurable readouts are missing. This means that the identification of functional readouts will increase the development of AONs and other therapies.

If we think about the future of AONs compared to other therapies, it is that they can be designed and tested relatively fast at a lower cost than gene augmentation for instance, therefore accelerating the development of potential treatments. As said, despite in our lab, we use them mainly for modulating splicing, the AONs can also be directed to degrade a transcript, block translation or modulate gene expression.


Once the preclinical phase is over, biotechnology companies move on to start the clinical phases in humans. What is the relationship between preclinical stages with the pharmaceutical companies in The Netherlands?

Developing any type of genetic therapy is costly. So preclinical work is often covered by grants, but the translation to the clinic cannot be easily made in an academic setting. Therefore, the interaction between academia and industry is crucial. In the field of the oligonucleotides, we are quite well connected. A great example is the Oligonucleotide Therapeutic Society, a scientific meeting in which academia and industry are equally represented. Also, another important player that we often forget are the patients and patient organizations. We should definitely have them also on board in the design of any strategy. Also, they support our research financially, both the work in academia and industry. And last but not least, regulators are also important. We need to design and identify new ways to:

  • Design these clinical trials: usually these therapies are for rare diseases. Therefore, it can happen that in a phase 1/2 study the majority of the patients are treated.
  • Decrease the price of the drug if it reaches the market, for example, Luxturna costs approximately 350.000 Euro per injection, so 700.000 euro per patient for the 2 eyes.
  • Increase interaction between academia and industry to speed up development.

Overall, the field is progressing fast, but we still have obstacles that we need to address.


What do you expect for the future of therapies in the field of IRD and other metabolic disorders?

The eye is the ideal organ for therapeutics, and I expect an increase of genetic therapies available in the coming years. For neurometabolic diseases, it is a bit different. These diseases are often multi-organ and therefore more challenging. For diseases affecting the liver, several approaches are already in advanced stages. For other diseases a combination of cellular and genetic therapy is used. For instance, taking cells of the patient, modifying them ex-vivo and implanting them again. In brief, many and many strategies are being developed and its therapeutic potential is being evaluated. One important aspect that is not easily covered in all countries is a good genetic diagnostic system. Many patients have not been diagnosed with the mutated gene, and this complicates the eligibility for or the design of treatment options.

Last but not least, I expect in the coming years an increase in the number of genetic treatments for ultrarare or unique variants. In fact, in the AON field there is an inspiring case published in 2019 [linexterior_2] in which a personalized AON for a single patient was developed in less than a year. This study has led to the creation of new initiatives to bring AON treatment to these unique mutations in a non-commercial context. Some examples of these initiatives are: n-lorem, 1M1M (one mutation one medicine) or the Dutch Center for RNA therapeutics (DCRT). [linkexterno_3]


Awards and Achievements:

  • 2023: Appointed full Principal Investigator, Radboudumc
  • 2022: Editor of the book Antisense RNA Design, Delivery, and Analysis (open access:
  • 2021: Mary Ann Liebert, Inc. publishers Young Investigator Award of the Oligonucleotide Therapeutics Society
  • 2021: Tenured position at the Department of Pediatrics in combination with the Department of Human Genetics.
  • 2020: Appointed junior Principal Investigator, Radboudumc
  • 2014-2019: Several travel grants including ARVO international, Oligonucleotide Therapeutics Society, Pro-Retina, Retina International, etc. to attend several international meetings
  • 2014: EMBO Short-term fellowship


Recommended Publications:

Antisense RNA Therapeutics: A Brief Overview. Arechavala-Gomeza V, Garanto A.Methods Mol Biol. 2022. 2434:33-49.

A look into retinal organoids: methods, analytical techniques, and applications. Afanasyeva TAV, Corral-Serrano JC, Garanto A, Roepman R, Cheetham ME, Collin RWJ. Cell Mol Life Sci. 2021. 78(19-20):6505-6532.

Antisense Oligonucleotide-Based Rescue of Aberrant Splicing Defects Caused by 15 Pathogenic Variants in ABCA4. Tomkiewicz TZ, Suárez-Herrera N, Cremers FPM, Collin RWJ, Garanto A. Int J Mol Sci. 2021. 22(9):4621.

Delivery of oligonucleotide-based therapeutics: challenges and opportunities. Hammond SM, Aartsma-Rus A, Alves S, Borgos SE, Buijsen RAM, Collin RWJ, Covello G, Denti MA, Desviat LR, Echevarría L, Foged C, Gaina G, Garanto A, Goyenvalle AT, Guzowska M, Holodnuka I, Jones DR, Krause S, Lehto T, Montolio M, Van Roon-Mom W, Arechavala-Gomeza V. EMBO Mol Med. 2021. 13(4):e13243.

Identification and Rescue of Splice Defects Caused by Two Neighboring Deep-Intronic ABCA4 Mutations Underlying Stargardt Disease. Albert S, Garanto A, Sangermano R, Khan M, Bax NM, Hoyng CB, Zernant J, Lee W, Allikmets R, Collin RWJ, Cremers FPM. Am J Hum Genet. 2018. 102(4):517-527

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