Periodic Reporting for period 1 - Organ-VIP (Genetically modified organoids for genetic variant interpretation in colorectal cancer)
Berichtszeitraum: 2020-09-01 bis 2022-08-31
Colorectal cancer (CRC) is the fourth leading cause of cancer death, being the second most common cancer in women and the third in men. Environmental and genetic factors play a key role in the development of this disease, and 10-20% of the cases correspond to familial or hereditary forms. Although several genes have been causally linked to a predisposition to CRC and/or polyposis, there is still a high proportion of patients with familial and/or early-onset CRC that do not carry pathogenic variants in any of the known hereditary cancer genes.
In the last decades, Next Generation Sequencing (NGS) technologies have boosted the discovery of candidate CRC predisposition genes, and the identification of variants of unknown significance (VUS) in known hereditary cancer genes. However, until the causal link of those candidate genes with cancer is confirmed, and the pathogenic or benign nature of the variants is dilucidated, they lack clinical value. The identification of a pathogenic variant in a cancer predisposition gene significantly improves the clinical management of the individual carrier, through the application of individualised surveillance protocols based on the corresponding cancer risks, and in some cases, of personalized oncologic treatments based on targeted therapies. Variant classification largely relies on clinical data (cosegregation data, tumour molecular features); however, these are often unavailable, leaving the functional experimental evidence to be decisive for variant classification.
The project to be developed by the MSCA-IF grantee is aimed to develop a platform based on genetically modified intestinal organoids for the functional characterization of variants and genes affecting Wnt signalling, BMP/TGF-beta pathway, or DNA repair mechanisms. The specific objectives are: (a) to implement and optimize the intestinal organoid culture, gene editing and gene-specific functional tests in the host lab to study VUS potentially involved in the development of hereditary CRC (hCRC); (b) to assess VUSs in hereditary CRC and polyposis genes affecting the different pathways/mechanisms mentioned above; and (c) to calibrate the obtained functional results for implementation of variant classification guidelines. On the other hand, this fellowship is also focused on the training and development of skills for the development of the Fellow’s professional career.
In the last decades, Next Generation Sequencing (NGS) technologies have boosted the discovery of candidate CRC predisposition genes, and the identification of variants of unknown significance (VUS) in known hereditary cancer genes. However, until the causal link of those candidate genes with cancer is confirmed, and the pathogenic or benign nature of the variants is dilucidated, they lack clinical value. The identification of a pathogenic variant in a cancer predisposition gene significantly improves the clinical management of the individual carrier, through the application of individualised surveillance protocols based on the corresponding cancer risks, and in some cases, of personalized oncologic treatments based on targeted therapies. Variant classification largely relies on clinical data (cosegregation data, tumour molecular features); however, these are often unavailable, leaving the functional experimental evidence to be decisive for variant classification.
The project to be developed by the MSCA-IF grantee is aimed to develop a platform based on genetically modified intestinal organoids for the functional characterization of variants and genes affecting Wnt signalling, BMP/TGF-beta pathway, or DNA repair mechanisms. The specific objectives are: (a) to implement and optimize the intestinal organoid culture, gene editing and gene-specific functional tests in the host lab to study VUS potentially involved in the development of hereditary CRC (hCRC); (b) to assess VUSs in hereditary CRC and polyposis genes affecting the different pathways/mechanisms mentioned above; and (c) to calibrate the obtained functional results for implementation of variant classification guidelines. On the other hand, this fellowship is also focused on the training and development of skills for the development of the Fellow’s professional career.
Within the framework of the fellowship, a human intestinal organoid biobank was successfully established, including seven lines originated from biopsies obtained in screening colonoscopies and fourteen lines derived from tissue pieces obtained during colorectal surgery.
The first purpose of this project was to generate knock out (KO) organoids of the main genes involved in hCRC using CRISPR/Cas9, for which a puromycin cassette was to be introduced in one of the first coding exons of the gene, thus disrupting its function and facilitating the selection of the edited organoids with puromycin treatment. Nucleofection and lipofection were tested to introduce the CRISPR/Cas9 machinery and, so far, lipofection appeared to be the most effective. However, some optimisation is still needed to increase transfection efficiency.
The second purpose of this project was to generate a battery of organoids containing different VUS in the genes of interest. Due to the low efficiency of the current transfection methods, lentiviral infection was used to generate organoid lines that endogenously express a base editor (a modified Cas9 protein that converts one base or base pair into another), thus only requiring the introduction of small sgRNA molecules to generate the desired point mutations and so facilitating the transfection. So far, organoid survival post-infection has been improved, but the editing efficiency is still low. Therefore, further tests are still needed to increase editing efficiency and to successfully isolate and expand Cas9-expressing organoids.
The project is ongoing, and we are currently working on producing a proof-of-concept paper that will be published shortly.
The first purpose of this project was to generate knock out (KO) organoids of the main genes involved in hCRC using CRISPR/Cas9, for which a puromycin cassette was to be introduced in one of the first coding exons of the gene, thus disrupting its function and facilitating the selection of the edited organoids with puromycin treatment. Nucleofection and lipofection were tested to introduce the CRISPR/Cas9 machinery and, so far, lipofection appeared to be the most effective. However, some optimisation is still needed to increase transfection efficiency.
The second purpose of this project was to generate a battery of organoids containing different VUS in the genes of interest. Due to the low efficiency of the current transfection methods, lentiviral infection was used to generate organoid lines that endogenously express a base editor (a modified Cas9 protein that converts one base or base pair into another), thus only requiring the introduction of small sgRNA molecules to generate the desired point mutations and so facilitating the transfection. So far, organoid survival post-infection has been improved, but the editing efficiency is still low. Therefore, further tests are still needed to increase editing efficiency and to successfully isolate and expand Cas9-expressing organoids.
The project is ongoing, and we are currently working on producing a proof-of-concept paper that will be published shortly.
The MSCA-IF fellow has contributed to the establishment of organoids from human normal intestinal tissue (colon and ileum) in the host laboratory, where a biobank of organoids has been created. This organoid collection is currently hosted by the biobank of the host institution. On the other hand, specific protocols for gene editing of the organoids using base editors are being optimized, and specific pilot tests have been performed for Wnt and TGF-beta-related genes. Once completed, the impact of the project will rely on the availability of a platform for the functional assessment of variants in genes involved in Wnt, TGF-beta or DNA repair pathways, with the implications that it will have for germline genetic testing in cancer and for precision medicine. Moreover, the ultimate goal of this project is to perform a high-throughput functional assessment of any potential variant in specific hereditary genes. The obtained results will be implemented into variant classification guidelines with the aim of providing new evidence that help effectively classify any variant identified in the clinical setting. In the long term, the technique developed may be adapted to other tissue-specific organoids and genetic diseases, widening its impact for the health systems and society.