Laboratory results will leverage partnerships with companies for clinical trials
Scientists from Brazil and the USA carried out a study on gene therapy for TCF4, a gene associated with several neurological and psychiatric conditions. In published article No. Nature Communications., researchers presented promising results for therapies for Pitt-Hopkins Syndrome, a condition caused by mutations in the gene TCF4 which shares some clinical characteristics with autism spectrum disorders (ASD). “Disorders such as schizophrenia, depression, post-traumatic stress and autism spectrum disorders are associated with changes in this genetic component”, explains Fabio Papes, professor at the Institute of Biology (IB) and one of the study coordinators.
Pitt-Hopkins Syndrome patients have cognitive impairment, motor delay and chronic constipation. Such symptoms may be accompanied by behaviors typical of autism spectrum disorders. The syndrome was described in the 1970s, but only in 2007 was a direct relationship established with mutations in the gene TCF4. "O TCF4 controls the production of Transcription Factor 4, which has a role that is still poorly understood in cells. Each transcription factor is a protein that controls the activity of several other genes, so the consequences of a mutation in a transcription factor can be very complex”, explains Papes.
It is already known that the gene TCF4 It is very active in neurons. Therefore, its relationship with neurodevelopment-related dysfunctions is not unexpected. However, how mutations in this gene affected the brain was poorly understood. Over the past five years, researchers have sought to uncover the effects of TCF4 in the development of patients' brain tissue, the first step towards gene therapy. In this process, cells from pediatric patients were used, which made it possible to verify the effects caused by the mutation in the gene TCF4 in the genetic context of the patients' own cells. The cells were donated by families of children with the syndrome, both in Brazil and the USA.
Using patients' skin cells, researchers obtained stem cells, which were then used to generate neurons and brain organoids – tissue kept in the laboratory that has similarities with the development of the human brain. “In general, genetic conditions of the nervous system are very difficult to study due to the lack of a non-invasive model. The organoids boosted research in the area”, says Alysson Muotri, professor at the School of Medicine at the University of California in San Diego, who supervised the study together with the professor at Unicamp.
Analysis of patient-derived organoids revealed that mutations in the gene TCF4 reduce the number of neurons. “We found evidence that some event interrupts the process of specialization of neural progenitor cells into neurons,” says Papes. Furthermore, the group found that the neurons studied in the laboratory have lower electrical activity, which may explain several of the patients' clinical symptoms.
Pharmacological therapy trials - Searching for pharmacological treatment methods for disorders linked to the studied gene, researchers uncovered how Pitt-Hopkins Syndrome works at the level of molecules, with the identification of targets that can be “attacked” by the application of drugs. The analysis of gene activity data in neurons and organoids allowed us to verify that mutations in TCF4 lead to changes in an important pathway for controlling cell proliferation, known as WNT. “Genes from this pathway are extensively studied due to their role in numerous types of cancer, which favored research due to the wide availability of substances capable of pharmacologically modifying the WNT pathway”, explains Antonio Camargo, one of the researchers on the Unicamp team.
The application of a specific compound to cells and organoids in the laboratory was able to activate the WNT pathway and correct the harmful effects of the genetic mutation, increasing the number of neurons in the affected tissue. “There was a remarkable recovery of the characteristics of the neural tissue derived from the patients,” explains Papes. The molecule used by the researchers cannot yet be used in patients, but pre-clinical and clinical studies will look for similar molecules that have effective action in patients. “For now, the use of this molecule helps us understand what happens inside the nerve cells of patients with the syndrome,” says Papes.
Gene therapy - Gene therapy is used to correct the activity or action of a mutated gene. Gene therapy methods include completely replacing the mutated gene in affected cells, introducing an extra, unaltered copy of the gene, or increasing the activity of one of the patient's own gene copies. “Complete replacement of the mutated gene is not always possible, due to the size of certain genes or the fact that patients present major changes in the chromosome”, explains Papes. Some children with Pitt-Hopkins Syndrome, for example, have major changes in the chromosome that contains the gene TCF4.
Researchers have tested other approaches, with methods that employ viruses to deliver the pieces of DNA needed for gene therapy to dysfunctional cells. “For the correct functioning of the gene TCF4, two copies are needed in each cell, that is, two alleles functioning normally. However, in the case of Pitt-Hopkins Syndrome, one of the alleles does not work. To compensate for the mutated gene, we added a third copy of the gene”, says Papes. “The syndrome is not caused by the defective allele, but because the cells do not have two alleles functioning sufficiently”, adds the researcher.
Another method causes the patient's own gene to have its activity increased in the affected cells. With CRISPR technology, researchers were able to target an activating protein to the region of the genome that controls gene expression. TCF4. This produced the activation of the TCF4, both in neural cells and in cerebral organoids, correcting the lower expression of the transcription factor in the tissue.
The results of both approaches excited researchers. Organoids from patients who underwent gene therapy showed a corrected number of neurons and the cells demonstrated electrical activity similar to that of unaffected tissue.
Expectations - Although the genetic approach has yielded promising results in proof-of-concept experiments, it is still necessary to test different variations of the method in extensive preclinical and clinical trials to ensure the safety of its application. “Gene therapy targeting the human brain has not yet been carried out, but the idea is to move forward to guarantee the safety of the therapy. We are pioneering gene therapy for the brain and hope to open doors for other neurological conditions,” says Muotri.
The next step of the study is the clinical stage. The researchers formalized a partnership with a company specialized in gene therapy, which licensed the technology and has already begun large-scale production of the reagents necessary for clinical testing of the methodology, with the quality control standards required for use on patients.
With funding from FAPESP, the National Institutes of Health (USA) and the Pitt-Hopkins Research Foundation, the research was developed at the Laboratory of Genomics and BioEnergy (LGE), No. Unicamp Medicinal Chemistry Center (CQMED) and the Sanford Consortium for Regenerative Medicine at the University of California, San Diego.
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