Researchers reverse the effects of autism-linked mutations with gene therapy

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In recent years, the use of organoids has led to many advances in medical research. Indeed, these miniature organs have repeatedly allowed scientists to better understand how diseases develop and test different therapeutic approaches. A team at UC San Diego School of Medicine recently made a major new discovery using these organ models: They identified a genetic mutation associated with a profound form of autism, and then successfully repaired the entire nervous system by correcting that mutation.

Several neurological and neuropsychiatric diseases, including autism spectrum disorders (ASD) and schizophrenia, are associated with mutations in transcription factor 4 (TCF4), a gene on chromosome 18 essential for brain development and neuronal function; Transcription factors are proteins that initiate or regulate the transcription of other genes. However, it is poorly understood how pathological TCF4 mutations affect neuronal tissues.

To learn more, the researchers studied neurons and brain organelles derived from skin fibroblasts taken from children with Pitt-Hopkins syndrome, a disease caused by certain specific mutations in TCF4. Pitt-Hopkins Syndrome is an ASD characterized by cognitive impairment, characteristic facial morphology, gastrointestinal problems, and respiratory rhythm disturbances. Through observation of the organoids, the team was able to identify underlying pathological molecular mechanisms and characterize the cellular abnormalities resulting from TCF4 mutations.

A mutation that limits cell proliferation and differentiation

Mouse models of Pitt-Hopkins syndrome cannot accurately mimic the neural characteristics of patients with this disorder; By converting skin cells taken from young patients into stem cells, the researchers were able to fabricate neural progenitor cells, neurons and “mini-brains” that more closely mimic the expected functions of a real organ. Thus, they could follow the development of tissues, as if they were studying the growth of a fetus.

By comparing the growth of tissues containing mutated versions of TCF4 with tissues with typical TCF4 genes, they were able to accurately map the changes caused by mutations in tissue structure and function. “Even without a microscope, we could tell which brain organoid is carrying the mutation,” said Alisson R. Muotri, director of the UC San Diego stem cell program and lead author of the study.

Significant differences in size and structure were observed between brain organoids obtained from patients with Pitt-Hopkins syndrome (right) and a normal organoid (left). © F. Papes et al.

To begin with, the team found that the syndrome-bearing organelles were aberrant in size and structure; they were significantly smaller than normal organelles and contained a higher percentage of neural progenitor cells and significantly fewer neurons. Indeed, progenitor cells cultured from stem cells showed reduced proliferation and a lower ability to differentiate into neurons, suggesting that the TCF4 mutation inhibits the proliferation and differentiation of neuronal progenitor cells. The latter also showed early aging.

The few cells that were still differentiated into neurons were less active than usual and remained clustered together rather than organized into neural networks. According to the researchers, this atypical architecture likely underlies the cognitive and motor deficits in Pitt-Hopkins syndrome. So they wondered if these structural modifications could be reversed by acting directly on TCF4 expression.

Possible recovery of motor and cognitive functions

A series of experiments have shown that TCF4 mutation results in reduced Wnt/β-catenin signaling and expression of SOX transcription factors, two important molecular signals that regulate embryonic cell reproduction, their maturation into neurons, and their migration into neurons. suspected area of ​​the brain. Thus, the decrease in neuronal differentiation appears to be related to this decrease in signaling.

The researchers then turned to pharmacological support for Wnt signaling (through a chemical called CHIR99021), a procedure that restored some of the diversity and neural activity in diseased organoids. In addition, direct correction of TCF4 mutations (by gene editing) reversed their effects: organelles with the syndrome became more similar to normal organelles used as controls. “The fact that we can fix this gene and restore the entire nervous system, even at a functional level, is amazing,” Muotri said.

wnt signaling pathway support

h) Live cell count after treatment of neural progenitor cells with Wnt pathway agonist CHIR99021. j) Quantification of senescent cells among neural progenitor cells treated with CHIR99021. k) CHIR99021 restores expression of proliferation genes in treated cells. l) Pitt-Hopkins syndrome organelles 4 weeks in vitro after treatment with CHIR99021, showing a marked increase in the number of neural progenitor cells (green) and neurons (pink), as well as the reappearance of neuronal rosettes (arrow). © F. Papes et al.

However, the team points out that these manipulations took place at a very early (prenatal) stage of brain development; however, children with Pitt-Hopkins syndrome, as with other ASDs, are not diagnosed until about 2–3 years of age. Therefore, clinical trials should be conducted to ensure that the same procedure can be performed safely and with the same efficacy at this age. Muotri and colleagues are optimizing their gene therapy tools for such trials. “For these children and their loved ones, any improvement in motor-cognitive function and quality of life is worth trying,” the specialist concludes.

F. Papes et al., Nature Communications.

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