Gene Editing Approach for PKU Children Shows Precise Correction in Mice

Gene Editing Approach for PKU Children Shows Precise Correction in Mice
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The gene editing approach being investigated by Homology Medicines to potentially treat children with phenylketonuria (PKU) is delivered to liver cells with high efficacy and without causing unwanted changes in DNA, a study in mice shows.

The findings suggest that a single dose of HMI-103, the company’s therapy candidate for children with PKU, may safely and effectively target the root cause of the disease.

Titled “Molecular characterization of precise in vivo targeted gene integration in human cells using AAVHSC15,” the study was published in PLOS One.

PKU is caused by mutations in the PAH gene, which provides instructions for an enzyme that converts the amino acid phenylalanine into another amino acid called tyrosine. Amino acids are organic compounds that combine to form proteins.

Mutations in PAH significantly increase the amount of phenylalanine in the blood and lower the concentration of other amino acids, some of which are needed in the brain to produce neurotransmitters – the chemical messengers used by nerve cells to communicate. As a result, patients typically experience neurological impairments.

Homology is developing a gene-editing therapy that uses a harmless adeno-associated vector developed from human blood stem cells (AAVHSC) to deliver a functional PAH gene and replace the mutated copy in liver cells. Because the liver divides rapidly throughout childhood, a gene-editing approach to permanently correct the genome could offer a potential cure for children with PKU, the company said in a press release.

This is done through a natural process of DNA repair called homologous recombination. It uses a template DNA sequence to integrate the PAH gene into the place it is needed. In the case of HMI-103, the sequence is inserted in a specific non-coding region of the mutated gene.

The approach is more attractive than other gene-editing technologies because it does not require DNA-cutting enzymes — or nucleases — for gene insertion. Those nucleases are often accompanied by unwanted changes in the DNA that can cause disease.

Researchers at Homology used molecular methods to characterize the integration of the PAH gene using the nuclease-free approach. Such assessments are important for understanding whether the gene is being incorporated in the right place, through the intended homologous recombination mechanism, and without unwanted genetic alterations.

“As we continue to develop our AAVHSC-based gene editing technology into new treatment options and potential cures for patients, we believe that it is important to employ quantitative molecular methods to characterize changes to the genome,” said Albert Seymour, PhD, chief scientific officer at Homology.

Experiments were done in mice whose livers were almost entirely composed of human cells (80%). That allows for studies of human cells in a physiological environment. “While not a completely human system, this mouse model is as close as we could come to a human model,” the researchers wrote.

The animals were infused into the back of the eye’s orbit with a single dose of the gene-editing construct used in HMI-103 — a viral vector developed, called AAVHSC15, that has a high affinity toward liver cells. After six weeks, the livers were collected for analysis.

Building on recent positive results, these findings showed that the AAVHSC15 vector entered both mouse and human liver cells, but the functional PAH gene was only integrated in the genome of the human cells. This was because the human and mouse PAH genes only share 66.4% sequence identity, which is not enough for homologous recombination to happen, the investigators said.

Two independent assessments showed a 6% efficiency in inserting the corrected copy of PAH in human liver cells. Such level of efficiency is better than seen with other gene-editing constructs, Homology said.

The insertion also was happening as expected, via homologous recombination, and without unintended mutations. In addition, no inverted terminal repeats (ITRs) were observed, the researchers said. That means that no pieces of viral DNA used in the construct were inserted in the liver cells’ genome, again supporting that AAVHSC15-mediated gene editing is due to homologous recombination.

“We used multiple molecular methods to assess our gene editing technology, which demonstrated precise PAH gene integration into the target location without introducing any unintended mutations or viral components,” Seymour said.

The findings not only provide critical information about the precision and integration mechanisms of Homology’s gene-editing approach for PKU. They also inform a new strategy to monitor gene insertions using other viral-based technologies.

“We believe that the framework described herein represents a major advancement in the field of AAV-based gene editing and, going forward, should now enable the ability to make direct data comparisons across studies,” Seymour added.

Inês holds a PhD in Biomedical Sciences from the University of Lisbon, Portugal, where she specialized in blood vessel biology, blood stem cells, and cancer. Before that, she studied Cell and Molecular Biology at Universidade Nova de Lisboa and worked as a research fellow at Faculdade de Ciências e Tecnologias and Instituto Gulbenkian de Ciência.
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José holds a PhD in Neuroscience from Universidade of Porto, in Portugal. He has also studied Biochemistry at Universidade do Porto and was a postdoctoral associate at Weill Cornell Medicine, in New York, and at The University of Western Ontario in London, Ontario, Canada. His work has ranged from the association of central cardiovascular and pain control to the neurobiological basis of hypertension, and the molecular pathways driving Alzheimer’s disease.

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Inês holds a PhD in Biomedical Sciences from the University of Lisbon, Portugal, where she specialized in blood vessel biology, blood stem cells, and cancer. Before that, she studied Cell and Molecular Biology at Universidade Nova de Lisboa and worked as a research fellow at Faculdade de Ciências e Tecnologias and Instituto Gulbenkian de Ciência.
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