Liver-targeting Gene Therapy Reduces Extra Phenylalanine in PKU Mice

Liver-targeting Gene Therapy Reduces Extra Phenylalanine in PKU Mice
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Delivering the human PAL gene — phenylalanine amino lyase — into the liver cells of phenylketonuria (PKU) mouse models via gene therapy can safely reduce excess phenylalanine in the long-term, a recent study found.

The researchers noted that, while prior approaches either reversed excess phenylalanine only in male animals or required lifelong injections, this gene therapy approach was effective in both male and female mice and its effects were durable, lasting at least six months.

The findings point to the viral delivery of the PAL gene as a promising strategy to treat PKU.

It may potentially eliminate the need for the current severe dietary restrictions for people with the rare genetic disorder.

The study, “Long-Term Metabolic Correction of Phenylketonuria by AAV-Delivered Phenylalanine Amino Lyase,” was published in Molecular Therapy: Methods & Clinical Development.

PKU is caused by mutations in the PAH gene that prevent individuals from breaking down the amino acid phenylalanine, one of the building blocks of proteins. The lack of a functional PAH enzyme causes phenylalanine to build up in the body, with toxic effects.

Restricting the amount of phenylalanine in a patient’s diet is an essential component of PKU management. While effective, it requires major lifestyle changes and can result in an individual not getting certain other needed nutrients.

Potential gene therapies targeting PAH have shown some promise but appear to be more effective in male mice, with little success in reducing excess phenylalanine in females.

Enzyme substitution therapy using the PAL enzyme has emerged as an alternative strategy. But it requires frequent and lifelong subcutaneous (under-the-skin) injections, which complicate adherence to a treatment regimen.

Now, scientists from Sichuan University, in Chengdu, China, tested a more stable PAL replacement therapy. The treatment used a virus to introduce a more active version of the PAL gene into the livers of PKU mice.

As a first step, the researchers verified that the new PAL made inside cells — in this case, cells grown in petri dishes — could effectively deplete phenylalanine.

Next, they showed that a single injection of a viral vector (called AAV8) carrying the PAL gene under the control of a genetic sequence that only allows it to be made in liver cells resulted in a stable reduction of phenylalanine levels in both male and female mice. Of note, the liver cells are where it is normally produced.

In this experiment, three groups of mice were selected to receive high, medium, or low doses of the AAV8-PAL particle. Blood phenylalanine levels of the high-dose mice dropped significantly over the course of two weeks. In two out of three mice, phenylalanine levels remained stably low for the full 24-week (about six-month) course of the study.

The loss of hair color, known as hyperpigmentation, caused by an inability to synthesize melanin is the most visible effect of excess phenylalanine in mice. Notably, high-dose treatment nearly completely reversed this effect.

To evaluate the safety of AAV-PAL, the researchers looked for evidence of liver toxicity in the mice.

The blood levels of liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. These enzymes are maintained at low levels in healthy individuals, but their production ramps up in the liver in response to injury.

Although AAV-PAL did increase the serum levels of ALT and AST at two weeks post-injection, these levels returned to normal and remained stable by 24 weeks (about six months).

The researchers also examined the levels of trans-cinnamic acid (TCA), a byproduct of PAL’s interaction with phenylalanine. Mice continued to metabolize this molecule, indicating that it did not accumulate in the liver and other organs or cause any damage.

Supporting this finding, the team observed no physical signs of damage to the liver, heart, kidneys, spleen, and lung of any of the mice.

As a final safety test, the researchers looked for evidence of antibodies against the newly-introduced PAL. No such evidence was found, suggesting that this treatment would not trigger an immune response.

“Taken together,” the researchers wrote, “our results demonstrated that PAL-based gene therapy is a promising option in the treatment of PKU syndrome.”

Forest Ray received his PhD in systems biology from Columbia University, where he developed tools to match drug side effects to other diseases. He has since worked as a journalist and science writer, covering topics from rare diseases to the intersection between environmental science and social justice. He currently lives in Long Beach, California.
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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. Inês currently works as a Managing Science Editor, striving to deliver the latest scientific advances to patient communities in a clear and accurate manner.
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Forest Ray received his PhD in systems biology from Columbia University, where he developed tools to match drug side effects to other diseases. He has since worked as a journalist and science writer, covering topics from rare diseases to the intersection between environmental science and social justice. He currently lives in Long Beach, California.
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