Blood Vessel Abnormalities May Explain Gene Therapy Limitations

Marta Figueiredo, PhD avatar

by Marta Figueiredo, PhD |

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The effectiveness of liver-directed, viral-based gene transfer — the most used experimental gene therapy approach for hemophilia A — is significantly reduced in mouse models of hemophilia A relative to healthy mice, a study shows.

This limitation was associated with a pronounced reduction in fenestrations — cell surface pores that allow molecules, but also small viruses, to pass through — in specialized blood vessel cells in the liver.

These findings suggest deficient levels of factor VIII (FVIII) — the blood clotting protein missing in hemophilia A — result in structural changes in specialized liver blood vessels that limit small viruses’ passage into the liver, and therefore their ability to deliver genetic material to liver cells efficiently.

Further studies are needed to understand how FVIII deficiency leads to such blood vessel abnormalities and whether these changes may also be limiting liver-directed gene therapy in hemophilia A patients, the researchers noted.

The study, “Defenestrated endothelium delays liver-directed gene transfer in hemophilia A mice,” was published in Blood Advances.

Hemophilia A is caused by mutations in the F8 gene, which provides instructions to produce FVIII. By delivering a healthy version of the F8 gene to liver cells, the body’s main producers of blood clotting factors, gene therapy has the potential to be a long-term therapeutic approach, or even a cure, for hemophilia A.

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Several gene therapies are currently being tested in clinical trials, including Phase 3 studies. Most use a modified and harmless adeno-associated virus (AAV) to carry and deliver the modified gene to liver cells.

While some of these approaches have shown promising results in hemophilia A patients, “limitations persist due to AAV-induced cellular stress, [immune responses], and reduced durability,” the researchers wrote.

To better understand what’s behind some of these limitations, researchers at the University of Pittsburgh assessed the effectiveness of liver-directed, AAV-mediated gene transfer in two mouse models of hemophilia A.

Both models lacked a working F8 gene, but had different genetic backgrounds. Healthy mice of each model served as controls.

Researchers used the AAV variant 8 (AAV8) to deliver the GFP gene, which provides instructions to generate a small fluorescent molecule with no known side effects in the liver. AAV8 is being used in some gene therapies currently in development for hemophilia A, including ASC Therapeutics’ ASC618.

The effectiveness of AAV8-GFP transfer in the liver was assessed through the number of GFP-positive liver cells, as well as GFP levels in liver samples.

Results showed AAV8-GFP transfer was significantly reduced in both mouse models of hemophilia A relative to the controls across all evaluated time points, up to two months.

Since the virus efficiently enters cells by passing across the permeable barrier of endothelial cells — those lining blood vessels — the team then evaluated the health and structure of liver sinusoidal endothelial cells (LSECs).

These specialized cells usually present clusters of fenestrations, or large cell surface holes, that allow bidirectional passive passage of molecules, therapeutic compounds, and small viruses between blood circulation and the liver.

Researchers found that FVIII-deficient mice had significantly fewer and smaller fenestrations in LSECs than healthy mice.

This was associated with the abnormal production of several proteins that are known to promote the transformation of LSECs into nonspecialized endothelial cells that lack fenestrations and therefore don’t allow easy bidirectional passage.

“This is the first study to link impaired liver-directed gene transfer to liver-endothelium maladaptive structural changes associated with FVIII deficiency in mice,” the research team wrote.

The researchers noted that a previous study reported abnormal endothelial function in people with moderate to severe hemophilia A and hemophilia B.

These findings may “inspire future investigations looking at the molecular mechanism underlying loss of endothelial fenestration associated with FVIII deficiency and whether or how this possibly could be affecting liver gene-therapy in hemophilia,” they wrote.