Vibrating Microbubbles May Improve Gene Therapy Delivery for Hem A

Marta Figueiredo PhD avatar

by Marta Figueiredo PhD |

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An ultrasound-mediated, non-viral gene therapy safely and effectively increased the levels of factor VIII (FVIII) — the missing clotting factor in hemophilia A — and lessened bleeding in a mouse model of the disease, a study shows.

The delivery of a modified, improved version of the disease-associated F8 gene using microbubbles resulted in sustained therapeutic FVIII levels, highlighting the potential benefits of this therapeutic approach for people with hemophilia A.

This proof-of-concept study supports further evaluation of this gene therapy strategy in larger animal models before moving to clinical trials, the researchers noted.

The study, “Ultrasound-mediated gene delivery of factor VIII plasmids for hemophilia A gene therapy in mice,” was published in the journal Molecular Therapy – Nucleic Acids.

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Cell-based Therapy Shows Promise in Mouse Model of Hemophilia A

The standard treatment for hemophilia A is FVIII replacement therapy, which delivers the missing FVIII directly into the bloodstream to prevent or treat bleeds. However, this approach often requires patients to receive regular infusions for the rest of their lives.

By delivering to cells a healthy version of the F8 gene, which provides instructions to produce FVIII, gene therapy has the potential to be a long-term therapeutic approach, or even a cure, for hemophilia A.

Gene therapy usually uses a modified and harmless version of a virus vector to carry and deliver the modified gene. However, viral-based therapies can only be given once due to the body’s natural immune response against the viral carrier, which would be enhanced in subsequent exposures.

In addition, several patients already have high levels of antibodies against frequently used viral carriers, which can render the therapy useless.

As such, non-viral gene therapies have gained increasing interest, and ultrasound-mediated gene delivery (UMGD) in the presence of microbubbles “has been considered a promising non-viral gene delivery method,” the researchers wrote.

This type of therapy combines gas-filled microbubbles with DNA molecules containing the gene of interest that naturally attach to the microbubbles’ fatty surface. Upon targeted exposure to ultrasound, the microbubbles vibrate in a way that promotes the release of the DNA molecules and their entry into cells in the vicinity.

Now, a team of researchers at Seattle Children’s Research Institute, along with colleagues at Indiana University and NuvOx Pharma, developed an optimized ultrasound-based, microbubble-mediated, non-viral gene therapy strategy for hemophilia A.

The approach was tested in a mouse model of hemophilia A that is prone to develop antibodies against FVIII within 10 to 14 days after treatment, which can limit its efficacy.

The non-viral gene therapy involved a mix of microbubbles and a DNA molecule containing a modified version of the F8 gene designed to promote increased FVIII production specifically in hepatocytes — the liver’s chief functional cells and the body’s main producers of clotting factors.

This mix was delivered directly into the animals’ portal vein, the main blood vessel delivering blood to the liver, and ultrasound was applied to the liver so that DNA molecules could enter hepatocytes. This process required surgery to gain access to the portal vein and the liver.

Results showed the gene therapy led to a rapid increase in FVIII activity levels, reaching 5%–20% of normal, and then dropping to undetectable levels within two weeks, coinciding with the detection of anti-FVIII neutralizing antibodies.

As such, the team conducted a similar experiment in mice pre-treated with immunosuppressive treatment to slow or prevent the development of such antibodies.

They found that half of these mice maintained 2%–10% of normal FVIII activity without neutralizing antibody formation for at least 84 days (nearly three months), and that blood loss was significantly reduced, reflecting a 57% therapeutic correction.

These results suggested that this strategy could be “successfully applied to treat 70%–80% of the [hemophilia A] patients who are not prone to [anti-FVIII antibody] formation,” the researchers wrote.

To achieve persistent and therapeutic FVIII levels, the team then used a DNA molecule containing a F8 version known to result in even higher FVIII levels.

The gene therapy delivering the optimized F8 version resulted in significantly higher FVIII activity levels at both day three (30%–77% vs. 2%–18%) and day seven (30%–150% vs. 3%–16%), compared with the previous approach.

In mice pre-treated with immunosuppressive treatment, the optimized approach was associated with initial high FVIII levels of up to 150%. After two months, half of the animals maintained 8%–20% FVIII activity levels without generating high levels of anti-FVIII antibodies.

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Benefits of Roctavian, Hem A Gene Therapy, Holding for 2 or More Years

In addition, on day one, treated mice showed a small increase in the levels of liver enzymes, suggestive of liver damage, that returned to normal within three days.

These findings highlight that “long-term and therapeutic FVIII [levels] can be achieved following UMGD in [hemophilia A] mice,” demonstrating “the potential of this novel technology to safely and effectively treat hemophilia A,” the researchers wrote.

This surgery-based approach was restricted for use in the mouse model and “a minimally invasive procedure has been developed instead for proof of clinical application and transference in large animal models,” the team wrote.

In this procedure, the microbubbles/DNA mix is delivered to a major vein in the neck, and the liver is subsequently exposed to ultrasound that is applied in the skin at the liver region.

This approach “without open surgery further reduced the procedure burden and [temporary] tissue damage, which will also be carefully evaluated in [hemophilia A] large animal models in future studies,” the team wrote.