Roctavian’s Effectiveness Tied to Molecules that Aid FVIII Production

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by Steve Bryson, PhD |

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A double helix DNA strand reclines on a couch in front of a therapist taking notes.

Factor VIII (FVIII) levels in the bloodstream of men with severe hemophilia A treated with the investigational gene therapy Roctavian appear to be influenced by regulatory molecules involved in F8 gene activity and FVIII protein folding and release, a study reveals.

Researchers also found a dose-dependent relationship between the percentage and activity of therapeutic DNA in their liver cells.

The study, “Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A,” was published in Nature Medicine.

Roctavian (valoctocogene roxaparvovec) by BioMarin is a one-time gene therapy that uses a harmless AAV5 virus to deliver a healthy copy of the F8 gene — defective in people with hemophilia A — to liver cells. Here, the blood-clotting protein FVIII is made and secreted into the bloodstream.

Clinical trials supporting the therapy’s development for severe hemophilia A show it generated clinically relevant FVIII levels, leading to fewer bleeding episodes and lower FVIII replacement therapy usage with effects lasting up to four to five years.

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Because multiple, complex biological processes are involved in achieving sustained FVIII levels by gene therapy, its effectiveness can vary from person to person. As such, it is important to understand the underlying cellular processes that lead to successful, long-term FVIII production.

Scientists at BioMarin recruited five men participating in a Phase 1/2 study (NCT02576795) evaluating increasing doses of Roctavian. The clinically stable patients donated liver samples by biopsy to determine the distribution and forms of therapeutic DNA and ongoing protein production that persisted for more than four years after treatment.

Tissue examination revealed a dose-dependent trend in the percentage of liver cells containing Roctavian DNA: 1.3% with the lowest dose, 32% with the medium dose, and up to 52% with the highest dose.

Although two medium-dose participants had a similar percentage of therapeutic DNA in their liver cells, FVIII levels in their bloodstream differed by more than ten times.

In the cell nucleus, there was also a dose-dependent trend in the levels of therapeutic DNA strands that formed closed circles as designed. Some of these circular DNA molecules carried one copy of the F8 gene, while others were fused together, carrying multiple F8 copies.

The research team also measured the Roctavian F8 gene’s activity by measuring the levels of messenger RNA (mRNA) — the molecule that carries the genetic message necessary to make the FVIII protein. Higher levels of therapeutically induced mRNA were seen at higher doses consistently.

Sample analysis from the two participants with widely different FVIII levels showed that one had a lower percentage of liver cells with therapeutic DNA and about 10 times less F8 mRNA. This participant also had five to seven times lower levels of a protein involved in converting the therapeutic F8 gene to mRNA (transcription).

“These findings are consistent with a marked difference between the two participants in plasma FVIII activity reported at the time of biopsy,” the scientists wrote.

Surprisingly, those with the highest levels of FVIII activity in the bloodstream had lower mRNA levels in liver cells. The team then demonstrated that these higher FVIII levels correlated with higher amounts of GRP78 — a protein that directly interacts with human FVIII during its production, aiding in its proper folding and secretion.

As a comparison, the team examined 32 healthy liver samples and found a high degree of variability in GRP78 levels between people, supporting the hypothesis that GRP78 in liver cells may play a role in Roctavian effectiveness, the scientists noted.

Because participants received FVIII replacement therapy at the time of biopsy as a precaution, measuring FVIII protein in liver tissues was unsuccessful. Instead, experiments showed FVIII production by Roctavian did not result in stress of the endoplasmic reticulum — a cellular compartment where proteins are made, located right next to the nucleus.

Under the microscope, tissue samples showed no significant scarring (fibrosis) and no signs of abnormal cell growth or death, or structural defects. Four participants had mild steatosis, or excess fatty tissue buildup.

“In this study, long-term expression of [FVIII] following a single [Roctavian] infusion was associated with the presence of circularized full-length genomes,” the authors wrote. Differences in F8 expression between people “may result from differences in expression of regulatory molecules involved in transcription and protein folding/secretion.”

The researchers said more “investigation into these complex mechanisms is warranted.”