Gene therapy without side effects
Hemophilia A gene therapy did not cause liver damage in the participants
Yulia Panchenko, PCR.news
BioMarin Pharmaceutical researchers and collaborators performed liver biopsy in five adult men with hemophilia A who received the coagulation factor VIII gene in an adeno–associated viral vector (roxaparvovec valoctocogen) 2.6-4.1 years ago. There were no signs of inflammation or abnormal histopathology. The authors found full-sized ring vector genomes in the samples. On average, a higher dose of the drug correlated with increased levels of DNA and RNA of the transgene.
Hemophilia A develops due to a violation of the activity of blood clotting factor VIII (FVIII). A gene therapy for this disease is under development with the drug valoctocogen roxaparvovek (AAV5-hFVIII-SQ), which is a human F8 gene without a B-domain (hFVIII-SQ) with a hybrid promoter for expression in the liver, packed in an adeno-associated viral vector. Thirteen adult men with hemophilia A received AAV5-hFVIII-SQ. As a result, the expression level of FVIII reached clinically relevant values, and bleeding decreased. The effect lasted for five and four years, respectively. However, the biological mechanism of transgene expression is not fully known. In particular, it is unclear why the transgene is expressed at different levels in different people. BioMarin Pharmaceutical researchers and collaborators decided to clarify these issues.
The authors performed liver biopsies of five participants in clinical trials. 2.6–4.1 years have passed since participating in the CI. First, the histopathology of the liver was investigated. No inflammation, fibrosis, apoptosis, necrosis or signs of chronic liver disease were detected. Four out of five participants were found to have moderate steatosis.
The distribution of hFVIII-SQ DNA in liver tissues was revealed by in situ hybridization. The higher the initial dosage, the greater the proportion of hepatocytes had copies of the vector genome. Interestingly, two people who received the same dose of the drug had a similar number of hepatocytes positively stained, while the activity levels of circulating FVIII differed by more than ten times. The authors also identified full-sized circular hFVIII-SQ vector genomes in the cells in the form of monomers and concatemers.
hFVIII-SQ RNA was shown in all participants. On average, a higher dose of the drug correlated with an increased level of expression, except for the above—mentioned case - the RNA level in one of the participants was ten times lower than in another participant who received the same dose of the drug. At the same time, the efficiency of transduction, apparently, was the same. To explain this phenomenon, the authors conducted RNA sequencing, but they did not receive an unambiguous answer. They suggested that the reason is the acetylation of transcription factors that bind to the AAV5-hFVIII-SQ promoter.
It was not possible to measure the level of FVIII protein in the sample directly. Probably the reason is that before the biopsy, the participants received recombinant hFVIII to prevent bleeding. However, there were no signs of endoplasmic reticulum stress associated with transgene expression.
The authors suggest that the long-term expression of FVIII in the liver is ensured by the formation of annular episomes after transduction. However, they do not exclude the possibility of integrating a transgene into the genome. Unexpectedly, the activity of FVIII in plasma was inversely correlated with the level of its RNA in three participants. The reason may be inefficient translation, folding, posttranslational modification and (or) protein secretion. The authors talk about the need for more extensive tests.
Article by Fong et al. Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A is published in the journal Nature Medicine.
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