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Europe’s first gene therapy

Scientists from the National Center for Tumour Diseases (NCT) have shown that adeno-associated viruses (AAV) do not pose a cancer risk. This conclusion is based on the analysis of several million patient cells treated with AAV and the finding that the viruses do not integrate into the patient genome. AAV is the first-ever gene therapy for clinical use in the Western world. AAV vectors could also potentially be used as prototypes for the treatment of other genetic defects.

The large number of leukaemia cases resulting from the application of gene therapy in the treatment of Wiskott-Aldrich syndrome (see article entitled "Successes and setbacks of clinical gene therapy") shows that the gene therapy of genetic diseases is associated with the risk that the viral vectors used for transferring a healthy gene into a diseased cell may trigger uncontrolled cell division and potentially also cancer. Each gene therapy trial must therefore also carefully investigate if the viral vectors used could lead to serious adverse effects of this kind or if there is no danger of this happening.

Dr. Manfred Schmidt, head of the Molecular and Gene Therapy research group in the Department of Translational Oncology at the National Center for Tumour Diseases in Heidelberg. © NCT

Dr. Manfred Schmidt and his group of researchers at the National Center for Tumour Diseases (NCT) in Heidelberg are renowned international experts in the field of gene therapy safety. Schmidt’s Molecular and Gene Therapy research group at the NCT (in the Department of Translational Oncology headed up by Prof. Dr. Christof von Kalle) is involved in most international gene therapy trials focused on the treatment of genetic immune diseases. As part of the trials, the group monitors the composition and growth of blood forming cells with regard to patients’ risk of developing some form of blood cancer.

Using a complex form of PCR (linear amplification-mediated PCR) along with next-generation sequencing technologies and bioinformatic data management, the researchers developed analytical methods to establish whether the virus integrates into the genome of the host cells. This was possible even on the single cell level. This technology platform has been used for assessing the safety of gene therapy trials in over 40 national and international cooperative projects. 

The first approval in the Western world

The scientists from Heidelberg have shown that the viruses used for the first ever gene therapy for clinical use in the Western world do not pose a cancer risk. The modified viruses (AAV-LPLS447X) were developed by the Dutch biotech company uniQure N.V. for the treatment of a very rare inherited metabolic disease called lipoprotein lipase deficiency (LPLD). The modified viruses are adeno-associated viruses (AAV) that carry an intact lipoprotein lipase gene into diseased cells. Prior to the European Medicines Agency granting authorisation for marketing Glybera® (alipogene tiparvovec) in the European Union in late 2012, only one gene therapy for the treatment of head and throat tumours had been given marketing authorisation, and that was in China. This was therefore the first European cancer gene approval to be granted in Europe.

Adeno-associated viruses (AAV) – electron microscope image. © DKFZ

AAV-LPLS447X viruses cannot usually integrate their DNA into the host cell genome because they lack a particular enzyme needed to do so. The vectors remain in the cytoplasm as small, separate ring structures (episomes, plasmids). However, such integration might occasionally occur. “We had to exclude that AAV-LPLS447X integrates at sites in the genome where the integration might lead to the activation of cancer-promoting genes. This is exactly what had been observed with a retrovirus used for the gene therapy of Wiskott-Aldrich syndrome.”

The researchers analysed the genomes of several million cells of five LPLD patients treated with AAV-LPLS447X. Treatment involved the injection of the virus into the leg muscles. The researchers found that AAV-LPLS447X rarely integrated into the genome of the muscle cells and, if it did, it was at random sites and showed no tendency for integration at particular sites in the genome. “The few cases where we have observed the integration of viral DNA in muscle cells are barely relevant given all the reconstructions and rearrangements that constantly take place in our DNA,” says Schmidt, the principal investigator of the study. The researchers’ thorough analyses, including the use of AAV-treated mice (the therapeutic virus was injected into the mice’s muscles or blood circulation), have shown that AAV-LPLS447X is safe and suitable as gene therapy for human application.

Integration in mitochondrial DNA

Genetic map of the ring-shaped human mitochondrial genome. © INSERM U889
The researchers from Heidelberg were surprised at the occasional discovery of the AAV-LPLS447X genome in the mitochondrial genome. The mitochondria of human and animal cells are the only cellular structures besides the nucleus that contain DNA. In contrast to nuclear DNA, mitochondrial DNA consists of 16,600 base pairs. Despite its small size, a growing number of diseases have been found to be linked to alterations in mitochondrial genes. “An adeno-associated virus has never before been seen to integrate into the mitochondrial genome on its own,” reported molecular biologist Christine Kaeppel, lead author of the paper published in the renowned journal Nature Medicine. The newly discovered property of the AAV vector may also prove useful for correcting genetic defects in human mitochondrial DNA, and AAV-LPLS447X (Glybera®) might also be a safe prototype vector for the gene therapy of other inherited diseases.

A breakthrough for gene therapy?

When Glybera® was granted authorisation for the European market, the newspaper Frankfurter Allgemeine Zeitung (FAZ) published an article (9th January 2013) stating its belief that this was the next phase in the development of the biotech industry and was a major scientific step towards society’s ideal in terms of medical treatment because the drug not only treated disease symptoms, but also the cause of the disease. But does it actually represent a major breakthrough in gene therapy?

Although the work done by Manfred Schmidt and his team has proven that the gene shuttle used is safe according to best available criteria, it remains to be seen whether the treatment is able to permanently cure LPLD patients. The European Medicines Agency had initially rejected the application on the grounds that the effectiveness of the new gene therapy had only been established in 27 people.

Jörn Aldag, CEO of uniQure, explained in an interview with FAZ: “In order to meet the formal requirements, we needed to have enrolled 350 people in our trial. However, as there are probably only around 500 LPLD patients in Europe, we would have had to treat more than half of all potential patients free of charge.” A similar argument also applies to other rare diseases, and the EMA eventually accepted uniQure’s application on the condition that treatment is restricted to adult patients with specific severe familial forms of LPLD. The company was also required to provide additional data to monitor outcomes in patients treated with Glybera®.

Price is another issue. Glybera® is administered to a patient just once or, at most, on several consecutive occasions. In order to recoup the huge development costs, the price of Glybera® will, according to Aldag, “be of a different magnitude than the kind of prices we are used to.” At 1.2 million euros per patient, Glybera® is the most expensive medicine currently available. However, Aldag regards this a fair price to pay for a therapy that restores natural body function and is not just a short-term fix. In addition, it helps save on the costs health insurance companies would normally have to pay for treating the adverse effects of other LPLD treatments. Aldag believes that the drug provides greater benefits than traditional LPLD therapy and should be appropriately compensated.

Kaeppel C, Beattie SG, Fronza R, van Logtenstein R, Salmon F, Schmidt S, Wolf, S, Nowrouzi A, Glimm H, von Kalle C, Petry H, Gaudet D, Schmidt M: A largely random AAV integration profile after LPLD gene therapy. Nature Medicine 2013, DOI: 10.1038/nm.3230

Schmidt M, Schwarzwaelder K, Bartholomae C, Zaoui K, Ball C, Pilz I, Braun S, Glimm H, von Kalle C: High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR). Nature Methods 2007 Dec; 4(12):1051-1057.

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/europe-s-first-gene-therapy