Gene therapy currently offers the only chance of curing genetic diseases such as cystic fibrosis and beta thalassaemia. Gene therapy is the replacement or correction of a mutated gene with DNA that encodes a functional gene. Intensive research has been going on in this field for many years, however only a handful gene therapies are available at this point in time. Dr. Michael Kormann, junior professor at the Children’s Hospital at the University of Tübingen, and his team of researchers deal with genes that have the potential to cure disease; their work focuses on gene therapy techniques and on the correction of genes that lead to severe, hereditary lung diseases. They were able to prolong the life of mice suffering from surfactant protein B deficiency using modified ribonucleic acids.
Gene therapy and gene correction are ultramodern, new treatment options for patients suffering from severe, hereditary diseases. Gene therapy refers to the replacement of a mutated gene with a healthy functional gene or the improvement of the protein production of genes that are not working properly. Dr. Michael Kormann, biologist and junior professor for translational genomics and gene therapy at the Children’s Hospital at the University of Tübingen, and his research group are specifically focused on genetic techniques of this kind. The researchers are developing new methods for the therapy and correction of genes involved in the pathogenesis of cystic fibrosis, certain forms of asthma and blood diseases such as beta thalassaemia.
One type of model the geneticists from Tübingen use is mouse models with surfactant protein B deficiency, so-called SP-B mice. This is a rare, autosomal recessive lung disorder with a bad prognosis. Infants born with the disease do not survive beyond a few months. The SP-B mice underwent gene supplementation therapy during which they received the missing gene product.
Treatment involved local application of an aerosol of modified SP-B mRNA into the lungs of the mice, and as a result, their protein synthesis apparatus immediately started producing correct SP-B protein. The researchers were thus able to correct the SP-B protein deficiency. “We modify the mRNA because the natural SP-B ribonucleic acid is very instable in vivo, i.e. in the body, and also causes a very strong immune response,” explains Michael Kormann.
“We modified the mRNA in such a way that it did not generate immune reactions and associated side effects.” The specific nucleic acids used for gene therapy were developed by Kormann in cooperation with PD Dr. Carsten Rudolph during his post-doctoral period at the University of Munich. The biologist has since transferred the patent to the Munich-based biotech company ethris GmbH. However, mRNA that is used for research purposes is still produced in the laboratories in Tübingen, where the method is being further developed.
The SP-B mice were successfully treated with gene supplementation therapy: in 2010, Kormann and his team successfully prolonged the life of mice using this type of therapy. “We only need a small amount of nucleic acid,” says Kormann. “For mice, that’s just a few microgrammes. For treating humans, we would need a few grammes.” However, the therapy has not yet been transferred to humans.
Kormann was appointed junior professor around three years ago and has since been working on transferring the techniques to other diseases such as asthma for example, in order to promote individualised treatment of patients in this field. Asthma is the most common lung disease in humans and the scientists have already been able to treat asthma mouse models using modified mRNA. They also successfully used the modified mRNA to prevent the animals from developing asthma.
The next major goal of the biologists from Tübingen is the transfer of the supplementation therapy to humans. They are currently working on the establishment of the techniques. Individualised therapy will start with the genetic analysis of the patient in order to determine which genes are affected. Asthma is caused by environmental factors, but its pathogenesis and course is also determined by a large number of genes. The variants of these genes will be identified in order to be able to specifically treat the dysregulated genes.
In addition, Kormann’s research group is not only investigating the possibility of supporting defective genes using supplementation therapy, but also the possibility of specifically correcting such gene sequences. An ex vivo therapy of this kind is given to patients with beta thalassaemia – a group of inherited blood disorders that is caused by the reduced synthesis of the haemoglobin beta chains and predominantly occurs in Arab countries. The researchers remove bone marrow stem cells from the patients and correct them outside the patients’ body. If this step is successful, the cells are then injected into immunocompromised mice in order to monitor the potential growth of healthy cells in the body.
In vivo gene correction is another gene therapy strategy in which the defective gene is corrected directly in the patient’s body. For this purpose, the biologists induce DNA double-strand breaks in the vicinity of the gene defect using specific enzymes, so-called nucleases that specifically recognise sequences and cut the DNA. The specifically prepared, correct nucleic acid can be exchanged with defective DNA by way of adeno-associated viral vectors (AAV). The gene is thus corrected. The advantage is that the gene remains under the control of its original promoter.
Viral vectors were initially used for this procedure. “However, viral components have the disadvantage that they might still be active years after therapy,” says Kormann. This is why the researchers from Tübingen are using a special type of mRNA (nec-mRNA, nuclease encoded chemically modified mRNA) which is efficiently taken up by the lung cells and associated with fewer side effects. In contrast to viral vectors, nec-mRNA is not integrated into the genome. The degradation of the mRNA is easy to control via modifications. In addition, it quickly and efficiently produces the required nucleases and easily enters both dividing and non-dividing cells,” explains Kormann.
The scientists from Tübingen corrected the genes of SP-B mice and have already achieved a successful outcome; the animals survived four to five times longer than untreated mice. In this case, treatment involved local application of an aerosol of nec-mRNA and AAV into the pulmonary trachea. The researchers are now working on the further optimisation of the technology. Amongst other things, they are exploring the use of single-strand DNA instead of AAVs and have already applied for a patent for the system. The next step will include testing the technique in human individuals. Over the next two years, the researchers plan to create humanised mouse models and design suitable nucleic acid cutting tools, i.e. nuclease systems. The technique will then become experimental treatment and, if successful, subsequently tested in clinical studies on cystic fibrosis patients.
Further information:Jun.-Prof. Dr. Michael KormannTranslational Genomics and Gene Therapy in PaediatricsLothar Meyer BuildingDepartment of Paediatrics (Dept. I) University Hospital of Tübingen Wilhelmstr. 56 (Laboratory: Wilhelmstr. 27)72074 TübingenTel.: +49 (0)7071/29-76776E-mail: michael.kormann(at)med.uni-tuebingen.de