Thesis supervisor: Róbert Katona
Location of studies (in Hungarian): HAS, BRC, Institute of Genetics, Mammalian artificial chromosome and stem cell laboratory Abbreviation of location of studies: SZBK
Description of the research topic:
X chromosome-linked severe combined immunodeficiency syndrome (X-SCID) is a rare inherited disorder, which affects boys. The X-SCID syndrome is caused by faulty expression of the -chain (c, gamma-c) of the interleukin-2 receptor (IL2RG), which results in the complete absence of mature T and natural killer (NK) cells, whereas B cells are frequently present in increased numbers. Disruption of IL-2 mediated signaling, however, could not account for the X-SCID phenotype, because IL-2 deficiency was compatible with T-cell development. Further studies established that c also participated in the receptors for IL-4, IL-7, IL-9, and IL-15. The X-SCID phenotype, therefore, results from combined defects in these 5 cytokine systems. Children born with X-SCID lack a working immune system, so their bodies are unable to fight off any infections. As a result, they suffer from severe infections since several months after birth, and without treatment these babies with X-SCID rarely survive beyond their first birthday. Until recently, the only cure for X-SCID was a bone marrow transplant. About 30% of children have the option of an HLA-identical bone-marrow transplant (BMT), which provide them a >90% chance for survival. BMT with a partially matched donor the survival rates are much lower and the complication rates higher. For children where no suitable donor is available at all, gene therapy is their only real hope. Alain Fischer’s team at the Necker Hospital for Sick Children (Paris, France) pioneered the X-SCID gene therapy treatment. First, bone-marrow cells were extracted from the patient and immature haematopoietic cells were selectively isolated. Then, the correct gene (c) was introduced to these cells by repeated infection of a retrovirus containing the normal version. The final step is to transplant these cells back into the patient. Using basically the same protocol similar successes have been seen in the USA, UK, Germany and Italy. A serious setback for gene therapy occurred in 2002 when it was reported that a child being treated in France for X-SCID showed signs of having developed leukemia after undergoing treatment. To date, T-cell acute lymphoblastic leukemia (T-ALL)-like disease occurred in 4 out of a total of 20 patients with X-SCID, treated in two separate gene-therapy trials. Occurrences of serious adverse events (SAEs) in otherwise successful gene therapy trials for X-linked severe combined immunodeficiency were somewhat unexpected, as it was not predicted from prior preclinical studies in mice. The development of leukemia after this gene therapy has been attributed to the upregulated expression of the oncogene LMO2 as a result of vector integration. To investigate the origin of these adverse events, IL2RG was expressed from a lentiviral vector in a murine model of X-SCID, and the fates of mice were followed for up to 1.5 years post-transplantation. Unexpectedly, 33% of these mice (n=15) developed T-cell lymphomas that were associated with a gross thymic mass. Most recently the death of a monkey 5 years after administration of CD34+ cells transduced by a retroviral vector containing marker genes was reported. Autopsy revealed that the monkey developed a fatal myeloid sarcoma, a type of acute myeloid leukemia. Tumor cells contained 2 clonal vector insertions. One of the insertions was found in BCL2-A1, in an antiapoptotic gene. This event suggests that currently available retroviral vectors may have long-term side effects, particularly in hematopoietic stem and progenitor cells. In the human gene-therapy trials, leukemias did not appear until 2–3 years after treatment, therefore SAE can be regarded as a long-term risk. At present, taking all the options into account, regulatory authorities allow to continue retrovirus vector-mediated c clinical trials if there were no acceptable alternative therapies for trial participants who would have died without this intervention. X-SCID gene therapy should be limited to patients who have failed identical or haploidentical stem cell transplantation or for whom no suitable stem cell donor can be identified. The procedure is still being reviewed and used on a case-by-case basis including appropriate risk-benefit analysis accompanied by implementation of informed consent and monitoring plans. At the same time, regulatory authorities urged for the development of safer vectors to reduce the risk of insertional mutagenesis and vectors capable of regulated therapeutic gene expression. Mammalian artificial chromosomes (MACs) are safe, stable, non-integrating vectors with large transgene carrying capacity. Previously, a method was discovered in our laboratory to produce stable, properly segregating and non-integrating artificial chromosomes in various mammalian cell lines. These chromosomes were called: SATACs (Satellite-DNA-based Artificial Chromosomes). To establish the basic principles of ex vivo stem cell and artificial chromosome X-SCID gene therapy, we plan to establish de novo SATACs as therapeutic artificial chromosomes (tSATAC) harboring and expressing the c gene for the treatment of NSG mice (c-tSATAC) as a preclinical model of the human disorder. Previously, we have demonstrated that the SATAC system and stem cell technologies can be combined and this combination offers a new strategy for stem cell-based therapy. The efficacy of this therapy was confirmed and validated by using a mouse model of a devastating monogenic disease (galactocerebrosidase deficiency, Krabbe’s disease). Therapeutic SATACs were generated by sequence specific loading of galactocerebrosidase transgenes into a SATAC. The lifespan of treated mice increased up to five-fold, verifying the feasibility of the development of MAC-stem cell systems for delivering therapeutic genes in stem cells in order to treat genetic diseases, cancers and to produce cell types for cell replacement therapies (Katona et al 2008). Based on our results, we propose that by using tSATAC as a gene therapy vector, we would able to offer a safer alternative to the presently used virus vectors for the treatment of X-SCID disorder in humans. In X-SCID therapy, substitution of retroviral/lentiviral vectors with therapeutic artificial chromosomes would eliminate the risk of SAE caused by insertional mutagenesis. This therapeutic approach was not tried before, which makes it original. This experiment can serve as a model for combined stem cell and artificial chromosome protocols tailored for various target diseases.
Required language skills: English, intermediate level Recommended language skills (in Hungarian): English, intermediate level Further requirements: It is a plus if applicant is already familiar with the following methods: molecular cloning, transformation of competent bacterial cells with DNA constructs, plasmid purification, restriction enzyme digestion, mammalian cell culture, transfection of
Number of students who can be accepted: 1
Deadline for application: 2018-02-28
2024. IV. 17. ODT ülés Az ODT következő ülésére 2024. június 14-én, pénteken 10.00 órakor kerül sor a Semmelweis Egyetem Szenátusi termében (Bp. Üllői út 26. I. emelet).
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