Background
Synovial sarcoma (SS) is a malignancy of the soft tissue especially affecting young adults, with a poor prognosis and limited therapeutic options. It originates as a result of a chromosomal translocation, t(X;18)(p11;g11), leading to the expression of the SS18-SSX fusion protein in permissive cells (including myoblasts) that display plasticity and are of mesenchymal origin.
Current treatment, which consists of surgery and of generic chemotherapy and/or radiotherapy, has limited success. Thus, more specific and effective therapeutic approaches are needed to manage the disease, which, in turn, require better understanding of its molecular pathogenesis. Considering that the early events leading to tumour formation are poorly defined, the purpose of this project is to lay the foundations for the creation of a new model based on a pluripotent cell of mesenchymal lineage in which the translocation can be induced and the early events observed.
Design
Mouse myoblast cells: C3HT101/2 cells, were chosen as a cell-based model because they were shown to harbour properties thought to be required for transformation by SS18-SSX, including permissiveness for its stable expression.
To create the translocation, first, two loxP sites were introduced at the breakpoints of chromosomes 18 and X of C3HT101/2 cells and subsequently the translocation was induced by treating the cells with the Cre recombinase. This first goal can be achieved by using homologous recombination generated by DNA damage and driven by two strategically designed templates. The latter were delivered by AAV viral vectors and designed to target the two breakpoints and provide the loxP sites. Those templates were generated by a separate master project. DNA damage is achieved by TALEN technology, which is initiated by the insertion of the TALEN molecular machinery into lentiviral vectors.
Results
In the present work, we prepared the TALEN constructs and produced the viruses. Then we transduced both TALENs, tested their expression and function, and created a tool to identify the best AAV serotype to deliver the donors to our cell model.
The next step will be to transduce the AAV viruses containing LoxP sites and select the cells for positive events of recombination. Finally, we will assess by genomic sequencing the ratio between true and false positive cells and eventually proceed to a more restrictive clonal selection.
The last phase of the project requires the induction of a chromosomal translocation at the loxP sites by Cre recombinase transduction and selection of the cells that have undergone the translocation. Western blots for SS18-SSX identification and genome sequencing will assess the ratio between real and false positive cells, and clonal selection can be used to isolate true positive cells. Finally, we will remove the genomic scar by transducing the cells with a flippase.