Lymphoma and Chromosomal Instability in Artemis Deficient Mice - A Challenge for Long Term Malignancy Free Survival and Choice of Optimal Conditioning Regimen in Artemis Gene Therapy Trials.

Correction of Artemis deficiency, which causes a T^−/−B^−/− negative and radiosensitive type of SCID, is subject of murine gene therapy trials as an alternative treatment protocol for Hematopoietic Stem Cell Transplantation (HSCT). A conditioning regimen consisting of irradiation or busulfan in mice is indispensable for a succesfull engraftment. Gene therapy related toxicity on the basis of vector integration and conditioning regimen has been observed previously. Herein we provide evidence that Artemis deficiency harbours an additional risk factor for the development of lymphoproliferative disease. We provide in vivo evidence, that non gene corrected Artemis mice are able to develop lymphoma facilitated by irradiation and/or aging. Artemis^−/− mice were irradiated at 3 Gy. Lymphoma were detected in 2 out of 10 Artemis^−/− mice at the age of 4 to 6 months. Lymphoproliferative disease were exclusively of T-cell origin and arose from the thymus carrying a highly CD4+ or CD4+/CD8+ phenotype. Additionally, increased chromosomal rearrangements were detected in bone marrow cells of irradiated Artemis^−/− mice compared to non irradiated control mice. Karyograms of bone marrow cells of 3 Gy-irradiated, 4 months old mice were stained with the Spectral Karyotyping (SKY) method. Analysis of 5 out of 20 metaphases revealed chromosomal translocations whereas in two out of 5 abnormal metaphases chromosome 15 was involved in the translocations. On contrary in karyograms of 4 to 6 weeks old Artemis^−/− Sca1+ cells, which were used for viral transduction twenty out of twenty metaphases showed no obvious rearrangement. Even stimulation with cytokines in the course of viral transduction (SCF, FLT3L, TPO, IL6 and IL11) for five days did not increase chromosomal instability. Here we provide evidence, that Artemis^−/− mice have an additional increased risk of developing hematopoietic malignancies, due to chromosomal instability which is independent of gene therapy. Human cells might not harbour the same riskfactor. However chromosomal instability in human Artemis^−/− cells needs to be analysed in more detail for the choice of an optimal conditioning regimen and long term malignancy free survival in clinical trials.