CORRECTION OF AN X-LINKED GENETIC DEFECT BY MICROCELL-MEDIATED CHROMOSOME TRANSFER

Several approaches to the correction of genetic defects have been investigated in the last two decades, including those based on retroviral and lentiviral vectors. Although viral vectors are a powerful instrument for gene therapy and are being used in human trials, they present drawbacks such as risk of insertional mutagenesis and limited insert size. Homologous recombination techniques, which has long been applied to mouse stem cell engineering, have recently been used in combination with novel tools exploited the activity of nucleases coupled to targeting methods such those based on zinc fingers, TALEN and CRISPR, which allow targeting to specific DNA sequences to a great extent. However some genetic defects, and in particular genomic abnormalities such as large deletions, are not suitable to correction with these tools. Here I suggest that large structural abnormalities of the X chromosome can be treated by chromosome transfer mediated by microcells (MMCT). The rationale underlying my work was the fact that by MMCT all the sequences and structures needed for proper expression of the affected gene(s) are transferred into the defective cell and no non-human sequence is added to the cell. Furthermore, additional X chromosomes are usually silenced and cells with more than two X chromosomes are essentially normal, as shown by XXX trisomic human organisms. Therefore, MMTV has the potential to correct several diseases whose underlying abnormality lies on the X chromosome. With this aim in mind, we devised an in vitro model to show the feasibility of the approach. Our plan was to transfer an exogenous X chromosome into the embryonic stem cell (ESC) line HM1 which is defective in the hprt gene, whose mutation in humans cause the Lesch-Nyhan syndrome, a severe neurological pathology. A mouse normal chromosome was transferred from normal MEF to the A9 cell line, a neoplastic cell which has a great capability to form micronuclei. The HM1 was the recipient cell line, while the A9-X was the donor one. The A9-X cells were micronucleated and microcells of small size were filtered and fused to HM1 cells in the presence of PEG. Cells were cultured in HAT medium, which selects for the expression of a correct HPRT protein by the fused cells. A few colonies were obtained and their karyotype was determined to be XXY. These cells showed the presence of a correct HPRT transcript and grow in the selective HAT medium. These results suggest that a single chromosome can be transferred to ESC line in order to correct the genetic anomaly and opens the way to the correction of more complex structural abnormalities of the X chromosome, such as that associated to the fragile X