The development of a neocentromere-based minichromosome gene delivery system

Abstract

© 2011 Dr. Yoshimi InabaGene therapy has the potential to cure human genetic diseases permanently by introducing correct sets of therapeutic genes into patient cells. Multiple vector systems have been generated for gene therapy applications. They include human engineered chromosomes (HECs) that can overcome many of the hurdles presented by other gene delivery systems such as viral vector systems. HECs are fully functional chromosomes containing the minimal genomic content required for correct mitotic function, and have the capacity to carry large transgene or gene clusters. The neocentromere of marker chromosome mardel(10) provides a highly desirable source for HEC construction. The mardel(10) neocentromere-based minichromosomes (NC-MiCs) were generated as a gene delivery vector system, and developed for use in gene therapy applications. This study focused on (1) the purification of NC-MiC to achieve efficient NC-MiC delivery into target cells, and (2) the integration of a therapeutic gene into NC-MiC to demonstrate the potential of NC-MiCs as gene delivery vectors. A chromosome soup was prepared from a HT1080-derived cell line containing NC-MiC1 (3.5Mb) and subsequently subjected to flow-sorting. A high purity level of NC-MiC1 chromosome was obtained. The same approaches were applied for purifying NC-MiC6.1 (1.4Mb), which has a greater potential for use in gene therapy because of its smaller size and the presence of a site specific sequence for targeted gene integration. By applying multiple-gates and parameters on the sorting of NC-MiC6.1, the chromosome was enriched further by 27 times in the sorted samples, opening the way for the preparation and use of minichromosomes carrying less background genetic material. Success in the isolation of NC-MiC minichromosome using flow-sorter has not been previously described, and there has been no published report on the isolation or enrichment of minichromosomes as small as 1.4Mb. While comparing NC-MiC6.1 quantity in the sorted samples, we developed a novel DNA quantification method. It is relatively simple and quick and, importantly, more sensitive than currently available DNA quantification methods. This method, which is based on DOP-PCR, can be widely used to measure any DNA source. It can also be used as internal controls for genomic qPCR experiments, such as ChIP and Microarray experiments. Additional chromosome labelling using the interaction of lac operator (lacO) and lac repressor (LacI) was attempted to improve the isolation of specific chromosomes by flow-sorting. This lacO-LacI interaction was also utilised in coupling with magnetic bead for affinity purification of target chromosomes. Although isolation of specific chromosomes was not fully achieved in this study using these approaches, both approaches showed a potential use for chromosome purification. To demonstrate the utility of NC-MiCs as gene delivery vectors, the frataxin gene (FXN gene) was chosen as a model therapeutic gene for treatment of Friedreich ataxia by gene therapy. Targeted integration of a BAC containing FXN gene (FXN-BAC) into NC-MiC6.1 can be achieved using FRT/Flp site-specific recombination. The incorporation of the FRT sequence into FXN-BAC was successfully modified by homologous recombination. Due to time constraint, a small number of attempts to co-transfect FXN-BAC and the Flp expression vector into NC-MiC6.1 line were performed but to date no positive clone was obtained. Time constraint has also did not allow the transfer of NC-MiC into various cell lines in this study. In summary, this study has established the feasibility of using high-resolution flow-sorting methods to purify or significantly enrich the purity of minichromosomes as small as 1.4-3.5Mb. The study has also tested additional potential methods for further improving the purification of the very small chromosomes, established a new DNA quantification method, and begun testing the targeted integration of disease-relevant genes into the minichromosomes