Mammalian artificial chromosomes and clinical applications for genetic modification of stem cells: an overview.

Modifying multipotent, self-renewing human stem cells with mammalian artificial chromosomes (MACs), present a promising clinical strategy for numerous diseases, especially ex vivo cell therapies that can benefit from constitutive or overexpression of therapeutic gene(s). MACs are nonintegrating, autonomously replicating, with the capacity to carry large cDNA or genomic sequences, which in turn enable potentially prolonged, safe, and regulated therapeutic transgene expression, and render MACs as attractive genetic vectors for "gene replacement" or for controlling differentiation pathways in progenitor cells. The status quo is that the most versatile target cell would be one that was pluripotent and self-renewing to address multiple disease target cell types, thus making multilineage stem cells, such as adult derived early progenitor cells and embryonic stem cells, as attractive universal host cells. We will describe the progress of MAC technologies, the subsequent modifications of stem cells, and discuss the establishment of MAC platform stem cell lines to facilitate proof-of-principle studies and preclinical development.

Transgene expression after stable transfer of a mammalian artificial chromosome into human hematopoietic cells.

OBJECTIVE: The transfer of mammalian artificial chromosomes (MACs) to hematopoietic stem and progenitor cells (HSPCs) presents a promising new strategy for ex vivo gene therapy that alleviates numerous concerns surrounding viral transduction along with a unique platform for the systematic study of stem cell biology and fate. Here we report the transfer of a satellite DNA-based artificial chromosome (an ACE), made in mouse cells, into human cord blood hematopoietic cells. MATERIALS AND METHODS: A GFP-Zeo-ACE encoding the genes for humanized Renilla green fluorescence protein (hrGFP) and zeomycin resistance (zeo) was transferred into CD34 positively selected cord blood cells using cationic reagents. RESULTS: Post ACE transfer, CFU-GM-derived colonies were generated in methylcellulose in the presence or absence of bleomycin. Bleomycin-resistant cells expressed GFP and contained intact autonomous ACEs, as demonstrated by fluorescent in situ hybridization. Moreover, when the cells from these plates were replated in methylcellulose, we observed secondary bleomycin-resistant CFU-GM-derived colonies, demonstrating stable chromosome retention and transgene function in a CFU-GM progenitor. CONCLUSION: To our knowledge this is the first report demonstrating the transfer of a mammalian artificial chromosome and the stable expression of an encoded transgene in human hematopoietic cells.