Carl F. Perez

Generation of transgenic mice and germline transmission of a mammalian artificial chromosome introduced into embryos by pronuclear microinjection.

We have generated transgenic mice by pronuclear microinjection of a murine satellite DNA-based artificial chromosome (SATAC). As 50% of the founder progeny were SATAC-positive, this demonstrates that SATAC transmission through the germline had occurred. FISH analyses of metaphase chromosomes from mitogen-activated peripheral blood lymphocytes from both the founder and progeny revealed that the SATAC was maintained as a discrete chromosome and that it had not integrated into an endogenous chromosome. To our knowledge, this is the first report of the germline transmission of a genetically engineered mammalian artificial chromosome within transgenic animals generated through pronuclear microinjection. We have also shown that murine SATACs can be similarly introduced into bovine embryos. The use of embryo microinjection to generate transgenic mammals carrying genetically engineered chromosomes provides a novel method by which the unique advantages of chromosome-based gene delivery systems can be exploited.

Stability of a functional murine satellite DNA-based artificial chromosome across mammalian species.

A 60-Mb murine chromosome consisting of murine pericentric satellite DNA and two bands of integrated marker and reporter genes has been generated de novo in a rodent/human hybrid cell line (mM2C1). This prototype mammalian artificial chromosome platform carries a normal centromere, and the expression of its β-galactosidase reporter gene has remained stable under selection for over 25 months. The novel chromosome was transferred by a modified microcell fusion method to mouse [L-M(TK−)], bovine (P46) and human (EJ30) cell lines. In all cases, the chromosome remained structurally and functionally intact under selection for periods exceeding 3 months from the time of transfer into the new host. In addition, the chromosome was retained in three first- generation tumours when L-M(TK−) cells containing the chromosome were xenografted in severe combined immunodeficiency mice. These data support that a murine satellite DNA-based artificial chromosome can be used as a functional mammalian artificial chromosome and can be maintained in vivo and in cells of heterologous species in vitro.

Development of mammalian artificial chromosomes for the treatment of genetic diseases: Sandhoff and Krabbe diseases

Mammalian artificial chromosomes (MACs) are being developed as alternatives to viral vectors for gene therapy applications, as they allow for the introduction of large payloads of genetic information in a non-integrating, autonomously replicating format. One class of MACs, the satellite DNA-based artificial chromosome expression vehicle (ACE), is uniquely suited for gene therapy applications, in that it can be generated denovo in cells, along with being easily purified and readily transferred into a variety of recipient cell lines and primary cells. To facilitate the rapid engineering of ACEs, the ACE System was developed, permitting the efficient and reproducible loading of pre-existing ACEs with DNA sequences and/or target gene(s). As a result, the ACE System and ACEs are unique and versatile platforms for exvivo gene therapy strategies that circumvent and alleviate existing safety and delivery limitations surrounding conventional gene therapy vectors. This review will focus on the status of MAC technologies and, in particular, the application of the ACE System towards an exvivo gene therapy treatment of lysosomal storage diseases, specifically Sandhoff (MIM #268800) and Krabbe (MIM #245200) diseases.

476. Human Mesenchymal Stem Cells Genetically Modified To Express High Levels of Erythropoietin Through Transfer of an Artificial Chromosome Engineered Using the ACE System

The ACE System is a versatile, reliable system for genetically modifying cell therapies, generating transgenic animals, and engineering mammalian cells for high expression of a recombinant protein. Key components of the ACE System include an artificial chromosome (Platform ACE) encoding multiple (>50) DNA site-specific integration sites (acceptor sites); a targeting vector (ACE Targeting Vector) encoding both a Platform ACE-specific DNA donor site and the therapeutic gene of interest; and a proprietary DNA recombinase (ACE Integrase) that catalyzes the site-specific recombination of ACE Targeting Vectors onto the Platform ACE. ACEs are a promising means of genetically modifying and engineering cells for cell therapy, as they are stably maintained, autonomous, non-integrating, easily purified by flow cytometry (de Jong et al, 1999) and readily transfected into a variety of cell types, including human adult stem cells (deJong et al, 2001; Vanderbyl et al, 2001; Vanderbyl et al, Stem Cells in Press).