Organica

Cell. Mol. Life Sci. DOI 10.1007/s00018-012-1113-3

Cellular and Molecular Life Sciences

REVIEW

A new generation of human artificial chromosomes for functional genomics and gene therapy
Natalay Kouprina • William C. Earnshaw Hiroshi Masumoto • Vladimir Larionov
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Received: 14 June 2012 / Revised: 25 July 2012 / Accepted: 30 July 2012 Ó Springer Basel AG (Outside USA) 2012

AbstractSince their description in the late 1990s, human artificial chromosomes (HACs) carrying a functional kinetochore were considered as a promising system for gene delivery and expression with a potential to overcome many problems caused by the use of viral-based gene transfer systems. Indeed, HACs avoid the limited cloning capacity, lack of copy number control and insertional mutagenesis due tointegration into host chromosomes that plague viral vectors. Nevertheless, until recently, HACs have not been widely recognized because of uncertainties of their structure and the absence of a unique gene acceptor site. The situation changed a few years ago after engineering of HACs with a single loxP gene adopter site and a defined structure. In this review, we summarize recent progress made in HACtechnology and concentrate on details of two of the most advanced HACs, 21HAC generated by truncation of human chromosome 21 and alphoidtetO-HAC generated de novo using a synthetic tetOalphoid DNA array. Multiple potential applications of the HAC vectors are discussed, specifically the unique features of two of the most advanced HAC cloning systems. Keywords Human artificial chromosomes Á HAC Á Geneexpression Á Gene delivery vector

Introduction Genetic manipulation with human cells aiming to complement a gene deficiency or to re-program the cells requires gene expression in a physiologically regulated fashion represents one of the most challenging tasks in modern medicine. It is generally believed that such expression can be achieved only with full-length genes containing all their regulatorysequences, and under conditions where only one copy of the gene is introduced into each gene-deficient cell. The majority of currently used gene delivery systems do not match these requirements. For example, the most advanced adenovirus-, lentivirus-, and retrovirus-derived vectors employ cDNA or ‘minigene’ constructs [1–9] that cannot recapitulate the physiological regulation of endogenous loci.Viral episomal vectors carrying herpes simplex virus type 1 (HSV-1) and Epstein-Barr virus (EBV) amplicons can deliver and express full-length genes up to *150 kb in size [10, 11]. However, HSV-1 and EBV viral vectors lack strong copy number control. In addition, the use of viral vectors may induce undesired immunological responses and occasional integration of the vector sequences into the hostgenome, causing insertional mutagenesis and gene silencing [12–16]. Human artificial chromosomes (HACs) represent an alternative system for gene delivery and expression with a potential to overcome many of the problems caused by the use of viral-based gene transfer systems [17–23]. All HACs by definition contain a functional centromere that provides them several advantages over currently usedepisomal viral vectors for gene function studies and gene therapy applications. Firstly, the presence of a functional centromere enables the long-term stable maintenance of HACs as single copy episomes without integration into the host chromosomes, thereby minimizing such complications as silencing of the therapeutic gene. Secondly, there is no

N. Kouprina (&) Á V. Larionov Laboratory of MolecularPharmacology, NCI, NIH, Bethesda, MD, USA e-mail: kouprinn@mail.nih.gov W. C. Earnshaw Wellcome Trust Centre for Cell Biology, University of Edinburgh, Scotland, UK H. Masumoto Kazusa DNA Research Institute, Kisarazu, Chiba, Japan

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upper size limit to DNA cloned in a HAC: entire genomic loci with all regulatory elements can be used that faithfully mimic the normal...