A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy

Abstract

Mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells' chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure. In order to extend the utility of ACEs, we have established the ACE System, a versatile and flexible platform for the reliable engineering of ACEs. The ACE System includes a Platform ACE, containing >50 recombination acceptor sites, that can carry single or multiple copies of genes of interest using specially designed targeting vectors (ATV) and a site-specific integrase (ACE Integrase). Using this approach, specific loading of one or two gene targets has been achieved in LMTK(-) and CHO cells. The use of the ACE System for biological engineering of eukaryotic cells, including mammalian cells, with applications in biopharmaceutical production, transgenesis and gene-based cell therapy is discussed.

De novo formed satellite DNA-based mammalian artificial chromosomes and their possible applications

“…Basically, any cultured mammalian cell line that contains rDNA region is suitable for the production of de novo formed SATACs. (Csonka et al 2000;Hadlaczky et al 1991;Hadlaczky 2001;Holló et al 1996;Keresö et al 1996;Lindenbaum et al 2004;Praznovszky et al 1991). There is no evidence in humans that amplification of these pericentromeric sequences is deleterious to an individual, as polymorphisms in the short arms of acrocentric chromosomes have been shown to consist of amplified pericentromeric heterochromatin and/or rDNA (Conte et al 1997;Friedrich et al 1996;Sands 1969;Sofuni et al 1980;Stergianou et al 1993;Trask et al 1989A;Trask et al 1989B;Verma et al 1977;Warburton et al, 1976) and have been inherited with no adverse effects (Bernstein et al 1981;Cooper and Hirschhorn 1962;Schmid et al 1994;Therkelsen 1964;Yoder et al 1974).…”

“…Furthermore, supernumerary chromosomes derived from amplified pericentromeric heterochromatin and/or rDNA sequences of acrocentric chromosomes have been shown to be stably inherited through 1-3 generations in humans without any deleterious effects of phenotypes (Adhvaryu et al 1998;Blennow et al 1994;Brøndum-Nielsen and Mikkelsen 1995;Crolla et al 1997;Hadlaczky et al 2002;Howard-Peebles 1979;Mukerjee and Burdette 1966). Additionally, SATACs have been engineered with multiple (>50) site-specific recombination acceptor sites (attP) which enable the unidirectional loading of heterologous DNA (encoding an attB recombination donor site) via a mutant lambda integrase (Carl 2004;Lindenbaum et al 2004) . The …”

“…Ren et al 2006;Kouprina et al 2013;Kouprina et al 2014). Four different strategies have been developed for the construction of artificial chromosomes (Irvine et al 2005): (i) in the synthetic approach the artificial chromosome is assembled from chromosomal components (Basu et al 2005a;Basu et al 2005b; Harrington et al 1997;Henning et al 1999;Ikeno et al 1998;Ikeno et al 2009;Kaname et al 2005;Kouprina et al 2003;Nakashima et al 2005;Suzuki et al 2006), (ii) the 'top down' method applies the in vivo telomere-associated fragmentation of existing chromosomes (Au et al 1999;Auriche et al 2001;Carine et al 1986;Choo et al 2001;Farr et al 1995;Heller et al 1996;Ishida et al 2000;Katoh et al 2004;Mills et al 1999;Shen et al 2000;Voet et al 2001;Wong et al 2002) , (iii) naturally occurring minichromosomes may be engineered to construct an artificial chromosome (Raimondi 2011), and (iv) de novo chromosome generation can be induced via targeted amplification of specific chromosomal segments (Carl 2004;Csonka et al 2000;Holló et al 1996;Keresö et al 1996;Lindenbaum et al 2004;Praznovszky et al 1991;Tubak et al 1991). To date, two technologies, the 'top down' and the induced de novo chromosome generation approaches have advanced to the point suitable for biotechnological applications.…”

“…The artificial chromosome technology exploits the unique properties of artificial chromosomes as being engineered chromosomes with defined genetic contents, capable of functioning as non-integrating vectors with large carrying capacity and stability (Duncan and Hadlaczky 2007;Hadlaczky 2001;Katona et al 2011). The incorporation of different integrase and recombinase recognition sequences and integrase/recombinase enzyme genes into artificial chromosomes greatly improved the feasibility of integrating a transgene into the artificial chromosome (Duncan and Hadlaczky 2007;Katona et al 2011;Kouprina et al, 2013;Lindenbaum et al 2004) . A MAC as a genetic vector should (1) possess large DNA payload capacity of 1 Mb or greater, (2) facilitate efficient gene(s) loading, (3) be capable of being transferred from one cell to another, and (4) enable stable, high-level transgene expression.…”

Generation of induced pluripotent stem cells by using a mammalian artificial chromosome expression system

“…Recently a platform mammalian artificial chromosome expression system (Platform ACEs) was developed ( Fig. 1) [4, 8,23]. This system is suitable for the high-level production of recombinant proteins in mammalian tissue culture cells [16,17].…”