Novel method to load multiple genes onto a mammalian artificial chromosome.

Anna Tóth,Vilmos Tubak,Tünde Praznovszky,Katalin Fodor,Robert L Katona,Andor Udvardy,Gyula Hadlaczky,Zoltan Rakonczay

doi:10.1371/journal.pone.0085565

Abstract

Mammalian artificial chromosomes are natural chromosome-based vectors that may carry a vast amount of genetic material in terms of both size and number. They are reasonably stable and segregate well in both mitosis and meiosis. A platform artificial chromosome expression system (ACEs) was earlier described with multiple loading sites for a modified lambda-integrase enzyme. It has been shown that this ACEs is suitable for high-level industrial protein production and the treatment of a mouse model for a devastating human disorder, Krabbe’s disease. ACEs-treated mutant mice carrying a therapeutic gene lived more than four times longer than untreated counterparts. This novel gene therapy method is called combined mammalian artificial chromosome-stem cell therapy. At present, this method suffers from the limitation that a new selection marker gene should be present for each therapeutic gene loaded onto the ACEs. Complex diseases require the cooperative action of several genes for treatment, but only a limited number of selection marker genes are available and there is also a risk of serious side-effects caused by the unwanted expression of these marker genes in mammalian cells, organs and organisms. We describe here a novel method to load multiple genes onto the ACEs by using only two selectable marker genes. These markers may be removed from the ACEs before therapeutic application. This novel technology could revolutionize gene therapeutic applications targeting the treatment of complex disorders and cancers. It could also speed up cell therapy by allowing researchers to engineer a chromosome with a predetermined set of genetic factors to differentiate adult stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells into cell types of therapeutic value. It is also a suitable tool for the investigation of complex biochemical pathways in basic science by producing an ACEs with several genes from a signal transduction pathway of interest.

Highlights

Gene therapy may be extremely promising for the treatment of various genetic and acquired disorders in the near future [1]
A Platform artificial chromosome expression system (ACEs) chromosome with multiple recombination recognition sites for a special lambda-integrase (ACE integrase) was earlier constructed and a vector system was established to deliver ‘‘useful genes’’ onto the Platform ACEs [52]. This system begins with an entry vector (Figure 1A; pSEV1R), which contains an expression cassette including a multiple cloning site for the insertion of a gene of interest, or briefly transgene [52]. This expression cassette is introduced into an ACEtargeting vector (ATV) by the action of the yeast homing endonucleases I-Ceu and PI-PspI (Figure 1B; pATVMin)
These ATV plasmids carry a mammalian selectable marker gene without a promoter

Summary

Introduction

Gene therapy may be extremely promising for the treatment of various genetic and acquired disorders in the near future [1]. The pCre-GFP plasmid (a gift from Jim Downing’s laboratory, St. Jude Children’s Research Hospital, Memphis, TN, USA) was used for the transient expression of Cre recombinase to remove the neomycin-thymidine kinase selectable marker gene cassette flanked by direct LoxP sites from the ACEs chromosomes.

Results

Conclusion