Novel method to load multiple genes onto a mammalian artificial chromosome

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.

Figure 1. The pST plasmid vector was produced to achieve the superloading of Platform ACEs chromosomes.

(A) The pSEV1R (shuttle expression vector-1R) entry vector map. The gene of interest is cloned into the multi-cloning site (MCS) of this vector. MCS is part of an expression cassette with a chicken beta-actin promoter and an SV40 polyA signal. The I-Ceu and PI-PspI restriction enzyme recognition sites flank the expression cassette. The whole expression cassette with the gene of interest can be moved from this plasmid into several different types of Platform ACEs targeting plasmids (ATVs) with the help of these enzymes. (B) A Platform ACEs-targeting plasmid, pATVMin. The I-Ceu and PI-PspI enzyme sites are used to transfer the expression cassette with the gene of interest into pATVMin. The attB site is the recognition site of ACE integrase. This enzyme performs site-specific recombination between the attB site and the attP site found on Platform ACEs. This process integrates the pATVMin plasmid together with the gene of interest in the expression cassette into the artificial chromosome. After integration, the promoterless neomycin resistance gene acquires the promoter of puromycin gene on the Platform ACEs and G418-resistant cell lines can be isolated with ACE chromosomes carrying and expressing the gene of interest. (C) The pmccKO14 plasmid map. The HSV-TK expression cassette with the LoxP site was removed from this plasmid by XbaI-SpeI restriction enzyme digestion and transferred into the pATVMin plasmid. (D) The map of the pST plasmid. This plasmid contains a promoterless neomycin gene and a HSV-TK expression cassette flanked by direct repeats of two LoxP sites. The HSVT-TK expression cassette came into this plasmid from the pmccKO14 vector by ligation into the unique XbaI site in the pATVMin plasmid. The LoxP site upstream from NeoR CDS was inserted by a PCR-based method. The gene of interest is transferred into the pST vector by the method described in (B). This transgene-carrying vector is then loaded onto the Platform ACEs chromosome as described in (B) and previously published⁷. The LoxP-flanked selectable marker gene cassette can be removed by the transient action of Cre recombinase. Through the use of ganciclovir selection, cell lines lacking the neomycin-thymidine kinase selectable marker gene cassette can be isolated.

Figure 2. The schematics of the site-specific integration process by which the ACE chromosome was targeted with therapeutic transgenes.

(A) The Platform ACEs contains multiple attP recombination acceptor sites for the ACE integrase. The attP site is situated between an SV40 promoter and the puromycin resistance gene, which is driven by this promoter. A Platform ACEs chromosome is also shown. The chromosome is counterstained by DAPI (blue). The green fluorescent staining demonstrates the presence of mouse major satellite sequences, major components of the ACE chromosome. Red fluorescent staining designates the attP sequence carrying sites suitable for the site-specific integration of transgenes. (B) The ATV vectors carry the attB recombination site for the ACE integrase. There is a promoterless neomycin resistance gene immediately after the attB site. The ACE integrase catalyzes the site-specific recombination between the attB and attP sites and integrates the ATV vector into the ACE chromosome. (C) The integration event disconnects the puromycin resistance gene from its promoter and replaces it with the promoterless neomycin resistance gene, which acquires the SV40 promoter. Targeted, transgene-carrying cell lines can be selected for G418 resistance. A transgene-loaded ACE chromosome is also present at the bottom of this figure. The green fluorescent staining demonstrates the presence of mouse major satellite sequences, major components of the ACE chromosome. Red fluorescent staining demonstrates the presence of the “loaded” transgene on the ACE chromosome. The chromosome is counterstained by DAPI (blue).

Figure 3. The assembly of the pSTRFP plasmid.

(A) The pSEV2 entry vector was digested with the BamHI and HindIII restriction enzymes. (B) The pmR-mCherry plasmid was digested with the BamHI and HindIII restriction endonucleases, the RFP CDS-containing DNA fragment was isolated and inserted into the pSEV2 plasmid and the pS2RFP vector was assembled. (C) The I-Ceu and PI-PspI enzymes were used to transfer the mCherry expression cassette from pS2RFP into the pST plasmid and the pSTRFP ACE targeting vector was produced.

Figure 4. The production of the pSTLZ plasmid.

(A) The pCH110 (Amersham) plasmid was digested with the BamHI and HindIII restriction endonucleases and the LacZ-LacY-containing DNA fragment was isolated. The pSEV1R plasmid (Figure 1A) was digested with the BglII and HindIII restriction enzymes. The appropriate DNA fragment was isolated and the LacZ-LacY was ligated together with this fragment to yield the pSLZ plasmid. (B) The LacZ-LacY expression cassette was removed from pSLZ plasmid with the I-Ceu and PI-PspI homing endonucleases. Subsequently, this cassette was ligated into the pST vector, previously digested with the same endonucleases, and the pSTLZ plasmid was constructed.

Figure 5. The “superloading” cycle.

The transgene is cloned into the pST plasmid and loaded onto the ACE chromosome as shown in Figure 1. In the third step the Cre recombinase is transiently expressed in the cell line carrying the transgene-loaded ACE. The Cre recombinase removes the neomycin-thymidine kinase selectable marker gene cassette found between the direct repeats of LoxP sites. Transgene-loaded ACE chromosome-carrying cell lines that lack the neomycin-thymidine kinase selectable marker gene cassette can be obtained by ganciclovir selection. The ACE chromosome is now suitable for the loading of a new transgene cloned into the pST plasmid vector.

Figure 6. Experimental demonstration of the superloading process.

(A) Site-specific integration of the ATV plasmid carrying the various transgenes is demonstrated by means of PCR experiments on purified genomic DNA samples. All the relevant transgene targeted cell lines are presented (1D-9-16, RFPG18 and RFPG18-12), each of which was positive for site-specific integration of the transgene, as demonstrated by the presence of the correct-sized PCR fragment (784 bp). (B) 1D9-16 cells emit the red fluorescence of the mCherry protein, expressed from the loaded ACE chromosome. (C) 1D9-16 cells carry the mCherry-loaded ACE chromosome, demonstrated by means of FISH experiments with a TRITC-labeled plasmid probe (magenta signal, magenta arrow). All chromosomes are counterstained with DAPI (blue signal) (M: 630x). (D) RFPG18 cells emit the red fluorescence of the mCherry protein, expressed from the loaded ACE chromosome (M: 200x). This cell line was treated with the transiently expressed Cre recombinase. The Cre protein removed the selection marker gene cassette (NeoTK) and left the mCherry expression cassette intact. The ACE is now suitable for re-loading with a new transgene. (E) The RFPG18 cells carry the mCherry-loaded ACE chromosome, demonstrated by means of FISH experiments with an FITC-labeled plasmid probe (green signal, green arrow). All chromosomes are counterstained with DAPI (blue signal) (M: 630x). (F) The RFPG18-12 cells emit the red fluorescence of the mCherry protein, expressed from the loaded ACE chromosome (M: 200x). The LacZ staining procedure also stains the RFPG18-12 cells blue. These results demonstrate that the second loading with beta-galactosidase was successful and beta-galactosidase is expressed from the superloaded ACE chromosome and functional (M: 200x). (G) The RFPG18-12 cells carry the mCherry and beta-galactosidase-loaded ACE chromosome, as demonstrated by means of FISH experiments with an FITC-labeled plasmid probe (green signal, green arrow). All chromosomes are counterstained with DAPI (blue signal) (M: 630x).