Gene Therapy (2005) 12, 1559–1572 & 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00 www.nature.com/gt RESEARCH ARTICLE Bacterial transfer of large functional genomic DNA into human cells

A Laner1,7, S Goussard2,7, AS Ramalho3, T Schwarz1, MD Amaral3,4, P Courvalin2, D Schindelhauer1,5,6 and C Grillot-Courvalin2 1Department of Medical Genetics, Childrens Hospital, Ludwig Maximilians University, Munich, Germany; 2Unite´ des Agents Antibacte´riens, Institut Pasteur, Paris, France; 3Centre of Human Genetics, National Institute of Health Dr Ricardo Jorge, Lisboa, Portugal; 4Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisboa, Lisboa, Portugal; 5Institute of Human Genetics, Technical University, Munich, Germany; and 6Livestock Biotechnology, Life Sciences Center Weihenstephan, Freising, Germany

Efficient transfer of chromosome-based vectors into mam- and transferred into HT1080 cells where it was transcribed, malian cells is difficult, mostly due to their large size. Using a and correctly spliced, indicating transfer of an intact and genetically engineered invasive vector, functional locus of at least 80 kb. These results demonstrate alpha satellite DNA cloned in P1-based artificial chromosome that bacteria allow the cloning, propagation and transfer of was stably delivered into the HT1080 line and efficiently large intact and functional genomic DNA fragments and their generated human artificial chromosomes de novo. Similarly, subsequent direct delivery into cells for functional analysis. a large genomic cystic fibrosis transmembrane conductance Such an approach opens new perspectives for gene therapy. regulator (CFTR) construct of 160 kb containing a portion of Gene Therapy (2005) 12, 1559–1572. doi:10.1038/ the CFTR gene was stably propagated in the bacterial vector sj.gt.3302576; published online 23 June 2005

Keywords: gene delivery; bacterial E. coli vector; human artificial chromosome; CFTR

Introduction either on or, to circumvent viral protein expres- sion, on natural host mechanisms. The most important of For functional analysis of genomic DNA of higher these elements is a functional centromere. Human eukaryotes, stable cloning of large genomic fragments artificial chromosomes (HACs) based on alpha satellite is well established in bacterial artificial chromosomes DNA as the only human component in a circular PAC or (BACs) and in P1-based artificial chromosomes (PACs) on linear, telomerized alpha satellite DNA, faithfully that are single-copy artificial chromosomes in Escherichia replicate and segregate during mitosis for many cell coli based on the F factor and P1 phage replicons, divisions even in the absence of selection.5 Specific alpha respectively.1,2 BACs and PACs can contain the majority satellite DNA arrays from various human chromosomes, of human genomic loci; most of them are comprised such as chromosomes 14, 17, 21, 22, X (and Y between 27 and 72 kb in size with just B4% larger than inefficiently), have been transferred as purified DNA in 140 kb.3 In addition, BACs and PACs allow the stable order to form centromeres on de novo HACS.5–13 The cloning of highly repetitive sequences, which have centromeres formed on the transferred DNA acquire proven to be unstable in other cloning systems, as chromatin proteins specific for functional centromeres demonstrated for larger than 100 kb of highly homo- such as CENP-A a histone H3 variant, only found within geneous alpha satellite tandem repeat arrays of human active centromere chromatin.13 Moreover, complete centromeres.4 genes can be incorporated into HACs and expressed, as Successful gene therapy requires persistent tissue- shown for human HPRT (42 kb),14,15 or GCH1 genes specific expression of the and could be (61 kb).16 optimally achieved by delivery of complete loci of However, most gene delivery systems do not allow genomic DNA of interest including native regulatory efficient transfer of large (4100 kb) DNA fragments into and promoter elements. To avoid random integration mammalian cells, thus limiting their functional analysis. into the host chromosomes and allow stable inheritance, The currently used viral derived vectors do not provide additional genetic elements are required. These are based sufficient packaging capacity. High-capacity herpes simplex -based amplicon vectors are an exception Correspondence: C Grillot-Courvalin, Unite´ des Agents Antibacte´riens, but still cannot accommodate loci larger than 150 kb.17 Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris cedex 15, Nonviral delivery systems based on lipofection, com- France and D Schindelhauer, Livestock Biotechnology, Life Sciences bined or not with polycations, have no size limits and Center, TUM, Hochfeldweg 1, 85354 Freising-Weihenstephan, Germany 7These authors contributed equally to this work have therefore been used with some success to transfer 18 Received 10 February 2005; accepted 22 May 2005; published online intact BAC DNA, both in vitro and in vivo. However, 23 June 2005 this technique requires production and purification of Bacterial transfer into human cells A Laner et al 1560 the DNA construct in significant amounts prior to These vectors included two PACs containing alphoid transfection, a step that can in some cases impair its DNA, TTE122 and B2T8, of 150 and 200 kb, respectively, physical intactness. which could form HACs upon cell transfer, and an We have shown that invasive E. coli, which undergo engineered 160 kb PAC construct, CGT21, which con- lysis upon entry into mammalian cells because of tains a large portion of the human locus containing the impaired cell wall synthesis due to diaminopimelate cystic fibrosis (CF) transmembrane conductance regula- (dap) auxotrophy, can efficiently deliver DNA to tor (CFTR) gene fused to the EGFP gene as a marker.23 host mammalian cells.19 Plasmid pGB2Oinv-hly contain- We demonstrate, for the first time, that bacterial gene ing the inv gene from Yersinia pseudotuberculosis and the delivery systems allow stable clone formation after hly locus from Listeria monocytogenes has been introduced transfer into cells of low copy number genetic elements into the dap auxotroph E. coli BM2710. The inv gene of large size, either through the formation of free confers to E. coli the ability to invade nonphagocytic cells episomal HACs or through their integration into the provided they express b1-integrins. The hly gene host chromosome. Moreover, bacteria allow transfer of product, listeriolysin O, is a pore-forming cytolysin that functional DNA of at least 80 kb, as demonstrated by allows escape of the bacteria, or of its cytoplasmic expression and correct splicing in stable cell lines of the content, from the vacuole of entry. Transfer of functional transferred construct. DNA into a variety of mammalian cell lines occurs after simple coincubation with the bacterial vector.20 Recom- binant-deficient E. coli DH10B is widely used for the Results propagation of BAC clones. Recently, a dapÀ DH10B derivative, similarly made invasive by transformation Invasive E. coli can stably propagate PAC-based with pGB2Oinv DNA was able to mediate transfer into constructs HeLa cells of a 200 kb BAC plasmid, as demonstrated by PAC vectors are usually stably maintained and propa- EGFP expression 48 h after transfection.21 gated in recA1 E. coli DH10B after introduction by We have used the recombination-deficient and dap electrotransformation. We tested if recA1 strains auxotroph strain E. coli BM2710 expressing the invasin BM2710 and BM4573, both derivatives of E. coli and listeriolysin O to deliver three large PAC-derived MM294,24 were also able to propagate stably PAC-based vectors into HT1080, a telomerase-proficient human lung constructs. The three P1-derived used, TTE1, sarcoma cell line often used to analyse HAC formation B2T8 and CGT21 (Figure 1), are compatible and can due to its relatively stable pseudotetraploid karyotype. therefore coexist with pGB2Oinv-hly in E. coli BM2710.

Figure 1 Maps of the plasmids used. (a) Plasmid TTE1 carries ca. 116 kb of a-satellite DNA from the centromeric region of human chromosome 5. The array was cloned as a NotI fragment from P1 artificial chromosome (PAC) E1 into the Bsp120 I site of the tetratelomeric PAC vector pTT. The pTT vector was derived from pTAT-BS by insertional duplication of vector portions, including telomeres, that resulted in tandem repeated vector sequences. An EGFP-

cDNA cassette (CMV/EGFP), two blasticidin S resistance genes (BS) and 2 pairs of telomeres (TTAGGG)n ¼ 135 (arrows) are present along with the unit copy replicon of phage P1 (P1). The location of primer pairs used to analyse the constructs by PCR are shown as black dots (5IF/5IR is present in multiple copies (ca. 340-fold) in the 0.34 kb higher order repeats throughout the array). (b) Plasmid B2T8, a ditelomeric PAC containing approximately 190 kb of a- satellite DNA from human chromosome 17, was constructed by subcloning the NotI cut PAC B2 in the Bsp120 I site of pTAT-BS. The location of primer pairs is indicated by black dots (17-1A/17-2A is present, ca. 70-fold, in the 2.6 kb higher order repeats). (c) Plasmid CGT21 was engineered by cloning from a PAC containing the human CFTR locus from approximately À60 kb 50 to the middle of intron 9. To this CFTR region, a synthetic CFTR-EGFP fusion, composed of CFTR intron 9/exon 10 sequences, the EGFP cDNA CFTR exon 24 and 30UTR and an additional 2 kb 30 region of the CFTR gene, was fused in frame. The construct was then cloned as a NotI fragment into the Bsp120 I site of the ditelomeric PAC vector pTAT-BS containing a blasticidin S resistance gene (BS).

Gene Therapy Bacterial transfer into human cells A Laner et al 1561 Circular DNA from the three PAC plasmids, TTE1, B2T8 multiplicities of infection (MOI) (bacteria/mammalian and CGT21 isolated from agarose plugs prepared from cells ratio) of 5 or 25 to infect HT1080. After a 2 h strain DH10B, was introduced by electrotransformation coincubation, the cells were washed and incubated in into E. coli BM2710/pGB2Oinv-hly and BM4573. Struc- complete medium containing 20 mg/ml of gentamicin to tural integrity and stable propagation of the plasmids in kill extracellular bacteria. After 45 min of incubation, the the new hosts were assessed, after 150 generations, by cells were trypsinized and analysed by flow cytometry. pulsed-field gel electrophoresis. Plasmid I-SceI fragments At an MOI of 5, 74.5% of the cells were fluorescent of the expected size were obtained from some of the (Figure 3, panel a), and at an MOI of 25, 97.6% of the cells clones studied, 124 kb for TTE1 (one out of two, clone F), were strongly fluorescent (Figure 3, panel b). Viable 212 kb for B2T8 (three out of eight, clones U4, U7 and U8) intracellular bacteria were enumerated after gentle cell and 151 kb for CGT21 (all clones studied, clones 20, 24, lysis; the number of viable bacteria per cell is calculated 34 and 38) (Figure 2). These data indicate efficient from the number of bacteria recovered per well. The transfer by electrotransformation of DNA isolated from number of viable intracellular bacteria recovered was E. coli DH10B to E. coli BM2710/pGB2Oinv-hly and 0.25 per cell at an MOI of 5 and 1.2 and at an MOI of 25. BM4573 and stable propagation of PAC-based constructs The discrepancy between the number of viable bacteria in these two bacterial vectors in the absence of major recovered from cells and the percentage of GFP-positive DNA rearrangements. Of note, the alphoid arrays tend to cells can be explained by the fact that dead or dying rearrange more than the CFTR region. bacteria can still be GFP positive. Altogether, the results indicate that, at an MOI of 25, nearly all cells had Optimal conditions for invasion of HT1080 by invasive internalized at least one GFP-producing bacterium. Cell E. coli viability, 48 h postinfection, ranged from 90 to 50% for an MOI of 5 and 25, respectively (data not shown). The first requirement for gene transfer from bacteria to mammalian cells to occur is efficient cell internalization of the bacteria, which varies depending on the cell line. Bacterial vectors can stably deliver PAC-derived The percentage of initially infected HT1080 cells was constructs into HT1080 cells determined by flow cytometry analysis of fluorescent Plasmid delivery into HT1080 cells was performed by cells after invasion with bacteria expressing GFP under simple coincubation for 2 h of HT1080 cells with E. coli the control of a prokaryotic promoter. Invasive E. coli BM2710/pGB2Oinv-hly harbouring plasmids B2T8 or harbouring pAT505 (pUC18Ogfpmut1), a plasmid that CGT21, or E. coli BM4573 harbouring plasmid TTE1 at directs synthesis of GFP in bacteria, was used at an MOI of 5 or 25. After 48 h in complete culture medium

Figure 2 PFGE of constructs in bacteria. PAC DNA preparations in agarose plugs were digested with I-SceI, run on a pulsed field gel and stained with ethidium bromide. Clones with apparently intact inserts of approximately 124 kb (TTE1, clone F), 212 kb (B2T8, clones U4, U7, U8) and 151 kb (CGT21, clones 20, 24, 34, 38) indicated stability of the constructs. Plugs from strain DH10B are shown for comparison. The sizes of the inserts (in kb) are indicated on the side. (M, PFG mid range marker II, New England BioLabs). The plasmids are indicated at the bottom, and the host strains at the top.

Gene Therapy Bacterial transfer into human cells A Laner et al 1562 with an average of 1.8 clones per 1 Â105 cells, B2T8 transfer by BM2710/pGB2Oinv-hly was less efficient with an average of 1.3 clone per 1 Â105 cells, whereas CGT21 delivery by the same bacterial vector was the least efficient with 0.25 clone per 1 Â105. The reasons for these differences could reside in the nature of the genetic element, HAC versus integration of a large DNA fragment for CGT21, or of the bacterial vector, BM4573 being more efficient than BM2710/pGB2Oinv-hly.Of note, bacterial delivery of pEGFP-C1 into HT1080 in similar conditions resulted, 48 h after transfer, in 0.3 and 1%, at an MOI of 5, and 1.2 and 2.9%, at an MOI of 25, EGFP-positive cells for BM2710/pGB2Oinv-hly and BM4573, respectively. A single internalized bacteria delivers ca. 500 copies of plasmid pEGFP-C1.

Characterization by PCR of BS-resistant HT1080 clones The cell clones obtained were further characterized by PCR using the primer pairs described in Table 4 and Figure 1: BemF/BemR, LPF/LPR and KF/KR amplified, respectively, 159, 109 and 381 bp fragments present in the PAC vectors pTAT-BS and pTT. Primers 17-2A/T7 amplified a 150 bp fragment from B2T8 containing the junction between a-satellite DNA and the pTAT-BS vector, and EGF/EGR primer pair amplified a 281 bp fragment present in plasmids TTE1 and CGT21. Table 3 summarizes the results of the PCR experiments per- formed on a fraction of isolated BS-resistant clones. As expected, nearly all clones harboured apparently com- plete cloning vectors. Most importantly, in the majority of the clones harbouring plasmids B2T8 (11/12) and CGT21 (23/40), fragments from the insert (generated with primer pairs 17-2A/T7 and EGF/EGR, respectively) could also be amplified. Rare deletant derivatives were observed with B2T8 (1/12) and more frequently with TTE1 (5/14) and CGT21 (17/40). The cell clones were further investigated by FISH for detection of extrachromosomal elements for B2T8- and TTE1-derived clones and for the expression of genes present in TTE1 and CGT21.

Centromere formation Figure 3 Efficiency of bacterial internalization by HT1080 cells. Cells Dual colour FISH analysis using the Rsf vector probe and infected with GFP-producing bacteria were analyzed by flow cytometry. cen 17 probe in B2T8 lines U10-3 and U10-6 and cen 5 E. coli BM2710 containing invasion pGB2Oinv-hly and reporter probe in TTE1 lines F100-1 and F100-4 was carried out pUC18Ogfpmut1 plasmids were added at an MOI of (a) 5 or (b) 25. after 30 days of growth with BS selection, which FL1-H fluorescence was monitored on cells trypsinized 45 min after cell invasion. corresponds to approximately 30 generations. Free episomal HACs hybridizing with both probes (vector and cen) were found in most of metaphases in the four with gentamicin to kill residual extracellular bacteria, the lines analysed (U10-3, n ¼ 13; U10-6, n ¼ 23; F100-1, cells were cultured in the presence of blasticidin S (BS) n ¼ 12; F100-4, n ¼ 15). None of the lines showed for up to 3 weeks. Resistant clones were detected as early integration into the host chromosomes. In the U10-3 as day 14 and were not isolated after day 19, to avoid and U10-6 lines grown for 30 additional days in the reseeding from existing clones. Clones were obtained, absence of BS, HACs that hybridized with the Rsf and albeit at a low frequency in every experiment and with the cen 17 probes were still detected (number of each PAC-derived vector (Table 2). The MOI of 25 metaphases studied: U10-3, n ¼ 30 and U10-6, n ¼ 42) yielded more clones for the same number of plated cells (Figure 4a); this demonstrates that transfer of B2T8 leads than that of 5. Of note, an MOI of 25 led to the to the formation of stably segregating HACs. Similarly, internalization of at least one bacterium per cell and dual colour FISH analysis with the Rsf and cen 5 probes, therefore of at least one PAC-derived vector molecule per of the two stable F100-1 and F100-4 lines, after 30 cell. Efficiency of transfer was variable depending on generations without BS, showed one, two or rarely three both the plasmid and the bacterial vector: the highest HACs in the metaphases studied (n ¼ 19 and 23, efficiency was obtained for delivery of TTE1 by BM4573, respectively), indicating mitotic stability in the absence

Gene Therapy Bacterial transfer into human cells A Laner et al 1563

Figure 5 Southern hybridization of U10-3 and U10-6 cell lines. To analyse the stability of HAC DNA, chromosomal DNA of the U10-3 and U10-6 lines (indicated at the top) was prepared 30 days after growth in the presence (BS) and 30 days in the absence (D) of BS. Equal amounts (3 mg) of HindIII digested genomic DNA were loaded on a 1% agarose gel, electrophoresed (left panel), blotted and hybridized with the 32P-labelled probe Rsf including the BS-resistance marker of pTAT-BS (right panel). The similar intensities of the characteristic HindIII bands specific to B2T8 (3.4, 2.81 and 1.97 kb) show mitotic stability of the HACs even in the absence of selection.

Figure 4 FISH analysis of stable HT1080 derived lines. Metaphases of B2T8 U10-6 (a) and TTE1 F100-4 (b) lines were prepared from cells grown Rsf probe hybridized with the bands corresponding to 30 days in the presence of BS and 30 days in the absence of BS. Free low the B2T8 construct (3.4, 2.81 and 1.97 kb) (Figure 5), copy episomal HACs (white arrowheads) could be detected by colocaliza- whereas no hybridization was detected in HT1080 (data tion of the vector probe rsf (red) and the centromere probe (green). In all not shown). Moreover, a similar pattern was observed metaphases analysed (n450 for both cell lines), no integration was with DNA prepared from clones grown for 30 additional observed with both probes. HACs at higher magnification are shown in the days in the absence of BS, indicating that the resistance inserts. marker was stably inherited even in the absence of selection. of selection (Figure 4b). Taken together, these data show that mitotically stable, low copy HACs have formed in Stability of TTE1-derived clones as assessed by EGFP all the cell lines analysed. expression The pTT PAC vector contains the EGFP expression cassette and lipofection of TTE1 in HT1080 resulted in Stability of HACs as assessed by Southern bright and stable expression of EGFP in the vast majority hybridization of B2T8 clones of the clones obtained (data not shown). This observation In addition to monitoring HAC stability directly by FISH, indicates that the EGFP is stably expressed from the indirect analysis by Southern blot was carried out on HAC constructs and can therefore be used to assess genomic DNA prepared from the U10-3 and U10-6 indirectly HAC stability. Four out of five cell lines clones grown 30 days in the presence of BS and for studied, F100-1, F100-4, F300-3 and F300-6, expressed another 30 days with or without BS. The Rsf fragment visible amounts of EGFP in more than 95% of the cells as (Table 4) spanning the BS resistance gene in the pTAT-BS assessed by FACS analysis (data not shown); in line F300- vector portion was used as a probe. Equal amounts (3 mg) 5, only 50% of the cells expressed EGFP. All cell lines of HindIII-digested genomic DNA fragments were were divided into two parallel cultures at day 35 and loaded onto a 1% agarose gel, separated, blotted and grown with or without BS for an additional 30 days. The hybridized with the 32P-labelled probe. As expected, the percentage of EGFP-expressing cells was determined by

Gene Therapy Bacterial transfer into human cells A Laner et al 1564 From two independent bacterial transfer experiments, a total of 40 individual cell clones were isolated and expanded. Although PCR with EGF/EGR primers amplified an EGFP-specific fragment in 23 lines (Table 3), EGFP expression was never detected in those clones by FACS analysis (data not shown). For transcript analysis, RNA was extracted from 14 out of these 23 lines, subjected to long RT-PCR (see Materials and methods) and nine out of the 14 clones expressed transcripts from the CGT21 construct (Figure 7a). Clones showing CGT21 expression, albeit at various levels, were: 24-2, 24-4, and 38-1 (faint amplification was confirmed by sequencing of the product), 20B2, 34A2, 34A3, 34A4, 34B1 and 38A1 (see Figure 7a). Parallel amplification of b-actin (Figure 7b) showing equivalent levels of RT-PCR amplification in samples from all clones demonstrated similar efficiency in RNA extraction. Although these RT-PCR results cannot be fully inter- preted in a quantitative manner, results in Figure 7a also suggest varying levels of expression of the CGT21 construct in different clones. The CGT21 RT-PCR product was sequenced in the Figure 6 Stability of EGFP expression from HACs in HT1080 lines nine expressing lines, and the sequences obtained during growth with (continuous lines) and without BS selection (dotted confirmed that it corresponded to the correctly spliced lines). Five cell lines (F100-1, F100-4, F300-3, F300-5 and F300-6) were CGT21 transcript spanning all exons between CFTR exon divided into two cultures on day 35, and were passaged in the presence or 1 and synthetic exon 10-EGFP (data not shown). in the absence of BS. The percentage of cells expressing EGFP was Detection of the CGT21 transcripts as well as the fact determined by flow cytometry at days 45, 55 and 65. they were correctly spliced in all nine cell lines indicates intact transfer of the CGT21 construct. Moreover, these data demonstrate maintenance of a genomic region of at least 80 kb spanning a minimal CFTR promoter. flow cytometry at 10, 20 and 30 days after the split into Two additional RT-PCR were performed to assess the two cultures (ie 45, 55 and 65 days after the initial relative levels of transcripts from the CGT21 construct transfection). As can be seen in Figure 6, the percentage and from the endogenous CFTR gene of the parental cell of EGFP-expressing cells remained stable while selection line. A semiquantitative approach was used consisting in was maintained for four of the five cell lines. The two amplifications yielding products of similar size, but expression of EGFP by line F300-5 was stable until day each being specific for one of the two transcripts. The 55, but started to decrease at day 65. When the cells were results obtained for the 34A2 and 34A3 clones are shown grown without selection, the percentage of EGFP- in Figure 7c and indicate that the endogenous CFTR gene positive cells decreased slowly over time in three cell is also transcriptionally active in the parental cell line. lines. There was a 50% drop after 30 days in a single line (F300-3), indicating an overall good mitotic stability in the absence of selection. Stable EGFP expression corre- lates with HAC formation in lines F100-1 and F100-4. Discussion Several laboratories have recently demonstrated that bacteria can transfer functional genes to a very broad Study of gene expression in CGT21-derived cell range of mammalian cells.25,26 Attenuated strains of clones Shigella,27 invasive E. coli,19,20,28 Salmonella29 and of To evaluate the expression of the CGT21 construct and Listeria30 are able to transfer plasmid DNA to mamma- further assess its integrity after transfer, HT1080 cells lian cells. The bacteria used for transfer to phagocytic were infected with E. coli BM2710/pGB2inv-hly harbour- and nonphagocytic cells are facultative intracellular ing this genomic expression construct and individual pathogens that have been engineered to lyse after cell clones were obtained after BS selection. The CGT21 PAC invasion. Until now, this property has been mainly construct results from the fusion of a portion of the exploited to develop DNA vaccines based on the ability human CFTR locus (exons 1–9 including 60 kb of of Shigella and Salmonella to target dendritic cells.27,29,31 upstream sequences) and of a synthetic exon made of More recently, invasive bacteria have also been used as CFTR exon 10, EGFP-cDNA, 30UTR of CFTR exon 24, DNA vectors for gene therapy. Administration to geneti- followed by the endogenous 30 CFTR genomic sequences cally immuno-deficient mice of avirulent Salmonella as described.23 The resulting transcript encodes an bearing the murine plasmid-borne IFN-g gene resulted artificial fusion protein without CFTR ClÀ channel in the expression of the transgene within macrophages function. This transcript, however, is easily distinguish- and dendritic cells and correction of the genetic defect.32 able from endogenous CFTR transcripts by reverse Taking advantage of the proximity of bowel mucosa to transcription (RT)-PCR using a reverse primer comple- luminal bacteria, we have successfully delivered a mentary to the EGFP sequence (see Materials and therapeutic gene to the colonic mucosa of mice. E. coli methods). BM2710/pGB2Oinv-hly (Table 1) was able to deliver the

Gene Therapy Bacterial transfer into human cells A Laner et al 1565

Figure 7 RT-PCR analysis of CGT21 clones. (a) Results of long RT-PCR of samples from 14 cell lines transfected with the CGT21 genomic construct. Analysis was performed using primers A1R (CFTR exon 1) and GFP1-AL (synthetic exon 10-EGFP, Table 4). NC, negative control. (b) b-actin amplification of the same samples as a control for RNA extraction and the relative RNA levels among the samples. (c) Relative expression of the CGT21 construct and of endogenous CFTR in two cell lines, as analysed by two RT-PCR; one amplification specific for short CGT21 transcripts using the primers B3F (CFTR exon 8) and GFP1-AL (synthetic exon 10-EGFP), spans exon 8 of the CFTR gene to the EGFP sequence in the engineered fusion exon 10 of CGT21, yielding the 474 bp product (right arrow). The other amplification using the primers B3F and CI6D (CFTR exon 10) specific for endogenous CFTR transcripts, produces a 391 bp fragment (left arrow). The size of the inserts (in bp) is indicated on the sides (M1, 1 kb ladder; M2, 100 bp ladder). plasmid-carried TGF-b1 gene into the colonic epithelial constructs and that the bacterial vector allows stable cell line CMT-93 and oral administration to mice of the cloning and propagation of the highly homogeneous bacteria significantly reduced the severity of experimen- tandem repeat arrays known to support de novo tal DNBS-induced colitis.33 centromere formation (Figure 2). Bacterial transfer led However, chromosome-based vectors are considered to formation of stable HACs, which faithfully segregated to be potentially useful to introduce exogenous genes in the absence of selection (Figure 4) and thus supported into mammalian cells, since they replicate autonomously centromere formation by alpha satellite DNA. Until now, and have unlimited cloning capacity allowing insertion de novo formation was observed only after transfection of of entire genes with their proper endogenous and tissue- purified DNA from constructs by lipofection or micro- specific regulatory elements.34,35 Thus, simple and injection. Since transfer occurs by direct cytoplasmic efficient techniques to transfer these very large molecules delivery upon lysis of intracellular dapÀ bacteria28 into mammalian cells need to be developed. without the need for DNA purification from E. coli,a We have demonstrated that BAC/PAC-based con- possible negative effect of prokaryotic proteins bound to structs can be transferred into mammalian cells by an the PAC-DNA on the initial steps of HAC formation invasive E. coli (Table 1) in which they can be introduced seems unlikely. and propagated (Figure 2) resulting in de novo formation Efficiency of stable clone recovery (Table 2) was in the of HACs (Figure 4) or stable gene expression (Figures 6 same range as that observed with other transfer methods and 7). (ie 10À5–10À6, Magin-Lachmann et al18), although these As shown previously, using an inducible homologous figures are usually not rigorously documented; most recombination system adapted for use in E. coli DH10B,36 studies consider early gene, such as egfp expression as an a dapÀ derivative of this strain was generated and human estimation of transfer efficiency. The optimal conditions genomic BAC clones were modified into suitable vectors for bacterial invasion defined in this study lead to more for mammalian cells by the introduction of an EGFP than one bacterium per cell (Figure 3) and therefore to neocassette.21 After transformation with plasmid more than one PAC-derived vector delivered intracellu- pGB2Oinv DNA, the resulting invasive bacteria were larly in each cell. Access to the cell cytosol of bacteria and able to deliver a 200 kb modified BAC to HeLa cells, plasmid DNA is an important requirement for successful resulting in 2.8% of cells expressing EGFP 48 h after gene transfer,37 but transfer to the nucleus is another bacterial invasion at high MOI (1500–4000); however, limiting step as is the case for all other methods. intactness of the transferred BAC and long-term BAC Detection of free episomal HACs after bacterial stability in the transfected cells were not studied. transfer of TTE1 or B2T8 (Figure 4) do not prove per se We demonstrate here that E. coli strain BM2710 can be transfer of intact centromere constructs. Owing to the transformed by large (from 140 to 220 kb) genomic highly homogeneous nature of alpha satellite arrays,

Gene Therapy Bacterial transfer into human cells A Laner et al 1566 Table 1 Plasmids and bacterial strains used

Designation Relevant characteristicsa Source or reference

Plasmids pAT504 pGB2Oinv-hly, inv from Y. pseudotuberculosis and hly from L. monocytogenes,SmR,SpR 20 pAT505 pUC18Ogfpmut1, ApR 20 R R R pTAT-BS ditelomeric PAC vector, PSV40-BS ;Ap ,Km 5 R R R pTT tetratelomeric PAC vector, PSV40-BS ; PCMV-egfp,Ap ,Km 22 R R R B2T8 pTAT-BSO190 kb a-satellite DNA array from human chromosome 17, PSV40-BS ;Ap ,Km 4, this work and unpublished data R R R TTE1 pTTO116 kb a-satellite DNA array from human chromosome 5, PSV40-BS , PCMV-egfp;Ap ,Km 22 R R R R CGT21 pTAT-BSOhuman CFTR locus with EGFP fusion, PSV40-BS ;Ap ,Km ,Tc 23

E. coli DH10B FÀ mcrA D(mrr-hsdRMS-mcrBC)(f80dlacZDM15) DlacX74 deoR recA1 araD139 D(ara-leu)7697 43 galU galK rpsL (SmR) endA1 lÀ nupG À + R BM2710 thi-1 endA1 hsdR17 (rK m K) supE44 DlacX74 recA1 DdapAOcat (Cm )19 BM4573 BM2710 DmsbB DdapAOPtac-inv DdapBOPtet-hly Unpublished data

aAbbreviations used for antibiotic resistance (R): Ap ¼ ampicillin; BS ¼ blasticidin S; Cm ¼ chloramphenicol; Km ¼ kanamycin; Sm ¼ strepto- mycin; Sp ¼ spectinomycin; Tc ¼ tetracycline.

Table 2 HT1080 clones obtained at days 17–19 with selection on BS nomenclature),39 indicating successful transfer of a large functional unit of at least 80 kb. A long RT-PCR spanning Experiment Plasmid transferred MOI Number of clones all CGT21 exons (from the CFTR start codon to the EGFP per total number of plated cells coding region fused to CFTR exon 10) showed that this construct is stably expressed from the CFTR promoter in 1 B2T8, clone U7 5 3/3 Â 106 the mammalian HT1080 cell line and that all exons are 25 B60/3 Â 106 correctly spliced. Assuming complete transfer of a given 2 B2T8, clone U7 25 13/1.5 Â 106 PAC construct, integration requires opening (ie linear- 3 B2T8, clone U4 25 11/1.5 Â 106 ization) of the circular molecules. If this process occurs clone U7 25 12/1.5 Â 106 randomly, any region of the construct, including the 6 clone U8 25 19/1.5 Â 10 functional unit (ie the gene), could be interrupted during 1 TTE1, clone F 5 3/5 Â 105 insertion. Within the 159 kb sized CGT21 construct, the 25 17/5 Â 105 functional unit covers at least 80 kb. Therefore, one 2 TTE1, clone F 5 0/5 Â 105 would expect that less than 50% of the integrants should 25 19/5 Â 105 have an intact functional CGT21 unit. Among the 40 3 TTE1, clone F 25 8/1.5 Â 106 clones resistant to BS obtained after transfer of CGT21, 23 harboured vector and insert portions as assessed by PCR 1 CGT21, clone 20 25 0/3 Â 106 clone 24 25 11/3 Â 106 (Table 3). In all, 14 candidate clones were analysed and clone 34 25 3/3 Â 106 nine (64%) were shown to express the correctly spliced clone 38 25 1/3 Â 106 transgene, as assessed by long RT-PCR and sequencing 2 CGT21, clone 20 25 17/3 Â 106 (Figure 7, and data not shown). Since stable cell lines clone 24 25 5/3 Â 106 were isolated with selection for the BS marker spanning 6 clone 34 25 17/3 Â 10 only 2 kb, there was no bias towards transfer of the intact clone 38 25 7/3 Â 106 80 kb unit. Taken together, these data suggest that transfer and maintenance of an unaltered large construct is not a rare event. Gene delivery systems play a central role in the transfer of a deleted construct could result, by subse- development of gene therapy strategies for monogenetic quent homologous recombination, in a final functional hereditary diseases, like CF, as they offer the potential of centromere; in fact, de novo formed HACs have been correcting the underlying cause when the responsible estimated to be larger than single input centromere gene is known.35 Since delivery of a large gene, like sequences implying DNA concatemerization7,6,14,38 and CFTR, to the relevant cells, at the proper expression level arrays as small as 70 kb of contiguous sequences have seems difficult to achieve with available vector systems, proven to be sufficient to form centromeres.5 alternative approaches should be considered. Engineered To test if bacteria allow transfer of large intact and bacteria have the potential to circumvent some of the functional genomic loci, we used an engineered genomic problems associated with construct/vector systems construct CGT21,23 which expresses a large transcrip- currently in use, such as the inefficiency of intact gene tional unit of the CFTR gene and is easily distinguishable delivery and lack of stable expression. In particular, this from the endogenous CFTR transcripts due to an EGFP system further extends the ability of introducing very fusion. Results indicated that the CGT21 construct after large DNA sequences into phenotypically inert and bacterial transfer into mammalian cells is expressed from nonintegrative autonomously replicating vectors such the CFTR promoter lying at a distance of 80 kb from the as artificial chromosomes. This is achieved by combining last of the 12 exons (exon 10 according to the CFTR their inherent advantages (ie stability and large carrying

Gene Therapy Bacterial transfer into human cells A Laner et al 1567 Table 3 PCR screening of BS-resistant HT1080 clones

Primers used for PCR

Plasmid transferred 17-2A/T7 EGF/EGR LPF/LPR BemF/BemR KF/KR Number of clones

B2T8 + NAa + + + 11/12 À NA + À + 1/12

TTE1 NA + + + + 9/14 NA À + + + 4/14 NA + + ÀÀ 1/14

CGT21 NA + + + + 23/40 NA À + + + 10/40 NA ÀÀ ÀÀ 4/40 NA À ++À 3/40 aNA ¼ not applicable.

capacity) with the ease of purification and transferability approximately 1–10 linearized copies and by lipofection into mammalian cells. The potential of this system is (data not shown), and similar alpha satellite arrays of demonstrated by the design of CGT21 that could be chromosome 17.6,12 modified by replacing EGFP by the remainder of CFTR Plasmid TTE1 contains a 116 kb a-satellite array of to encode a functional CFTR or by insertion of the other chromosome 5, belonging to a subtype of the dimeric elements required for HAC formation, opening new alpha satellite family, which is present on human perspectives for gene therapy of CF or for any other chromosomes 1, 5 and 19.41 The homogeneous array human genomic-based cell therapy. present in TTE1 contains 0.34 kb EcoRI higher-order A number of genetic techniques can be used to modify repeats as revealed by restriction analysis (not shown). It in bacteria the genomic inserts cloned in BACs and direct was isolated from PAC library RPCI 704 by array-specific transfer of the engineered BAC from the host strain into PCR using primers 5IF and 5IR. The primer sequences the target cell can be performed without DNA purifica- were derived from that of the 0.7 kb insert in plasmid tion. This approach allows functional analysis of pZ5.1 (Hulsebos et al42; http://www.biologia.uniba.it/ large structural and regulatory genomic regions in cell rmc/5-alfoidi/alfoidiplasmids.html). The NotI insert culture assays and has been successfully adapted for the (116 kb) of PAC E1 was cloned into the Bsp120I site of analysis and rapid phenotypic screening of the murine pTT (26 kb), a tetratelomeric PAC vector derived from cytomegalovirus genome.40 Combined with the efficient pTAT-BS by insertional duplication of the portion transfer of low copy intact large functional DNA between the telomeres, insertion of a prokaryotic demonstrated here for the human CFTR locus, these white/blue selectable marker derived from pUC19 data suggest that, in principle, bacteria can be engineered (Gibco BRL, Gaithersburg, MD, USA) and insertion of a to transfer artificial chromosomes that express human eukaryotic CMV/EGFP expression cassette derived by genes. PCR from plasmid pEGFP-N1 (Clontech, Palo Alto, CA, USA).22 Construction of the genomic CFTR expression plasmid Materials and methods CGT21 (EMBL/Genbank accession number BN000167) is described elsewhere.23 In brief, an unaltered PAC PAC-based plasmids containing the 50 portion of the human CFTR locus The maps of the PAC vectors are shown in Figure 1. (À60 kb to CFTR intron 9) was joined by cloning with a Plasmid B2T8 contains a 190 kb a-satellite DNA array of synthetic exon fused from sequences of CFTR intron 9, human chromosome 17 composed of 2.6 kb sized, 16- exon 10, the EGFP coding region, CFTR exon 24 plus meric EcoRI higher-order repeats belonging to the 30UTR and further CFTR 30 downstream region. Se- pentameric family of alpha satellite sequences also found quences of the CF-PAC exons are available online from on human chromosomes X and 11. It was constructed by the VR of the European working group on CFTR inserting NotI cut PAC B2 (isolation described in expression (http://central.igc.gulbenkian.pt/cftr/vr/) Schindelhauer and Schwarz4) in the Bsp120I site of the and under Genbank/EMBL accession numbers ditelomeric 17 kb PAC vector pTAT-BS, which contains a AJ574939–AJ575055. The resulting NotI fragment of BS resistance gene. End-sequence pairs of 10 randomly 142 kb was cloned in the Bsp120I site of pTAT-BS subcloned EcoRI higher-order repeats showed a high (17 kb) to include the BS selectable marker and for future similarity (497%). The sequences are provided in the incorporation into HACs. This PAC plasmid designated EMBL/Genbank database under accession numbers CGT21 (159 kb) was functionally analysed in stable AJ563631–AJ563650. Partial restriction and pulsed field HT1080 cell lines. RT-PCR spanning all exons revealed mapping showed the absence throughout the array of correct splicing of all exons, including the synthetic one fragments other than the 2.6 kb EcoRI fragments (data (the sequence of the resulting cDNA is deposited in the not shown). De novo centromere proficiency of the EMBL/Genbank database under accession number plasmid was shown by intranuclear microinjection of AY299332).23

Gene Therapy eeTherapy Gene 1568

Table 4 Oligodeoxynucleotides used in this study

Primer Sequence (50–30) Locationa Size of the Reference Used for PCR product (bp)

17-2A ATAACTGCACCTAACTAAACG B2T8, chr. 17 a-sat DNA, present in the 2.6 kb higher-order repeats Ca. 150 45 PCR cells human into transfer Bacterial T7 TAATACGACTCACTATAGGG Cloning boundary of the NotI insert of chr.17 a-sat DNA derived This work, 4 from PAC B2 EGF AGGGCGAGGAGCTGTTCAC TTE1, CGT21, EGFP cDNA fragment 281 This work, 23 PCR EGR GTGCGCTCCTGGACGTAGC LPF GAAACGGCCTTAACGACGTAGTCG B2T8, TTE1, CGT21, vector fragment, adjacent to the loxP site 109 This work, 23 PCR LPR ATGATAAGCTGTCAAACATGAGAATTG Laner A BemF CATGCTCACGGCAATGCCGG B2T8, TTE1, CGT21, vector fragment, close to Bsp120I 159 This work, 23 PCR BemR TGGCACTTTGCGTATCGTCCA KF GGGAAAACAGCATTCCAGGTATTAG B2T8, TTE1, CGT21, KmR fragment 381 This work PCR al et KR CCATGAGTGACGACTGAATCCGGT RsF AGCGGTCGGACCGTGCTC pTAT-BS vector fragment, includes the loxP site and the BSR 3160 This work, 23 Probe for selectable marker Southern and FISH 5IF GTGAGGAAACAGTCTGTTTGTC TTE1; chr. 5 a-sat DNA type I, present in the 0.34 kb higher-order 275 41,22 Probe for repeats FISH 5IR GAATCATTCTGTCTAGTTTTTATAC 17-1A TGTTTAGTCAGCTGAAATT B2T8; chr. 17 a-sat DNA, present in the 2.6 kb higher-order repeats Ca. 1900 with 45 Probe for 17-2A FISH A1R CGAGAGACCATGCAGAGGTC CFTR exon 1 (forward) 1668 with 49 RT-PCR GFP1-AL C16D GTTGGCATGCTTTGATGACGCTTC Endogenous CFTR exon 10 (reverse) 391 with B3F 47 RT-PCR GFP1-AL GAACTTCAGGGTCAGCTTG Synthetic exon 10 in CGT21, EGFP portion (reverse) 474 with B3F 23 RT-PCR B3F AATGTAACAGCCTTCTGGGAG CFTR exon 8 (forward) 47 RT-PCR ActS GCACTCTTCCAGCCTTCC b-Actin (forward) 228 48 RT-PCR ActAS GCGCTCAGGAGGAGCAAT b-Actin (reverse) 48 RT-PCR AC1L GAAACCAAGTCCACAGAAGGC CFTR exon 6a — This work Sequencing CFex5.F CTCCTTTCCAACAACCTGAAC CFTR exon 5 — This work Sequencing B2R GGAAGGCAGCCTATGTGAGA CFTR exon 7 — 49 Sequencing

a KmR ¼ resistance to kanamycin; BSR ¼ resistance to blasticidin S. Bacterial transfer into human cells A Laner et al 1569 Bacterial strains, plasmids and growth conditions 6 V/cm, ramped switch time 1–30 s, 16 h run in 0.5 Â The bacterial strains and plasmids used in this study are TAE buffer at 121C. The gels were stained with ethidium listed in Table 1. Bacteria were grown in brain heart bromide and the bands visualized under UV light. infusion (BHI, Difco Laboratories, Detroit, MI, USA) broth or agar. The dap auxotroph strains E. coli BM2710 Bacterial invasion of cells and BM4573 (a BM2710 derivative in which the inv and The HT1080 (human lung sarcoma) cell line was hly genes have been inserted in the chromosome, maintained in complete culture Dulbecco’s modified manuscript in preparation) were grown with 0.5 mM of Eagle’s medium (DMEM; ICN Biomedicals, Aurora, OH, diaminopimelic acid (dap, Sigma, St Louis, MO, USA). USA) supplemented with 2 mML-glutamine and 10% Bacteria harbouring pGB2Oinv-hly were selected with fetal calf serum (FCS, Gibco-BRL, Gaithersburg, MD, 25 mg/ml of spectinomycin. Bacteria harbouring B2T8 USA). The cells were incubated overnight in six-well and TTE1 were selected with both 25 mg/ml of kanamy- plates (2.5 Â 105 cells/well) or in 100 mm Petri dishes cin, 50 mg/ml of ampicillin; bacteria harbouring CGT21 (0.65 Â 105–1.5 Â 106 cells/dish). Bacteria were grown were selected with kanamycin, ampicillin and 2 mg/ml of overnight at 301C in BHI broth supplemented with tetracycline. Ampicillin (50 mg/ml) was used to select 0.5 mM dap and the appropriate antibiotics, harvested by strains containing pAT505. centrifugation and resuspended in DMEM containing 0.5 mM dap at 2.5 Â 108 bacteria/ml. Bacteria were added to HT1080 cells in 2 ml (six-well plates) or 6 ml (100 mm Overnight cultures of E. coli BM2710/pGB2Oinv-hly or E. Petri dishes) of DMEM with dap to obtain an MOI of ca. coli BM4573 were diluted 1:50 in 40 ml of BHI broth 5 or 25 and the plates or dishes were incubated for 2 h at containing 0.5 mM dap. Bacteria were grown at 371C with 371C. The cells were then washed three times with shaking to an OD600 of 0.5–0.6, chilled 15 min on ice, DMEM and incubated in complete medium containing harvested by centrifugation for 5 min at 6000 r.p.m. at 20 mg/ml of gentamicin to remove extracellular bacteria 41C, washed twice in 10 ml of cold sterile water and for 2 days. collected by centrifugation for 5 min at 6000 r.p.m. at 41C. The bacteria were resuspended in 10 ml of 10% cold Generation of stable BS-resistant HT1080 clones glycerol, centrifuged 10 min at 6000 r.p.m. and the pellet The infected cells were then incubated in complete was resuspended in 0.2 ml of 10% glycerol. Circular medium containing 4 mg/ml of BS (ICN Biochemicals, DNA of PAC plasmids B2T8, TTE1 and CGT21 was Aurora, OH, USA) for at least 2 weeks and individual 43 isolated from E. coli DH10B in agarose plugs containing resistant HT1080 clones (containing 250–1000 cells) were approximately 1 mg/100 ml of DNA. From 30 to 50 mg of isolated using cloning cylinders (Sigma, St Louis, MO, low melting point agarose containing plasmid DNA was USA). Colonies were trypsinized and transferred into 1 melted by incubation at 65 C for 10 min with 1/10 six-well plates and subsequently expanded in 25 and volume of 10 Â b-Agarase I buffer (New England 75 cm2 flasks for analysis. BioLabs, Beverly, MA, USA). The melted agarose was cooled to 401C and incubated from 1 to 1 h 30 with b- Agarase (one unit for 100 ml). Electrocompetent bacteria Counts of internalized bacteria 1 (25 ml) and 3 ml of plasmid DNA were added to After invasion, cells were incubated 30 min at 37 Cin microcentrifuge tubes placed on ice, mixed and trans- complete medium containing 20 mg/ml of gentamicin ferred to cuvettes with a 0.1 cm gap (Bio-Rad Labora- and washed three times with DMEM. The bacteria were tories, Richmond, CA, USA) chilled on ice. released from the cells with 0.25% deoxycholate, and Electroporation (Bio-Rad gene Pulser) conditions were viable counts were determined on BHI agar plates 100 O, 1.6 kV and 25 mF. A measure of 1 ml of BHI containing 0.5 mM dap and antibiotics. medium with 0.5 mM dap was immediately added to the cuvette and the content was then transferred to sterile Flow cytometric analysis glass cultured tubes for growth for 1 h with moderate HT1080 cells were trypsinized, washed once with 2% shaking at 371C. Bacteria (0.25 ml) were spread on BHI FCS in phosphate-buffered saline (PBS), resuspended in agar containing 0.5 mM dap and appropriate antibiotics, the same medium at 106 cells/ml and 3 Â 104 cells were and the plates were incubated for a minimum of 24 h at analysed by flow cytometry using a FACScan flow 371C. cytometer with CellQuest software (Becton-Dickinson, Mountain View, CA, USA). Analysis of plasmid DNA by pulsed field gel electrophoresis (PFGE) PCR screening of resistant HT1080 clones To verify integrity of the PAC plasmids after transfer in Cell clones were screened by PCR using primer pairs the bacteria, agarose plugs were prepared from E. coli described in Table 4. Cells (1–2 Â 106) were trypsinized, BM2710 harbouring pGB2Oinv-hly+B2T8 or CGT21, E. and washed once with 5 ml of PBS; the pellet was coli BM4573 harbouring TTE1 and from parental E. coli resuspended in 50–100 ml of TTE buffer (0.01% Triton DH10B strain. Circular DNA was purified after frag- X-100, 20 mM Tris-HCl (pH 8), 2 mM EDTA, pH 8), mentation of E. coli chromosome by restriction digestion incubated 10 min at 1001C and centrifuged for 3 min at and PFGE purification as described.44 The size of the 10 000 r.p.m. A measure of 2 ml of supernatant was used plasmids was determined by I-SceI cleavage, removing in a final volume of 50 ml for a PCR of 35 cycles with vector sequences outside of the telomeric repeats, and annealing temperatures of 601C (LPF/LPR; BemF/BemR; PFGE with a CHEF DRII apparatus (Bio-Rad Labora- KF/KR; EGF/EGR) or 531C (17-2A/T7). Untransfected tories, Richmond, CA, USA) with the following settings: HT1080 cells were used as a negative control.

Gene Therapy Bacterial transfer into human cells A Laner et al 1570 Southern hybridization exon 1) and GFP1-AL (EGFP sequence at the CGT21 Genomic DNA of stable cell lines was prepared using fusion) (Table 4). The expected size of the correctly pronase A, salt precipitation of proteins and ethanol spliced RT-PCR product was 1668 bp. Each reaction precipitation. Approximately 3 mg of genomic DNA was contained 10 ml of cDNA and 1 U of AmpliTaq DNA digested with HindIII and separated on a 1% agarose gel Polymerase (Applied Biosystems, Foster city, CA, USA); at 30 V for 16 h in TBE buffer and probed with the 3.15 kb 1  buffer I (Applied Biosystems); 200 mM of each dNTP PCR fragment obtained with Rsf primer (Table 4) (Amersham Bioscience, Uppsala, Sweden); and 0.2 mM of spanning the BS resistance marker. The PCR product each primer in a final volume of 50 ml. In all, 40 was labelled using random primed polymerization with amplification cycles were used as follows: denaturation Klenow enzyme (Roche, Mannheim, Germany) and 32P- at 941C for 1 min, annealing at 581C for 1 min and dCTP (Amersham, UK). Hybridization was carried out at extension at 721C for 3 min. A final extension at 721C was 651C overnight in the presence of dextransulphate and carried out for 12 min. salmon sperm DNA. Efficiency of RNA extraction and the RNA levels in the various samples were controlled by RT-PCR with 2 ml of each cDNA sample to amplify a 228 bp b-actin FISH analysis fragment using primers ActS (forward) and ActAS m Cells enriched for metaphases using 0.4 g/ml colcemid (reverse) (Table 4) at an annealing temperature of 581C. (Roche, Mannheim, Germany) for 4 h were treated with 1 To assess the relative levels of transcripts from the hypotonic solution at 37 C for 40 min in 0.8% (w/v) CGT21 construct and from endogenous CFTR, two sodium citrate. Nuclei were fixed in methanol/acetic additional RT-PCR reactions were performed. The first À 1 acid (3:1) at 20 C for standard chromosomal spreads. of these reactions used primers B3F (CFTR exon 8) and RNase A- (Roche, Mannheim, Germany) and pepsin- GFP1-AL (described in Table 4) amplifying a fragment of (Boehringer, Mannheim, Germany) treated chromosome 471 bp, specific of CGT21 transcripts. For the second preparations were dehydrated in ethanol. Chromosomal reaction, amplifying a 391 bp fragment specific of DNA and probes were simultaneously denaturated at endogenous CFTR, we used primers B3F (CFTR exon 1  72 C for 5 min in 50% (v/v) formamide, 2 SSC, 20% 8) and C16D (in region of CFTR exon 10 that is absent in (w/v) dextran sulphate and incubated in a humid m 1 CGT21, see also Table 4). A measure of 5 l of each cDNA chamber at 37 C for 72 h. FISH hybridization was carried sample was added to the PCR reaction mixture, which out in the presence of 1 mg/ml salmon testes DNA was heated at 941C for 5 min, and then subjected to 35 (Sigma, St Louis, MO, USA). Posthybridization washes 1 23 cycles of denaturation at 94 C for 1 min, annealing at were carried out as described. After blocking with 3% 601C for 1 min and extension at 721C for 2 min, followed (w/v) BSA, Cy3.5-conjugated Avidin (Rockland, Gil- by a final extension at 721C for 12 min. bertsville, PA, USA) or FITC-conjugated anti-dig anti- The RT-PCR products were analysed by 2% (w/v) body (Roche, Mannheim, Germany) was bound in 1% agarose gel electrophoresis. The gel images were  (w/v) BSA, 4 SSC and 0.2% (v/v) Tween-20. Slides registered on an UV DBT-08 digital analyser (UVItec were washed (4  SSC, 0.2% Tween-20, 421C) and 0 Ltd, Cambridge, UK). counterstained with DAPI (4 ,6-diamidino-2-phenylin- Products from the long RT-PCR amplification were dole; Sigma, St Louis, MO, USA), mounted with antifade purified using the Jet Quick PCR product purification (PPD) and analysed on a Leica DM RXA with a CCD spin kit from Genomed (Lo¨hne, Germany) and se- camera controlled by Q-FISH software (Leica Microsys- quenced with the ABI PRISMt Dye Terminator Cycle tems, Switzerland). The vector probe was obtained by sequencing system V1.1 (Applied Biosystems), according amplifying a 3.15 kb fragment with primer Rsf (Table 4) to the manufacturer’s instructions. Three primers (see from pTAT-BS. The product was labelled by PCR Table 4) were used in different sequencing reactions to incorporation of biotin-16-dUTP (Roche, Mannheim, obtain the complete sequence of the long RT-PCR Germany). The centromeric probes cen 5I and cen 17 products, namely: AC1L (reverse primer located in CFTR were amplified using primers 5IF/5IR and 17-1A/17-2A exon 6a), CFex5.F (forward primer located in CFTR exon (Table 4), respectively, from the corresponding PACs. 5) and B2R (forward primer located in CFTR exon 7). 5IF/5IR amplified a 275 bp region including the CENP-B Sequences of these three products were obtained on an box of the 0.34 kb higher-order repeat of alpha satellite t 41 ABI Prism 3100 capillary electrophoresis automatic array type I of chromosome 5 (Z5.1, Archidiacano et al ), sequencer (Applied Biosystems). and 17-1A/17-2A amplified a 1.9 kb segment of the 2.6 kb higher-order repeats of the alpha satellite DNA array of chromosome 17.45 Acknowledgements RT-PCR analysis Total RNA was isolated from BS-resistant cell lines using This work was supported in part by two grants from the RNeasy extraction kit (Qiagen, Hilden, Germany) l’association Vaincre la Mucoviscidose in 2002–2003 and according to the manufacturer’s instructions. RT was 2003–2004, the latter together with the German Associa- carried out using random hexamers and Superscript II tion Mukoviszidose e.v. D Schindelhauer was supported RnaseH-reverse transcriptase (Invitrogen, Paisley, UK) as by the Deutsche Forschung Gemeinschaft and MD described.46 The cDNA obtained was used as a template Amaral by research Grant POCTI/1999/MGI/35737 in a long RT-PCR to assess expression and correct from FCT Portugal. AS Ramalho was a recipient of splicing of the CGT21 construct. The reaction was PhD fellowship SFRH/BD/3085/2000 from FCT, Portu- performed between CFTR exon 1 and CGT21 synthetic gal. We thank C Klein and M Speicher for FISH fusion of CFTR exon 10-EGFP using primers A1R (CFTR equipment.

Gene Therapy Bacterial transfer into human cells A Laner et al 1571 References 23 Schindelhauer D et al. An engineered genomic CFTR construct is expressed and correctly spliced in the human lung sarcoma cell 1 Shizuya H et al. Cloning and stable maintenance of 300-kilobase- line HT1080. (submitted). pair fragments of human DNA in Escherichia coli using an 24 Bachmann BJ. Derivations and genotypes of some mutant F-factor-based vector. Biochemistry 1992; 89: 8794–8797. derivatives of Escherichia coli K-12. In: Brooks LK, Ingraham JL, 2 Ioannou PA et al. A new P1-derived vector for the Magasanik B, Neidhardt FC, Schaechter M, Umbarger HE (eds). propagation of large human DNA fragments. Nat Genet 1994; 6: Escherichia coli and Salmonella Typhimurium, Cellular and Molecular 84–89. Biology. ASM: Washington, 1987, pp 1190–1219. 3 International Human Genome Mapping Consortium. A physical 25 Grillot-Courvalin C, Goussard S, Courvalin P. Bacteria as gene map of the human genome. Nature 2001; 409: 934–941. delivery vectors for mammalian cells. Curr Opin Biotechnol 1999; 4 Schindelhauer D, Schwarz T. Evidence for a fast, intrachromo- 10: 477–481. somal conversion mechanism from mapping of nucleotide 26 Weiss S, Chakraborty T. Transfer of eukaryotic expression variants within a homogeneous alpha-satellite DNA array. plasmids to mammalian host cells by bacterial carriers. Curr Genome Res 2002; 12: 1815–1826. Opin Biotechnol 2001; 12: 467–472. 5 Ebersole TA et al. Mammalian artificial chromosome formation 27 Sizemore DR, Branstrom AA, Sadoff JC. Attenuated Shigella as a from circular alphoid input DNA does not require telomere DNA delivery vehicle for DNA-mediated immunization. Science repeats. Hum Mol Genet 2000; 9: 1623–1631. 1995; 270: 299–302. 6 Harrington JJ et al. Formation of de novo centromeres and 28 Fajac I et al. Recombinant Escherichia coli as a gene delivery construction of first-generation human artificial microchromo- vector into airway epithelial cells. J Control Rel 2004; 97: 371–381. somes. Nat Genet 1997; 15: 345–355. 29 Darji A et al. Oral somatic transgene vaccination using 7 Ikeno M et al. Construction of YAC-based mammalian artificial attenuated S. typhimurium. Cell 1997; 91: 765–775. chromosomes. Nat Biotechnol 1998; 16: 431–439. 30 Dietrich G et al. Delivery of antigen-encoding plasmid DNA into 8 Grimes BR, Rhoades AA, Willard HF. Alpha-satellite DNA and the cytosol of macrophages by attenuated suicide Listeria vector composition influence rates of human artificial chromo- monocytogenes. Nat Biotechnol 1998; 16: 181–185. some formation. Mol Ther 2002; 5: 798–805. 31 Paglia P et al. Gene transfer in dendritic cells, induced by oral 9 Henning KA et al. Human artificial chromosomes generated by DNA vaccination with Salmonella typhimurium, results in modification of a yeast artificial chromosome containing both protective immunity against a murine fibrosarcoma. Blood human alpha satellite and single-copy DNA sequences. Proc Natl 1998; 92: 3172–3176. Acad Sci USA 1999; 96: 592–597. 32 Paglia P et al. In vivo correction of genetic defects of monocyte/ 10 Schueler MG et al. Genomic and genetic definition of a functional macrophages using attenuated Salmonella as oral vectors for human centromere. Science 2001; 294: 109–115. targeted gene delivery. Gene Therapy 2000; 7: 1725–1730. 11 Kouprina N et al. Cloning of human centromeres by transforma- 33 Castagliuolo I et al. Engineered Escherichia coli deliver tion associated recombination in yeast and generation of therapeutic genes to the colonic mucosa of mice. Gene Therapy functional human artificial chromosomes. Nucleic Acids Res 12: 1070–1078. 2003; 31: 922–934. 34 Lipps HJ et al. Chromosome-based vectors for gene therapy. Gene 12 Meija JE et al. Efficiency of de novo centromere formation in 2003; 304: 23–33. human artificial chromosomes. Genomics 2002; 79: 297–304. 35 Klink D et al. Gene delivery systems-gene therapy vectors for 13 Ohzeki J, Nakano M, Masumoto H. CENP-B box is required for cystic fibrosis. J Cystic Fibrosis 2004; 3: 203–212. de novo centromere chromatin assembly on human alphoid 36 Narayanan K et al. Efficient and precise engineering of a 200 kb DNA. J Cell Biol 2002; 159: 765–775. b-globin human/bacterial artificial chromosome in E. coli 14 Meija JE et al. Functional complementation of a genetic DH10B using an inducible homologous recombinant system. deficiency with human artificial chromosomes. Am J Hum Genet Gene Therapy 1999; 6: 442–447. 2001; 69: 315–326. 37 Grillot-Courvalin C, Goussard S, Courvalin P. Wild-type 15 Grimes BR et al. Stable gene expression from a human artificial intracellular bacteria deliver DNA into mammalian cells. Cell chromosome. EMBO Rep 2001; 21: 910–914. Microbiol 2002; 4: 177–186. 16 Ikeno M et al. Generation of human artificial chromosomes 38 Rudd MK, Mays RW, Schwartz S, Willard HF. Human artificial expressing naturally controlled guanosine triphosphate cyclohy- chromosomes with alpha satellite-based de novo centromeres drolase I gene. Genes Cell 2002; 7: 1021–1032. show increased frequency of nondisjunction and anaphase lag. 17 Wade-Martins R et al. An infectious transfer and expression Mol Cell Biol 2003; 23: 7689–7697. system for genomic DNA loci in human and mouse cells. Nat 39 Zielenski J et al. Genomic DNA sequence of the cystic fibrosis Biotechnol 2001; 19: 1067–1070. transmembrane conductance regulator (CFTR) gene. Genomics 18 Magin-Lachmann C et al. In vitro and in vivo delivery of intact 1991; 10: 214–228. BAC DNA – comparison of different methods. J Gen Med 2004; 6: 40 Brune W, Me´nard C, Heesemann J, Koszinowski UH. A 195–209. ribonucleotide reductase homolog of cytomegalovirus and 19 Courvalin P, Goussard S, Grillot-Courvalin C. Gene transfer endothelial cell tropism. Science 2001; 291: 303–305. from bacteria to mammalian cells. CR Acad Sci 1995; 318: 41 Archidiacano N et al. Comparative mapping of human alphoid 1207–1212. sequences in great apes using fluorescence in situ hybridization. 20 Grillot-Courvalin C et al. Functional gene transfer from Genomics 1995; 25: 477–484. intracellular bacteria to mammalian cells. Nat Biotechnol 1998; 42 Hulsebos TJ et al. Assignment of the beta B1 crystallin gene 16: 862–866. (CRYBB1) to human chromosome 22 and mouse chromosome 5. 21 Narayanan K, Warburton PE. DNA modification and functional Genomics 1995; 29: 712–718. delivery into human cells using Escherichia coli DH10B. Nucleic 43 Grant SGN, Jessee J, Bloom FR, Hanahan D. Differential plasmid Acids Res 2003; 31: e51. rescue from transgenic mouse into Escherichia coli 22 Laner A, Schwarz T, Christan S, Schindelhauer D. Suitability methylation-restriction mutants. Genetics 1990; 87: 4645–4649. of a CMV/EGFP cassette to monitor stable expression from 44 Schindelhauer D, Cooke HJ. Efficient combination of large DNA human artificial chromosomes but not transient transfer in vitro: in gel site specific recombination (IGSSR) of PAC in the cells forming viable clones. Cytogenet Genome Res 2004; fragments containing alpha satellite DNA and the human HPRT 107: 9–13. gene locus. Nucleic Acids Res 1997; 25: 2241–2243.

Gene Therapy Bacterial transfer into human cells A Laner et al 1572 45 Warburton PE, Willard HF. Genomic analysis of sequence severity of pulmonary disease in cystic fibrosis. Am J Respir Cell variation in tandemly repeated DNA. Evidence for localized Mol Biol 2002; 27: 619–627. homogeneous sequence domains within arrays of a-satellite 48 Mickle JE et al. A mutation in the cystic fibrosis transmembrane DNA. J Mol Biol 1990; 216: 3–16. conductance regulator gene associated with elevated sweat 46 Ramalho AS et al. Methods for RNA extraction, cDNA chloride concentrations in the absence of cystic fibrosis. Hum Mol preparation and analysis of CFTR transcripts. J Cystic Fibrosis Genet 1998; 7: 729–735. 2004; 3: 11–15. 49 Chalkley G, Harris AA. Lymphocyte mRNA as a resource for 47 Ramalho AS et al. Five percent of normal cystic fibrosis detection of mutations and polymorphisms in the CF gene. J Med transmembrane conductance regulator mRNA ameliorates the Genet 1991; 28: 777–780.

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