Gene Therapy (2004) 11, 1195–1204 & 2004 Nature Publishing Group All rights reserved 0969-7128/04 $30.00 www.nature.com/gt RESEARCH ARTICLE Infectious delivery of the 132 kb CDKN2A/CDKN2B genomic DNA region results in correctly spliced gene expression and growth suppression in glioma cells

R Inoue, KA Moghaddam, M Ranasinghe, Y Saeki, EA Chiocca and R Wade-Martins1 Molecular Neuro-Oncology Laboratories, Neurosurgery Service, Massachusetts General Hospital-East and Harvard Medical School, Charlestown, MA, USA

The expression of genes from genomic loci can be relatively these proteins are expressed from overlapping genes, complex, utilizing exonic, intronic and flanking sequences utilizing alternative splicing and promoter usage. The to regulate tissue and developmental specificity. Infectious delivered locus expresses each gene at physiological levels bacterial artificial chromosomes (iBACs) have been shown to and cellular responses (apoptosis versus growth arrest) deliver and express large genomic loci (up to 135 kb) into occur dependent on cellular p53 status, as expected. The primary cells for functional analyses. The delivery of large work further demonstrates the potential of the iBAC system genomic DNA inserts allows the expression of complex loci for the delivery of genomic loci whose expression is mediated and of multiple splice variants. Herein, we demonstrate for by complex splicing and promoter usage both for gene the first time that an iBAC will deliver and correctly express in therapy applications and functional genomics studies. human glioma cells the entire CDKN2A/CDKN2B genomic Gene Therapy (2004) 11, 1195–1204. doi:10.1038/ region, which encodes for at least three important cell-cycle sj.gt.3302284; Published online 27 May 2004 regulatory proteins (p16INK4a, p14ARF and p15INK4b). Two of

Keywords: infectious genomic locus transfer; HSV-1 amplicon; CDKN2; p16INK4a; p14ARF; p15INK4b

Introduction maximum of 30 kilobases (kb),9 a value that is less than the size of the average human genomic locus (see The complexity of gene expression includes alternative http://www.ensembl.org/Homo_sapiens/martview). splicing mechanisms,1 alternative promoter–enhancer In this respect, we have recently developed the usage,2 intronic gene expression,3 expression of inhibit- infectious bacterial artificial chromosome (iBAC) method ing RNAs,4 and effects of DNA polymorphisms.5 Such that allows for the efficient delivery into primary cells of mechanisms likely account for the large diversity of genomic loci up to B150 kb, a size that should permit proteins (greater than 100 000 per cell) derived from a expression of 95% of all human genes from their genomic relatively small number of human genes (around 30 000) loci (http://www.ensembl.org/Homo_sapiens/martview). and for their expression with tissue and developmental The iBAC takes advantage of the packaging capacity specificity.6–8 Despite the increasing appreciation of the of herpes simplex virus type 1 (approximately 150– subtle complexities of gene expression, most current 160 kb)10,11 and of recent advances in packaging of foreign gene therapy viral vector systems employ cDNA or DNA into the virus in a ‘gutless’ manner without helper ‘minigene’ constructs that cannot easily recapitulate the virus contamination.12 Genomic sequences delivered context of a genomic locus. into cells by the iBAC persist as extrachromosomal The delivery of a gene as a genomic locus would allow elements.10,11 As such, they can be easily recovered and gene expression to be controlled by the native promoter reanalyzed and/or re-engineered in bacteria, thus allow- and regulated by the endogenous enhancer and silencer ing for potentially exquisite experimental manipulations. elements in the correct genomic locus context. However, As proof-of-principle of the iBAC technology, we have expression of large foreign genomic sequences in cells previously shown that the genomic locus for human and/or animals remains inefficient with current methods hypoxanthine phosphoribosyltransferase (HPRT) con- of transfection/electroporation. The packaging constraints tained within a 115 kb BAC insert could be efficiently of most viral vectors limit the size of foreign DNA to a expressed in primary cells.10 We have further shown that transfer and expression of the human low-density lipo- protein receptor (LDLR) gene contained within a 135 kb Correspondence: Dr EA Chiocca, Molecular Neuro-Oncology Laboratories, BAC insert resulted in the expected and correct regulation Neurosurgery Service, Massachusetts General Hospital-East and Harvard of the LDLR gene promoter by sterols in primary human Medical School, Building 149, 13th Street, Charlestown MA 02129, USA 1 fibroblasts taken from familial hypercholesterolemia (FH) Current address: The Wellcome Trust Centre for Human Genetics, 11 University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK patients. This result thus showed that iBAC-delivered Received 3 October 2003; accepted 3 February 2004; published genes retained physiological regulation of gene expression, online 27 May 2004 even in their extrachromosomal environment. Infectious delivery of the CDKN2A/CDKN2B locus R Inoue et al 1196 Further validation of the iBAC method to recapitulate glioblastomas (GBMs) in particular.20–24 In fact, animal genome complexity requires demonstration of the reten- models have shown that the combined loss of INK4a and tion of alternative splicing and differential promoter ARF from neural stem cells or astrocytes predisposes to usage. The chromosomal region 9p21.3 spanning the GBM formation.25,26 Therefore, the genomic manipula- CDKN2A and CDKN2B loci demonstrates complex tions permitted by the iBAC methodology could provide splicing and alternative promoter usage by overlapping an avenue for further study of this interesting locus and genes.2,13 The region contains only six exons. Never- the development of novel therapeutic strategies. theless, it encodes five proteins involved in cell-cycle To show that the CDKN2A/CDKN2B genomic region control. The major three proteins are p16INK4a, p14ARF is correctly spliced and retains its functionality when and p15INK4b. The CDKN2A locus generates two distinct delivered as a transgene, we have generated iBAC- important proteins, p16INK4a and p14ARF, from different CDKN2 vectors. We report that glioma cells, originally promoters, both of which function in the cell cycle to deleted for the CDKN2A/CDKN2B locus, show expres- regulate proliferation and apoptosis.14 The generation of sion of p16INK4a, p14ARF and p15INK4b after infection with these two proteins from this locus is relatively complex. iBAC-CDKN2. Functionally, this results in an apoptotic There are two alternative first exons, 1a for p16INK4a and or growth-arrest response, dependent on cellular p53 1b for p14ARF. Each of these two first exons is spliced to status. Therefore, alternative splicing and promoter common second and third exons to make two function- usage mechanisms are retained within the context of ally different proteins with no amino-acid homology to iBAC expression. one another. The p16INK4a protein binds and inhibits Cdk4 and Cdk6, which phosphorylate the retinoblastoma protein (pRB), an event that results in G1 arrest.13 In Results contrast, p14ARF interacts directly with Mdm2 to stabilize and activate p53 to induce growth arrest and apopto- Construction of iBAC-CDKN2 vectors sis.15,16 There is also an additional splice variant of The combined CDKN2A and CDKN2B region on chro- p16INK4a, termed p12, produced specifically in the mosome 9p21.3 contains some of the most complex pancreas, in a tissue-specific manner.17 Finally, an splicing seen in the human genome.2 The region contains alternative form of p15INK4b, termed p10, is known.18,19 six exons encoding five proteins by differential promoter The 9p21.3 deletion has been reported to be one of the usage and alternative splicing, the best characterized most frequent mutations found in cancer and in human being p16INK4a, p14ARF and p15INK4b (Figure 1a). We first

Figure 1 Construction of the iBAC-CDKN2 vectors carrying the complex CDKN2A/CDKN2B genomic DNA region. (a) The CDKN2A/CDKN2B genomic region contains six exons encoding p16INK4a, p14ARF and p15INK4b expressed by differential promoter usage and alternative splicing. (b) PCR analysis of the 195 kb insert of RP11 BAC149I2 confirmed the presence of all six exons from the region. (c) Outline of the strategy for vector construction involving removing 63 kb of extreme 30 DNA by RARE cleavage followed by Cre/loxP-mediated recombination to retrofit the shortened BAC with pHG and pEHHG to produce pHSV-CDKN2 and pHSV/EBV-CDKN2, respectively. (d) PCR analysis verifies the precise nature of the RARE cleavage process and the formation of the new junction.

Gene Therapy Infectious delivery of the CDKN2A/CDKN2B locus R Inoue et al 1197 identified and characterized a fully sequenced BAC library clone from the RPCI-11 library containing the genomic region of interest (RCPI-11 149I2, Genbank AL449423). PCR analysis of the 195 kb BAC insert confirmed the presence of all six CDKN2A and CDKN2B exons encoding p16INK4a, p14ARF and p15INK4b (Figure 1b). The HSV-1 amplicon-based iBAC system has been shown to have a total packaging capacity of approxi- mately 150–160 kb.10,11 To modify the CDKN2 insert to fit within this packaging capacity, we used EcoRI RecA- assisted restriction endonuclease (RARE) cleavage27 to remove 63 kb of extreme flanking DNA 30 of the CDKN2A locus (Figure 1c). We then confirmed the precise nature of the RARE manipulation by PCR analysis of the newly formed junctions (Figure 1d). The modified CDKN2 BAC clone carrying an insert of 132 kb was then retrofitted with HSV-1 amplicon elements using a Cre/loxP-based method.10–12 The methodology exploits the presence of a single wild-type loxP site in all BAC library clones. First, retrofitting the modified 132 kb CDKN2 BAC with pHG (carrying the oris and pac elements from HSV-1) created pHSV-CDKN2, a 148 kb HSV-1 amplicon. Second, retrofitting with pEHHG (containing the episomal retention elements from Epstein–Barr virus (EBV) in addition to the oris and pac sequences) created pHSV/EBV-CDKN2, a 154 kb HSV-1/EBV hybrid amplicon. Lastly, we reconfirmed the presence of all the p16INK4a, p14ARF and p15INK4b exons in the final pHSV-CDKN2 and pHSV/EBV-CDKN2 constructs (not shown).

Characterization of the CDKN2A/CDKN2B region within human glioma cells We wished to identify null cell lines lacking the CDKN2A/CDKN2B region to use as model systems to test the functional consequence of physiological levels of gene expression from our iBAC-CDKN2 vectors. To identify cell lines carrying homozygous deletions of the CDKN2A/CDKN2B region, we tested 10 glioma cell lines from the Massachusetts General Hospital Brain Tumor Bank for the presence or absence of p16INK4a, p14ARF and p15INK4b by screening for the presence of each exon by PCR. Four of the 10 glioma cell lines showed a complete absence of all six exons and presumably carry a homozygous deletion of the entire region (Figure 2a). We selected Gli-33 and Gli-36 for further study as an example of a primary and immortalized glioma line, respectively. We designed Figure 2 Characterization of CDKN2A/CDKN2B null cell lines. (a) PCR primer pairs to detect specifically expression of analysis of ten glioma cell lines for the presence of each of the six CDKN2A/CDKN2B exons. Glioma cell lines identified as carrying a each spliced gene and confirmed that the Gli-33 and homozygous deletion of the region are marked with asterisk. Detection of Gli-36 cell lines do not express any of the CDKN2 the HPRT locus was performed as a positive control for PCR; HP10 is a mRNAs, as expected (Figure 2b). human fibroblast cell line deleted for the HPRT locus. (b) RT-PCR analysis In addition, we also chose to work with the U87 confirms the Gli-33 and Gli-36 cell lines do not express p16INK4a, p14ARF glioma-immortalized cell line, which is known to have a and p15INK4b mRNA. complete deletion of the CDKN2 genomic region,28 and confirmed that U87 cells do not express any of the CDKN2A or CDKN2B mRNAs (not shown). The U87 Intact packaging and infectious delivery of the 132 kb glioma line was, however, relatively resistant to HSV-1 CDKN2A/CDKN2B region amplicon infection. To overcome this, we used a retro- We packaged the pHSV-CDKN2 and pHSV/EBV-CDKN2 viral construct expressing the herpesvirus entry protein amplicons in Vero 2-2 cells using an improved helper C (HveC)29 to create a highly infectable HveC þ -U87 virus-free HSV-1 amplicon packaging system,12 as derivative cell line, hereafter referred to simply as U87 described previously.10,11 Amplicon titers were measured (see Materials and methods for details). as green fluorescent protein (GFP)-transducing units on

Gene Therapy Infectious delivery of the CDKN2A/CDKN2B locus R Inoue et al 1198 G16-9 cells, a derivative of the human glioma cell line Gli-36 (see Materials and methods). Amplicon titers of B2–3 Â 107 transducing units per milliliter (t.u./ml) were routinely obtained after concentration. pHG and pEHHG were also packaged and used as vector-only control amplicons in CDKN2A and CDKN2B expression analysis. To demonstrate intact infectious delivery of the 132 kb CDKN2A/CDKN2B genomic region by both pHSV- CDKN2 and pHSV/EBV-CDKN2, we infected the human glioma cell line U87 with each amplicon. At 48 h after infection, total genomic DNA was prepared from the infected cells and a small aliquot was used to transform DH-10B Escherichia coli cells by electroporation. The HSV-1 amplicon DNA is linear inside the virion, but recircularizes upon entry into the nucleus. The plasmid rescue assay shuttles recircularizes BAC plasmid from the infected U87 cells back into the bacterial host. After selection of transformed bacteria on chloramphenicol/ ampicillin plates, we prepared plasmid DNA from surviving colonies. The plasmid DNA was digested by NotI, analyzed by pulsed-field gel electrophoresis and compared to the original starting DNA preparation used for packaging (Figure 3a and b). The 148 kb pHSV- CDKN2 and the 154 kb pHSV/EBV-CDKN2 were both found to be delivered to and rescued intact from U87 cells with 80% efficiency. This high level of intact delivery mediated by the iBAC system is in-line with previous reports.10,11

iBAC-CDKN2 infectious delivery leads to correctly spliced gene expression We then infected glioma cell lines null for the CDKN2A/ CDKN2B region with the pHSV-CDKN2 and pHSV/EBV- CDKN2 vectors to assay gene expression and function. Levels of infection efficiency of B50% were readily obtained with a multiplicity of infection (MOI) of 3. Using RT-PCR primers carefully designed to differentiate between expressions of each gene, we then assayed the infected cells for expression of p16INK4a, p14ARF and p15INK4b (Figure 4). Each of the three genes was correctly expressed and spliced after delivery with each iBAC- CDKN2 vector. Moreover, the RT-PCR results indicate the levels of p16INK4a, p14ARF and p15INK4b expression were approximately equal to those of normal human fibroblasts, when the efficiency of vector delivery (B50%) is taken into account. This confirms that iBAC- mediated delivery of CDKN2 resulted in broadly physiological levels of re-expression of these three genes in glioma cells that were previously deleted for this Figure 3 pHSV-CDKN2 and pHSV/EBV-CDKN2 transfer an intact genomic region, consistent with our previous expression 132 kb genomic DNA insert by infectious delivery in U87 glioma cells. data for BAC-based vectors.10,11,30 Plasmid rescue of (a) pHSV-CDKN2 and (b) pHSV/EBV-CDKN2 demonstrates intact delivery of both vectors at an efficiency of 80%. Plasmid DNA was prepared from bacterial colonies from each rescue Glioma cell growth inhibition following iBAC-CDKN2 assay, digested with NotI and analyzed by PFGE. Plasmids (16/20 and infectious delivery 8/10) rescued represent intact pHSV-CDKN2 and pHSV/EBV-CDKN2, respectively. Following the demonstration of correct expression of the three known cell-cycle inhibition genes from the CDKN2A/CDKN2B loci, we examined the effect of iBAC-CDKN2 gene expression on tumor cell growth in pHG or pEHHG control amplicons showed no difference vitro. The immortalized tumor cell lines Gli-36 and U87 in growth from uninfected cells, whereas cells infected were infected with the pHSV-CDKN2 and pHSV/EBV- with pHSV-CDKN2 or pHSV/EBV-CDKN2 showed sig- CDKN2 vectors and the control pHG and pEHHG nificant inhibition of cell growth by day 3 (Po0.01 by amplicons at the same MOI. Cell growth was then Student’s t-test; Figure 5a and b). We then extended this followed using the WST growth assay over 3 days for result to a primary tumor line by performing a pHSV/ Gli-36 and 5 days for U87. Glioma cells infected with the EBV-CDKN2 infection of the Gli-33 cells that resulted in

Gene Therapy Infectious delivery of the CDKN2A/CDKN2B locus R Inoue et al 1199

Figure 4 iBAC-CDKN2 amplicons express multiple correctly spliced CDKN2A/CDKN2B genes following infectious delivery to tumor cells. RT-PCR analysis demonstrates correctly spliced expression of p16INK4a, p14ARF and p15INK4b mRNA in Gli-36 and U87 cells infected with iBAC-CDKN2 vectors. MRC5-V2 is an immortalized human fibroblast line used as a positive control for CDKN2 expression. GAPDH expression acts as an RT-PCR internal control.

Figure 5 Infectious delivery of iBAC-CDKN2 suppresses tumor cell growth. The upper panels show all the glioma cell lines are readily infected by iBAC- CDKN2 amplicons as judged by GFP reporter gene expression. The lower panels show cell growth at various time points as evaluated by the WST-1 colorimetric absorbance assay. All cells infected with pHSV-CDKN2 and pHSV/EBV-CDKN2 showed a significantly reduced growth by day 3 for Gli-36 (Po0.01 by Student’s t-test), day 3 for U87 (Po0.01) and day 5 for Gli-33 (Po0.01), respectively. Data at each time point represents the mean of three wells. significant growth repression by day 5, again not seen in firefly to renilla luciferase expression for Gli-36, U87 a control pEHHG infection (Figure 5c). and Saos2 were 0.318, 27.76 and 0.149, respectively. The data confirmed previous reports that U87 cells express functional p5332 and indicated that Gli-36 cells do not Analysis of apoptosis in glioma cells infected express functional p53 protein. with iBAC-CDKN2 amplicons We therefore used iBAC-CDKN2 infection of the Gli-36 To understand further the mechanisms leading to the and U87 lines to compare the mechanisms of growth inhibition of cell proliferation following infectious repression of p53-negative and p53 wild-type cells in delivery of the iBAC-CDKN2 vectors, we assayed the response to the combined expression of physiologically p53 status of the immortalized tumor lines Gli-36 and relevant levels p16INK4a, p14ARF and p15INK4b. We looked U87 in our cultures. Gli-36 and U87 cells were cotrans- for evidence of apoptosis in infected glioma cells. Gli-36 fected with a plasmid containing the firefly luciferase and U87 cells were infected with iBAC-CDKN2 vectors reporter gene controlled by a p53-responsive element or empty control amplicons at an MOI of 10 and cellular and a plasmid expressing the renilla luciferase reporter morphology was observed following infection. At 3 days gene driven by the thymidine kinase promoter. Cotrans- postinfection, U87 cells infected with CDKN2 amplicon fection of the two plasmids into the human osteosarcoma were sparse, aggregated and rounded up, suggestive cell line, Saos2, which is known to have both p53 alleles of cells undergoing apoptosis, whereas cells infected deleted,31 served as a negative control. The ratios of with empty amplicons grew rapidly and showed no

Gene Therapy Infectious delivery of the CDKN2A/CDKN2B locus R Inoue et al 1200 morphological changes (Figure 6a). Furthermore, we strated apoptosis in U87 cells (Figure 7). The traces in performed TUNEL analysis 2 days following infection. A Figure 7a clearly show an increased number of U87 cells high proportion of U87 cells infected with iBAC-CDKN2 in the pre-G1 (sub-G0) phase following infection with amplicons showed positive TUNEL staining indicating pHSV-CDKN2 and pHSV/EBV-CDKN2 as compared to cells undergoing apoptosis, whereas U87 cells infected pHG and pEHHG at 48 and 72 h postinfection. The data with control amplicons showed negative TUNEL stain- are represented graphically in Figure 7b and show ing (Figure 6b). In contrast Gli-36 cells infected with the increase of cells in the pre-G1 phase to be highly iBAC-CDKN2 did not produce positive TUNEL staining significant by Student’s t-test (Po0.01). Again, flow (not shown), as expected from their absence of functional cytometric analysis of Gli-36 did not show an increase in p53. the number of cells in the pre-G1 phase, but showed Flow cytometric analysis of cell-cycle progression of evidence for G1 arrest (not shown), as expected from infected cells stained with Hoechst 23342 also demon- their absence of functional p53. Taken together, these

Figure 6 iBAC-CDKN2 amplicon infection leads to cell growth suppression and apoptosis in p53 wild-type U87 tumor cells 48–72 h postinfection. (a) At 3 days postinfection, U87 cells infected with pHSV-CDKN2 and pHSV/EBV-CDKN2 show a marked reduction in cell growth as compared to cells infected with pHG and pEHHG at the same MOI. (b) TUNEL staining of infected U87 cells 2 days postinfection shows U87 cells undergoing apoptosis after infection with pHSV-CDKN2 and pHSV/EBV-CDKN2. pHG and pEHHG infections used as negative controls show no sign of apoptosis. The positive control for apoptosis detection by TUNEL consists of cells treated with DNase I to cause DNA fragmentation and the negative control omits the TdT enzyme.

Gene Therapy Infectious delivery of the CDKN2A/CDKN2B locus R Inoue et al 1201

Figure 7 Cell-cycle analysis of infected U87 cells by flow cytometry. (a) Analysis of the pre-G1 (sub-G0) peak in infected U87 cells shows an increased number of cells in the pre-G1 phase following infection by pHSV-CDKN2 and pHSV/EBV-CDKN2 compared to infection by pHG and pEHHG. Cells were stained with the nuclear staining agent Hoechst 23342 48 and 72 h postinfection. (b) Quantitation of the flow cytometry data demonstrates pHSV-CDKN2 and pHSV/EBV-CDKN2 infection of U87 cells leads to a significant increase in the percentage of cells in the pre-G1 phase as compared to pHG and pEHHG infection both 48 and 72 h postinfection. Means are from three independent infections; error bars are þ 1 s.e.m. **Po0.01 by Student’s t-test.

data show that restoration of physiological levels of gene the delivery of one locus. In this report, for the first time, expression from the CDKN2A and CDKN2B suppresses we have demonstrated that iBAC-mediated gene deliv- cell growth in glioma cells, but induces apoptosis only in ery and expression leads to functionally correct complex cells with functional p53. splicing and promoter usage of 132 kb of CDKN2A/ CDKN2B genomic locus. The work further demonstrates the ability of the iBAC system to deliver a genomic locus Discussion that functions like an endogenous chromosomal region. There has been no previous report on the delivery of Recent reports have clearly demonstrated the advantages the entire CDKN2A/CDKN2B genomic locus and its gained in gene expression utilizing a genomic DNA effects on tumor suppression. Data presented in this locus over cDNA-based expression cassettes. These paper are the first demonstration of multiple genes advantages include prolonged vector retention and gene exhibiting complex splicing being expressed simultaneously expression,10,30,33,34 correct native promoter regulation,11 from a large genomic DNA insert following infectious and, now, the expression of multiple splice variants from delivery. The correction of genetic abnormalities in

Gene Therapy Infectious delivery of the CDKN2A/CDKN2B locus R Inoue et al 1202 human neoplasm is an interesting strategy to cure or to analysis. In contrast, p53-mutant Gli-36 cells underwent control cell growth. Human gliomas, especially glioblas- growth arrest after infectious delivery of CDKN2 tomas, are one of the most difficult tumors in human to genomic region without a significant increase in apop- treat effectively because of their location within the brain tosis. In the Gli-36 cells, thus, expression of p14ARF in the and invasiveness into brain parenchyma. Such tumors absence of functional p53 did not result in a apoptotic remain untreatable despite a combination of surgery, response. These findings are in agreement with the radiation and chemotherapy.35–37 Recent genetic analyses overexpression studies,42,44 and thus confirm the general of human gliomas have identified them as a disease mechanism of cell response to recorrection of p16INK4a, caused by the accumulation of genetic mutations, p14ARF and p15INK4b expression and, importantly for our including deletions or amplifications, in genes related objectives, validate the iBAC’s ability to provide pheno- to cell-cycle regulation.38,39 Among the many genes typically correct gene expression even in the context of involved in pathways of cell-cycle regulation, p16INK4a a locus with complex splicing and promoter usage and p14ARF are well known to play a critical role in cell- mechanisms. cycle progression.13 These two genes are encoded from a The work provides a basis for future studies of complex region of chromosome 9p21.3 and are tran- gene expression from this genomic region. First, the scribed from overlapping reading frames. vector system will be used to investigate potential A number of studies have been recently published therapeutic applications of simultaneous expression of describing the antitumor effects of expressing p16INK4a the three cell-cycle control genes under physiological and p14ARF in tumor cells.40–45 These reports describe control, and second the system is well suited to assess overexpression of tumor suppressor genes by viral the importance of polymorphisms, methylation events, transfer of the cDNA of interest. Such studies find that and chromatin acetylation in the maintenance of the cellular response to overexpression of p16INK4a and this region in tumors.47 Finally, we have previously p14ARF depends on the p53 status of cells.42,44 There have shown that episomal BAC vectors are highly stable been no studies that have addressed the cellular response in long-term cell cultures and are retained in dividing to re-expression of p16INK4a or p14ARF at physiological cells at B96–98% efficiency.30,48 This makes the levels, nor to the simultaneous re-expression of p16INK4a system suitable for functional genomics expression and p14ARF and p15INK4b. Restoration of expression of studies requiring long-term maintenance of unrear- physiological levels of tumor suppressor genes may have ranged exogenous genomic loci. distinct advantages compared to overexpression, as overexpression of p16 may induce aberrant differentia- tion processes.43,46 Materials and methods Using the iBAC system, the level of expression of each of the three genes (p16INK4a, p14ARF and p15INK4b) occurs Vector construction from a genomic locus, under the control of the native The fully sequenced BAC 149I2 clone (Genbank promoters, and at approximately physiologically rele- AL449423) from the RCPI-11 library was identified as vant levels when the number of infected cells is containing the complete CDKN2A/CDKN2B region by considered (see Figure 4, where iBAC expression is screening publicly available databases (http://www. compared to fibroblast expression). We use the term ncbi.nlm.gov) and was obtained from Research Genetics ‘physiological’ to refer to expression levels obtained from (Huntsville, AL, USA). EcoRI RARE cleavage was used as our vectors using endogenous promoters, which are described previously27 to remove 63 kb of flanking broadly equivalent to levels seen in wild-type cells. This sequence 30 to the CDKN2A locus by using 60-mer oligos contrasts with the high levels of gene overexpression (50-agcaagtgtattcaataGAATTCaattgaattctattctataattcaattg seen from convectional vectors in which a cDNA is ttcttaatt-30 and 50-ctaatatgtaagtca-ttttcttttccaGAATTC expressed from a strong, heterologous viral promoter. ctgctcttccagttttcactatctggg-30; EcoRI site shown in The data in this paper confirms previous work in which capitals). The retrofitting vector pHG contains the we have shown quantitative analysis of gene expression HSV-1 amplicon elements (oris and pac) and the GFP from genomic loci carried by BAC-based vectors. We reporter gene from pHSV-GFP (a gift from C Fraefel), recently showed physiological expression levels and the R6Kg bacterial replication origin from pSG76-A transcriptional regulation of the human LDLR gene from (a gift from G Posfai) and a loxP site. pEHHG contains an iBAC-LDLR vector in primary human cells.11 Previous in addition the EBV episomal retention cassette (oriP/ to that, we showed physiological expression levels of the EBNA-1/hygr) from pH300 (a gift from J-M Vos). The HPRT gene from each copy of a BAC vector using two retrofitting protocol used Cre-mediated recombination different assay measurements.10,30 Taken together, these in bacterial cells and was performed as described data demonstrate physiologically appropriate expression previously.10,12 levels from genomic loci carried by our BAC-based expression vectors. Characterization of CDKN2A/CDKN2B null cell lines We were interested to compare the functional effect of PCR of genomic DNA was used to amplify each exon in physiological levels of the expression of the tumor the CDKN2A/CDKN2B region using the following pri- suppressor genes with the reported results from over- mers pairs, listed as (forward primer; reverse primer): expression studies. The genetic status of the glioma cells p14 exon 1b (50-GAGGCG-GCGAGAACATGGTG-30;50- was important in determining the cellular response to GGGCCTTTCCTACCTGGTCT-30); p15 exon 1 (50-TC the vector-derived CDKN2A/CDKN2B locus expression. CGCGCTAGGCGCTTTTTC-30;50-GCCTCCCGAAAC We find that only glioma cells expressing p53 wild-type GGTTGACT-30); p15 exon 2 (50-CTCTCACCCGA protein, such as U87 cells, were able to undergo CCGGTGCAT-30;50-GGTGGGTGGGGGTGGGAAAT-30); apoptosis as assayed by flow cytometry and TUNEL p16 exon 1a (50-ATGGAGCCTTCGGCTGAC-TG-30;50-

Gene Therapy Infectious delivery of the CDKN2A/CDKN2B locus R Inoue et al 1203 AGGAGGTCTGTGATTACAA-ACC-30); p16 exon 2 (50- CDKN2A/CDKN2B genomic region. The following pri- CCCTGGCTCTGACCATTCTG-30;50-ACCACCAG-CGT mers pairs, listed as (forward primer; reverse primer), GTCCAGGAA-30); and p16 exon 3 (50-AAAAA- were used to detect specifically each transcript from the GAAAAACACCGCTTC-30;50-TTCCATGCGATGAAA CDKN2A/CDKN2B region: p14ARF (p14 exon 1b:50- TTGTT-30). GAGGCGGCGAGAACATGGTG-30; p14 exon 2: 50-AC CACCAGCGTGTCCAGGAA-30); p16INK4a (p16INK4a exon Tissue culture 1a:50-CCCAACGCACCGAATAGTTA-30; p16 exon 3: 50- MRC-5V2 (an SV40-immortalized human male lung TTCAATCGGGGATGTCTGCAG-30); and p15INK4b (p15 fibroblast cell line), Gli-33, Gli-36 and U87 cells were exon 1: 50-CGAGGAGAACAAGGGCATGC-30; p15 exon cultured in Dulbecco’s modified Eagle’s medium 2: 50-GCCCATCATCATGACCTGG-30). As an RT-PCR (DMEM) supplemented with 10% fetal bovine serum internal control, we analyzed GAPDH expression using (FBS), penicillin (100 U/ml) and streptomycin (100 mg/ primers (50-GGTCGGAGTCAACGGATTTGGTCG-30;50- ml) (P/S). HveC þ U87 cells were derived by infection CCTCCGACGCCTGCTTCACCAC-30). with the pBABEHveCpuro (VSV pseudotyped) retro- virus and selection of puromycin-resistant cell popula- tions as described previously.11 Vero 2-2 cells (a Analysis of cell proliferation and apoptosis derivative of Vero African green monkey kidney cells For assays of cell growth and proliferation, the colori- constitutively expressing the HSV-1 ICP27 protein) were metric WST-1-based assay was used. On day 0, 500 (Gli- grown in DMEM, 10% FBS, P/S and G418 (Geneticin; 36) or 1000 (Gli-33 and U87) cells were seeded per well LifeTechnologies, Rockville, MD, USA) at 500 mg/ml. into 96-well plate. On day 1, wells were infected with G16-9 cells (a derivative of the human Gli-36 glioma cell iBAC or control amplicons at MOI 20 (Gli-36) or 10 (Gli- line expressing the HSV-1 VP-16 protein to enhance 33 and U87) in 200 ml of DMEM supplemented with 2% expression from the amplicon pIE4/5-GFP promoter FBS and P/S. At various time points following infection, cassette (Y Saeki and EA Chiocca, unpublished materi- 10 ml of WST-1 Cell Proliferation Reagent (Roche, als)) were cultured in DMEM, supplemented with 10% Indianapolis, IN, USA) was added to the cultures and FBS, P/S and hygromycin-B (200 mg/ml). All cells were the absorbance read at 450 nm by a microplate reader. cultured at 371C in 5% (v/v) CO2. Two methods were used to measure apoptotoic cell death. First, for TUNEL staining, U87 and Gli-36 cells HSV-1 amplicon production were seeded in 4-well chamber slides, infected with HSV-1 amplicons were produced using an improved iBAC or control amplicons at the same MOI as for the HSV-1 helper virus-free system as described pre- WST assays, fixed in 2% paraformaldehyde in PBS 2 days viously.10–12 Typically, the supernatant from nine 6 cm postinfection and subjected to TUNEL staining using the dishes was concentrated by ultracentrifugation and DeadEnd Colorimetric TUNEL assay (Promega, Madi- resuspended in 500 ml Hank’s balanced salt solution to son, WI, USA). As a positive control for TUNEL staining, give a stock of 2 Â 107 transducing units/ml when titered uninfected cells were treated with DNase I, according to for GFP expression on a confluent G16-9 cell layer. the manufacturer’s protocol. Flow cytometric analysis was used as a second measure of apoptosis. At 2 and 3 Genomic analysis of infected cells days postinfection, cells were stained with Hoechst 23342 The plasmid rescue assays were performed as follows.30,48 dye solution (5 mg/ml in DMEM) for 45 min. The cells For each rescue assay 1.5 Â 105 U87 cells were seeded into were then harvested by centrifugation at 1500 rpm for a 6 cm dish. After 1 day, the cells were infected with 10 min, washed once with PBS and finally fixed in 2% amplicon vectors pHSV-CDKN2 and pHSV/EBV-CDKN2 paraformaldehyde. at an MOI of 3. After 48 h, genomic DNA was prepared from the infected cells and resuspended in 200 ml TE. DH- 10B ElectroMax E. coli cells (Invitrogen, Carlsbad, CA, USA) were electroporated with 2 ml of genomic DNA Acknowledgements extract and plated onto LB agar containing chloramphe- We thank Dr David Louis (Massachusetts General nicol (12.5 mg/ml) and ampicillin (100 mg/ml). Plasmid Hospital, Charlestown, MA, USA) for providing samples DNA was prepared from the resulting bacterial colonies, from the Massachusetts General Hospital Brain Tumor digested with NotI and the digests were resolved by Bank, for initially suggesting this experiment and for pulsed field gel electrophoresis. helpful discussion and advice. This work was supported by grants from The Wellcome Trust, the National Cancer Amplicon infection and RT-PCR expression analysis 5 4 Institute (CA692460) and the Berkowitz-Knott Fund for A total of 1 Â10 (U87) or 2.5 Â 10 (Gli-36) cells were Brain Tumor Research to EAC. RW-M was a Wellcome seeded per well of a 6-well plate. After 1 day, the cells Trust International Prize Travelling Research Fellow, and were infected with amplicons at an MOI of 3 (U87) or 10 thanks the Trust for their continued support. (Gli-36). 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Gene Therapy