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Human Artificial Chromosome (HAC) Vector with a Conditional Centromere for Correction of Genetic Deficiencies in Human Cells

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Human Artificial Chromosome (HAC) Vector with a Conditional Centromere for Correction of Genetic Deficiencies in Human Cells Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells Jung-Hyun Kima, Artem Kononenkoa, Indri Erliandria, Tae-Aug Kimb, Megumi Nakanoc, Yuichi Iidad, J. Carl Barrette, Mitsuo Oshimurad, Hiroshi Masumotoc, William C. Earnshawf, Vladimir Larionova, and Natalay Kouprinaa,1 Laboratories of aMolecular Pharmacology and bTumor and Stem Cell Biology, National Cancer Institute, Bethesda, MD 20892; cKazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan; dInstitute of Regenerative Medicine and Biofunction, Tottori University, Tottori 683-8503, Japan; eTranslational Sciences for Oncology Innovative Medicine, AstraZeneca, Boston, MA 02451; and fWellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH14 4AS, Scotland Edited* by John Carbon, University of California, Santa Barbara, CA, and approved October 27, 2011 (received for review September 6, 2011) Human artificial chromosome (HAC)-based vectors offer a promis- 1 to 3 Mb (7, 8, 10, 19). Several groups have reported successful ing system for delivery and expression of full-length human genes expression of full-length genes in HACs (9–11, 20, 21). However, in of any size. HACs avoid the limited cloning capacity, lack of copy most cases, genes to be expressed were cotransfected with an number control, and insertional mutagenesis caused by integra- alphoid DNA array into human cells and were incorporated into tion into host chromosomes that plague viral vectors. We pre- the forming HAC in vivo. Therefore, gene copy number and lo- viously described a synthetic HAC that can be easily eliminated cation of the gene on the HAC were not predetermined. These from cell populations by inactivation of its conditional kineto- factors greatly limited the application of de novo-generated HACs chore. Here, we demonstrate the utility of this HAC, which has as gene delivery vectors. a unique gene acceptor site, for delivery of full-length genes and Recently, this limitation was overcome by construction of an alphoidtetO-HAC carrying loxP sites (22, 23). This circular HAC correction of genetic deficiencies in human cells. A battery of contains approximately 6,000 copies of the tetracycline operator functional tests was performed to demonstrate expression of NBS1 VHL (tetO) sequence embedded in a synthetic alphoid DNA array. and genes from the HAC at physiological levels. We also Although the HAC is mitotically stable, tethering certain tetra- show that phenotypes arising from stable gene expression can be cycline repressor fusion proteins (tetR-KAP1 or tTS) (22, 24) “ ” reversed when cells are cured of the HAC by inactivating its inactivates the centromere, resulting in HAC loss. Thus, the kinetochore in proliferating cell populations, a feature that pro- alphoidtetO-HAC and any genes it carries can be eliminated from vides a control for phenotypic changes attributed to expression of cell populations, allowing for a regulated “hit-and-run” induction HAC-encoded genes. This generation of human artificial chromo- of phenotypic changes. somes should be suitable for studies of gene function and To minimize problems caused by the lack of certain genes from therapeutic applications. libraries of BAC and YAC clones or the presence of genes fragmented on multiple vectors, we developed a method for di- gene delivery vector | gene therapy | transformation-associated rect gene cloning from genomic DNA. Transformation-associated recombination cloning recombination (TAR) cloning allows isolation of any specificallele of a gene of interest as a predetermined DNA fragment (25, 26). Here, we describe the combination of the TAR gene-cloning ost advanced current gene therapy systems, such as ade- tetO Mnovirus-, lentivirus-, and retrovirus-derived vectors, use technology with the alphoid -HAC vector for gene delivery. We cDNA or “minigene” constructs (reviewed in refs. 1–5) that report a complete cycle starting with selective gene isolation, followed by gene loading into the HAC, and eventually leading to cannot recapitulate the physiological regulation of complex en- fi dogenous loci. Viral episomal vectors carrying HSV-1 and EBV complementation of gene de ciencies in a human cell line (Fig. 1). amplicons can deliver and express full-length genes as large as This approach is useful for studies of gene function and potentially for gene therapy. As a proof of principle, genomic copies of two approximately 150 kb in size (1, 2). However, several concerns —VHL limit the use of HSV-1 and EBV viral vectors: absence of strong average-size cancer-associated genes mutated in von Hip- pel–Lindau syndrome (VHL; MIM 193300) and NBS1 mutated in copy number control, undesired immunological responses, and — occasional integration into the host genome, causing insertional Nijmegen breakage syndrome (NBS; MIM 251260) were iso- lated by TAR cloning and loaded into the unique loxP site of the mutagenesis and gene silencing (6). tetO fi alphoid -HAC in CHO cells. HAC transfer into human cells Human arti cial chromosomes (HACs) represent another ex- fi NBS1 VHL trachromosomal gene delivery and gene expression vector system de cient in and reveals that both genes are expressed (7–11). Although this technology is less advanced than virus- normally and complement the defective endogenous alleles. derived vectors, HACs have several potential advantages over Results currently used episomal viral vectors for gene therapy applica- tions. First, the presence of a functional centromere provides Isolation of Genomic Regions Containing NBS1 and VHL Genes from long-term stable maintenance of HACs as single-copy episomes Human Genome by TAR Cloning. The TAR cloning scheme for without integration to the host chromosomes. Second, there is no isolating the NBS1 and VHL genes is shown in Fig. 1A. For upper size limit to DNA cloned in a HAC: entire genomic loci with all regulatory elements can be used. Finally, HAC vectors minimize adverse host immunogenic responses and the risk of Author contributions: J.-H.K., T.-A.K., H.M., V.L., and N.K. designed research; J.-H.K., A.K., I.E., M.N., Y.I., and N.K. performed research; T.-A.K., J.C.B., M.O., H.M., W.C.E., V.L., and N.K. cellular transformation. analyzed data; and N.K. wrote the paper. Recent advances have produced a variety of HACs via two fl different approaches. The “top-down” approach involves modi- The authors declare no con ict of interest. fication of human chromosomes in living cells to produce chro- *This Direct Submission article had a prearranged editor. mosome derivatives (11–18). The “bottom-up” approach involves Freely available online through the PNAS open access option. de novo kinetochore assembly from 50- to 100-kb blocks of 1To whom correspondence should be addressed. E-mail: [email protected]. centromeric alpha-satellite (i.e., alphoid) DNAs. These are multi- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. merized in human cells, forming HACs with sizes of approximately 1073/pnas.1114483108/-/DCSupplemental. 20048–20053 | PNAS | December 13, 2011 | vol. 108 | no. 50 www.pnas.org/cgi/doi/10.1073/pnas.1114483108 Downloaded by guest on September 28, 2021 cloning purposes, TAR vectors were designed containing short clones contain all exons (Fig. 1 F and H) and did not reveal any targeting hooks specificto5′ and 3′ sequence ends of the genes structural rearrangements. Analysis of the NBS1 genomic segments (SI Materials and Methods). Human genomic DNA was trans- is shown in Fig. S1. formed into yeast spheroplasts along with linearized TAR vectors. Recombination between the hooks and genomic DNA fragments Conversion of NBS1- and VHL-YACs into BACs with a LoxP Cassette for tetO results in isolation of NBS1 and VHL genes as circular YACs with Gene Loading into Alphoid -HAC. A yeast-bacteria-mammalian fi insert sizes of approximately 55 kb and 25 kb, respectively. Five in- cell shuttle vector, pJBRV1, was constructed to retro t YAC dependent TAR YAC isolates were obtained for each gene. The gene isolates into YAC/BACs (Figs. S2 and S3). The vector NBS1 contains a 3′ HPRT-loxP-eGFP cassette, allowing gene loading cloned locus contains approximately 5 kb sequence upstream tetO of the ATG codon and 1.5 kb sequence downstream of the stop into a unique loxP site of the alphoid -HAC in CHO cells. An F′ factor origin of replication allows YAC propagation as a BAC codon. The cloned VHL locus contains approximately 10 kb se- molecule. Conversion of the YAC into a BAC is advantageous quence upstream of the ATG codon and 7.3 kb sequence down- because purification of circular DNA molecules is much easier stream of the stop codon. PCR analysis and physical char- Escherichia coli fi from than from yeast cells. The protocol for acterization of the TAR isolates for each gene con rmed that the retrofitting is shown in Fig. 1B and Fig. S2. Integrity of NBS1- and VHL-containing BACs was confirmed by PCR amplification of all exons (Fig. 1 G and I) and analysis of their inserts by pulse- gel electrophoresis (Fig. S1D). Loading of NBS1 and VHL Genes into AlphoidtetO-HAC by Cre-loxP– Mediated Recombination in CHO Cells. The alphoidtetO-HAC with a unique gene loading site was used for this purpose. This HAC was identified among several HAC clones carrying a loxP cassette (s) (23) by Southern blot hybridization. To insert genomic copies of the NBS1 and VHL genes into the alphoidtetO-HAC, the ap- propriate BAC constructs and a Cre-recombinase expression vec- tor were cotransfected into hprt-minus CHO cells carrying the HAC and HPRT-plus colonies were selected on HAT medium GENETICS (Fig. 1C). Insertion of genes into the HAC was confirmed by PCR by using specific primers (Table S1) to detect reconstitution of the HPRT gene, which accompanies Cre/Lox targeting. FISH images of alphoidtetO-HAC/NBS1 and alphoidtetO-HAC/VHL are shown in Fig.
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