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Replicating Minicircles: Overcoming the Limitations of Transient and Of Replicating Minicircles: Overcoming the Limitations of Transient and of Stable Expression Systems In "Minicircle and Plasmid DNA Vectors - The Future of non-viral and viral Gene-Transfer", Schleef (Ed.) Wiley-VCH Verlag K. Nehlsen 1) , S. Broll 1,2), R. Kandimalla 3), N. Heinz 4), M. Heine 5), S. Binius 1) , A. Schambach 4) and J. Bode4*) 1) Helmholtz Center for Infection Research, Department Molecular Biotechnology, Inhoffenstraße 7, D-38124 Braunschweig 2) Leibniz Universität Hannover, Dezernat 4 - Forschung und Technologietransfer / Nationale Forschungsförderung. 3) Department of Pathology, Josephine Nefkens Institute Erasmus MC 3000 CA, Dr. Molewaterplein 50 Rotterdam, The Netherlands Germany 4) Hannover Medical School (MHH), Carl-Neuberg-Strasse 1, D-30625 Hannover, Institute for Experimental Haematology OE 6960, Room J11 01 6530; Tel.: +49 511-532-5136; Fax: +49 3212 106 7542; [email protected] ; *) Corresponding Author 5) Rentschler Biotechnologie GmbH Erwin-Rentschler-Straße 21, 88471 Laupheim Keywords: minicircles; nonviral episomes; ARS assay; oriP; S/MAR Abbreviations used: BPV, bovine papillomavirus; BUR, DNA base-unpairing region; CHO, Chinese hamster ovary; CUE Core-Unpairing Element; CS, constitutive S/MAR; DS, dyad symmetry element; EBV, Epstein–Barr virus; eGFP, enhanced green fluorescent protein; egfp , the corresponding coding region; FACS, fluorescence-activated cell sorting; FISH, fluorescence in situ hybridization; Flp, flippase (site specific recombinase); FR, Family of Repeats (OriP); FRT , Flp- recognition target; GANC, ganciclovir; GOI, gene of interest; GOD, gene on duty; HDACi, histone deacetylase inhibitor; HMT, histone-methyltransferase; IR, initiator of replication; IRE, inverted repeat, LRT, long terminal repeat; LUC, luciferase; MC, minicircle; MP, miniplasmid; Ori, origin of replication; MRE, mirror-repeat; Ori, origin of replication; OriP, origin of plasmid replication; ORC, origin-recognition complex; PD, population doubling; pEpi, plasmid-episomal; pFAR, plasmid free of antibiotic resistance genes; PP, parental plasmid / educt for MC preparation; RMCE, (Flp- )recombinase-mediated cassette exchange; S/MAR, scaffold/matrix attachment region; SIDD, stress-induced duplex destabilization; SV40, simian virus 40; UE, DNA Unpairing Element; TIC, teratoma-initiating cell. 1 ABSTRACT A - Gene therapy: Call for new vector vehicles • Nonviral vectors avoiding genomic disturbances • Independent expression units: chromatin domains o S/MARs: a unifying principle o S/MAR actions are multifold and context-dependent o Stress-induced duplex destabilization (SIDD), a unifying property of S/MARs o Chromosome-based expression strategies: Episomes and/or predetermined integration sites (RMCE) B - Replicating nonviral episomes • Can the yeast-ARS principle be verified for mammalian cells? • ARS and S/MARs: common (SIDD-) properties • S/MAR plasmids: verification of the concept o Transcription into the S/MAR: directionality and rate o Cell and nuclear permeation ° Transduction principles o Nuclear association sites o RMCE-based elaboration following establishment • Remaining shortcomings and their solution o Establishment and maintenance: the EBV paradigm ° Complementarity of “molecular glue” and initiator of replication (IR-) functions ° Two variants of the L1 transposon system ° Can replication-support elements be shuffled between the EBNA1- and S/MAR vectors? ° Selection principles overcoming the need of antibiotics ° Targets for DNA methylation: role of CpGs ° pEPIto o Vector-size limitations (?) C - Minimalization approaches • Oligomerizing S/MAR modules: pMARS and its properties • Replicating minicircles, a solution with great promise o Establishment and maintenance parameters o Clonal behavior o Bi-MC systems o MC-size reduction: “In vivo evolution” o Transcriptional termination and polyadenylation: an intricate interplay o Episomal status: Proof and persistence • Emerging extensions and refinements o Combination of excision- and RMCE-strategies o MC withdrawal at will o Pronuclear injection and somatic cell nuclear transfer o From cells to organs SUMMARY AND OUTLOOK 2 ABSTRACT Based on a 2 kb S/MAR- (Scaffold/Matrix Attachment Region) element, the first nonviral autonomously replicating nonviral episome could be introduced in 1999. S/MAR-binding proteins such as SAF-A/hnRNP-U were shown to act as „molecular glue” to provide maintenance functions. These actions enabled the association with replication factories of the host cell and thereby a once-per-cell-cycle replication of the supercoiled DNA circles. In case of the plasmid episome the requirement of a selection agent for its establishment, its continued silencing, and a limited cloning capacity remained the limiting parameters until 2006, when these restrictions could be overcome by deleting the prokaryotic vector backbone. The remaining ~4 kb ´minicircle´ (“MC”, later reduced to a ~3 kb derivative, “M18”), consists of only one active transcription unit in addition to the S/MAR and is devoid of prokaryotic CpGs. In contrast to the “parental plasmid” precursors (PPs) it can be established in the absence of drug selection, and it replicates stably without signs of integration. Other than conventional minicircles that are maintained only in non-dividing tissues, this is the first example suitable for the modification of dividing cells due to its authentic segregation. Supported by its minimized size, and in accord with the “pFAR”-principle, the vector is no target for epigenetic defense mechanisms; after its establishment it is efficiently retained in the host cell nucleus. Stable clones can be derived, stored for subsequent purposes and used to generate cell lines with predictable characteristics. In addition, several MCs can be established side-by side allowing the regulated expression of multi-subunit proteins. While the minicircle preparation process could continuously be refined in various cooperations, MC generation has also become possible in situ , i.e. in the recipient cell itself. At present this "all-in-one” concept mainly serves exploratory purposes to pre-select suitable candidates for MC production routines leading to MCs of unprecedented purity and and with an authentic superhelical (ccc-)status. 3 A – GENE THERAPY: CALL FOR NEW VECTOR VEHICLES General problems that have hampered gene therapy approaches concern the inability of targeting vectors to appropriate genomic sites. Such an option would guarantee adequate gene expression, and tolerance by the host. In the absence of certain drawbacks viruses might be the preferred systems. Although they have the natural inclination to invade human cells and to deposit their genome in highly expressed loci their cloning capacity is usually restricted while their preparation is demanding and evaluation is laborious. For retroviruses (except the genus Lentiviridae) gene transfer is restricted to dividing cells and expression is difficult to maintain over extended times. To circumvent unanticipated complications of this kind chromosomal organization principles gain increasing attention for an appropriate design of second generation nonviral “chromosome-based vectors” [1]. • Nonviral vectors avoiding genomic disturbances In this field the limited performance and shutdown of conventional transgene expression units are important limitations that have to be overcome for many potential gene therapy applications [2,3]. Until recently, virtually all stable transfection procedures involved the transfer of linearized DNA. The integration of these specimens depends on the eventual occurrence of a genomic break in processes that are often associated with unpredictable rearrangements due to cell-intrinsic nonhomologous end-joining (NHEJ-) related repair activities. Silencing phenomena have been attributed to host defense mechanisms directed against the bacterial backbone of traditional vectors that include elements such as unmethylated CpG motifs [2], a prokaryotic origin of replication and antibiotic resistance genes [3]. While these sequences are required for the production of plasmid DNA (pDNA), each raises serious biological safety problems due to the dissemination of antibiotic resistance genes via horizontal gene transfer and a residual activity of bacterial genes in the recipient [4]. This becomes particularly obvious in animal models for which intramuscular injections of pDNA raise immune responses. The corresponding findings led regulatory agencies to restrict the co-transfer of these components, especially antibiotic resistance 4 markers. These facts have motivated developments considering the organization of vector backbones into host-like chromatin structures [5-10]. • Independent expression units: chromatin domains Eukaryotic chromosomes are organized into a series of discrete higher order chromatin domains, each of which is delimited by two boundary elements, so called scaffold/matrix attachment regions (S/MARs; Fig. 1). These S/MARs associate with ubiquitous protein components of the nuclear skeleton (listed in Fig. 1B), most prominently complexes of scaffold attachment factor A (SAF-A), which form the base of a chromatin loop creating independent units of gene activity [10]. S/MARs, a unifying principle Naked transgenes are known to preferentially integrate into heterochromatic areas [11]. However, if transfected as a domain, (S/MAR1 – GOI – S/MAR2) , the resulting clones show elevated, comparable expression levels that are maintained for extended periods of time [12]. This effect
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