Nuclear Transfer and Establishment of Replicating Nonviral Episomes (Minicircles) Sandra Broll 1 , André Oumard 1 , Markus Heine 1 , Axel Schambach 2 & Juergen Bode 1,2 INTRODUCTION While there is significant progress in the modification by episomal DNA of slowly-dividing tissues like liver, muscle and brain, maintenance problems have so far limited the use of nonviral episomes for dividing cells. For liver, the most advanced vehicles appear to be “minicircles”, small circular vectors that are exclusively composed from eukaryotic sequences. In contrast to linear DNA, minicircles are less prone to integration and, owing to their superhelical status, they are better transcriptional templates. The essential components of a minicircle derivative that replicates once per cell cycle have been defined, i.e. an active transcription unit and a S/MAR – so far the upstream bordering element from the huIFN-β chromatin domain and its derivatives. Present work explores the requirements to be met by the S/MAR in association with regulatory elements and it investigates alternative transfer routes to optimize gene expression 1 HZ I / MBIO / Epigenetic Regulation 2 MHH / HBZ / Experimental Hematology HZI, Molecular Biotechnology/ Epigenetic Regulation D - 38124 Braunschweig MHH Hannover Medical School, HBZ/ Experimental Hematologie, D - 30625 Hannover Scaffold/Matrix Attachment Regions (S/MAR) Scaffold/Matrix Attachment Regions (S/MAR) Scaffold/Matrix Attachment Regions (S/MAR) Scaffold/Matrix Attachment Regions (S/MAR) S/MARs are DNA elements 300 to several thousand bp long, typically comprising 70 % A+T. They have a marked propensity to undergo strand separation (Bode et al., 1992), which is evident from the presence of base-unpairing regions (BURs) hat can be predicted from computer-assisted programs (stress induced duplex destabilization of profiles, SIDD; Benham et al., 1997, Bode et al, 2006; cf. Fig. 8) and traced by ssDNA specific agents within the living cells. S/MARs do not have a clear-cut consensus sequence. The characteristics that define their activity are rather thought to be secondary structures enabled by their strand separation potential in addition to associated factor binding sits The transcriptional augmentation effect of S/MARs arises only after rounds of replication and is absent during the transient expression phase. It is not restricted to integrated gene copies but also valid for replicating episomes. Usually, S/MARs counteract de novo methylation and mediate histone hyperacetylation whereby they support the long term expression of a gene. For references see http://pubjbo.de.vu HEK293 Parental av. copy No Plasmid 4.1+/-2.8 7.1+/-2.6 CHO-K1 CHO-K1 wt HEK293 Minicircle av copy No. 6.7+/-3.5 2.6+/-0.9 CHO-K1 HEK293 Doublet in 40% of cells Parental Plasmids and Minicircles: Episomal Status Parental Plasmids and Minicircles: Episomal Status Reprogramming events in early embryos are reflected by circular plasmid encoded marker genes Iqbal, K. 1 , Barg-Kues, B. 1 , Broll, S. 2 , Bode., J. 2 , Niemann, H. 1 , Kues, W.A. 1 1Institute of Farm Animal Genetics, Friedrich-Loeffler-Institute, Mariensee, D-31535 Neustadt 2HZI, D-38124 Braunschweig, Germany Reprogramming events in early embryos are reflected by circular plasmid encoded marker genes Iqbal, K. 1 , Barg-Kues, B. 1 , Broll, S. 2 , Bode., J. 2 , Niemann, H. 1 , Kues, W.A. 1 1Institute of Farm Animal Genetics, Friedrich-Loeffler-Institute, Mariensee, D-31535 Neustadt 2HZI, D-38124 Braunschweig, Germany Viral Routes of MC-Establishment.... Viral Routes of MC-Establishment.... Percentage of Cells Expressing eGFP from the MC after Sorting and Butyrate-Treatment 50 60 70 80 g Cells (%) Day 0 Day 7 1 kb 293 Clone 6 Clone 7 Fig. 1: Preparation of minicircles (MC) from a parental plasmid (PP) – traditional procedure. The PP is amplified in E. coli (strain MM294Flp, a kind gift of. F. Strewart, Dresden) and processed after thermal induction of a genomic copy of flp recombinase Fig. 2: Copy number determination via FISH; Confirmation of the episomal status. 4-7 copies can be stably established per nucleus. Contrary to the PP, for which, upon G418 selection, up to 40% of cells show integration events (intense doubletts across the chromosome arms), establishment efficiencies for MCs increase with decreasing size (Nehlsen et al., 2006) 88/55 zygotes ; ; ; ; 14/13 blastocysts = 16/24% ; 10/ ; 10/ ; 10/ ; 10/3 fluorescent = 71/ fluorescent = 71/ fluorescent = 71/ fluorescent = 71/ 21 21 21 21% (cattle/ % (cattle/ % (cattle/ % (cattle/mouse mouse mouse mouse) 88/55 zygotes ; ; ; ; 14/13 blastocysts = 16/24% ; 10/ ; 10/ ; 10/ ; 10/3 fluorescent = 71/ fluorescent = 71/ fluorescent = 71/ fluorescent = 71/ 21 21 21 21% (cattle/ % (cattle/ % (cattle/ % (cattle/mouse mouse mouse mouse) LEDGF LEDGF, lens epithelium-derived growth factor Coop. Christopher Baum/ Axel Schambach (MHH) George Dixon/ Rafael Yanez (RHUL) 0 10 20 30 40 H 2O H 2O + B Clone 6 Clone 6 + B Clon 7 Clone 7 + B eGFP Expressing 1LTR 2LTR Minicircle-Transfection into Synchronized Cells Minicircle-Transfection into Synchronized Cells Percentage of eGFP Expressing Cells at the Transient/Stable Phase 0 10 20 30 40 50 60 Constructs eGFP Expressing Cells (%) 09.02.08 12.02.09 16.02.09 19.02.09 05.03.09 12.03.09 19.03.09 - sort - Fig. 3: The compact, superhelical properties of MCs triggers a transport mechanism permitting their pronuclear expression even after delivery into the cytosol of the zygote. Expression of the superhelical template is in accord with the kind of promoter and the developmental stage (Iqbal et al., 2009, submitted) Fig. 4: Retroviral life cycle. In the absence of a functional integrase the cycle terminates at the stage of a circular intermediates (2-LTR and 1-LTR circles). We have constructed retroviral genomes such that these intermediates resemble our replicating minicircles with potential benefit for gene therapeutic applications (no integration; Figure: courtesy of M. Galla, MHH Fig. 5: 2-LTR mimetics permit investigation of MC-establishment and maintenance. As a performance model 2-LTR and 1-LTR PPs have been synthetized and processed, by Flp-mediated excision, to minicircles. These MCs are replicated as their nonviral counterparts but they undergo intense silencing. The silenced state can be reversed, transiently, by histone hyperacetylation (dark green bars at day 7) Fig. 6: Parameters promoting episome establishment. Histone deacetylase inhibitors such as butyrate can be used to open chromatin structures. An obligatory consequence of this treatment is G1 arrest. As a control, therefore, cell cycle arrest was also induced by a confluence block in order to be able to assign any effect to either or both of these parameters (Fig. 7) Fig. 7: Butyrate pretreatment significantly increases rates of establishment. Cells were kept, for 48 h, in the presence of 4 mM butyrate. They were then kept for 8 or 24 hours in the absence of this agent before episomes were transfected. This procedure permitted the recovery, by FACS, of significantly higher numbers of stably expressing cells for either MCs (center) or PPs (right). This effect is acetylation-specific as it is not found for mere synchronization. Fig. 8: A spontaneous deletion event within the S/MAR gives raise to a microcircle with authentic polyadenylation (A) and superior expression properties. Deletion reduces the original 2 kb S/MAR to a 733 bp sub-fragment, which augments transcription ~5 fold (D). A likely reason for this is the complete transcription of the 733 bp sequence and authentic termination at the downstream poly(A) site (B). The difference between PP and MC (lanes 1 and 2 in B, respectively) is ascribed to the fact that for the MC the poly(A) site is more destabilized (see SIDD profile in (C)). * Termination within S/MAR * Polyadenylation (SV40; ~200 nucleotides) PSV40 S/MAR eGFP polyA * !! * S/MARs: Artificial Termination or Authentic Polyadenylation S/MARs: Artificial Termination or Authentic Polyadenylation ∆1.2 kb ∆1.2 kb ∆1.2 kb ∆1.2 kb A B C D Outlook: One of the major S/MAR attributes is their recombinogenic property, which in our hands triggered the ´in vitro evolution´ of a ´microcircle´ in a process that reduced the element, from 2 kb to 733 base pairs (Fig. 8). This event improved both cloning capacity and expression characteristics enabling a novel nonviral mammalian vector system with an exceptional potential for cell modifications. These principles entered the industrial production of highly defined and pure minicircles and their use for systematic transgenesis (cooperation with PlasmidFactory and several academic partners). A critical parameter for episomal persistence is an at least partial transcription of the S/MAR since only this situation renders the DNA in a replication- competent state. While this opens the chance to control episomal maintenance, the obligatory extension of mRNA may impair the gene expression potential. There are indications that this can be overcome by approaches to control the termination- and polyadenylation steps. A major goal of present efforts is the implementation of the system for gene therapeutic purposes, the more as it leaves open uses for both the transient and/or the stable expression of the relevant factors. An area of active research is the rational, SIDD-based design of artificial S/MARs by established rules (cf. Fig. 8C) . Besides the generation of authentically terminated mRNAs with maximum stability these efforts comprise the parallel establishment of different episomes for the regulated expression of multi-subunit proteins. They will be extended by the addition of nuclear matrix targeting signals (NMTS). Once established in the nuclear architecture, minicircles can be further modified by RMCE.