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. Nehlsen1), S. Broll1,2), R. Kandimalla3), N. Heinz4), M. Heine5), S. Binius1), A. Schambach4) 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 / Forschungsförderung.
Nationale
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, Flprecognition 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.
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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 has been called “transcriptional augmentation” [5] as it is different from enhancement by the following criteria: -
traditionally, S/MAR actions have only been observed after integration, whereas an
enhancer is active in transient and stable expression systems; -
the presence and activity of S/MARs in episomes suggests their dependence on an
authentic chromatin structure, which can only be attained during replication. Since the same principles should exist for nonviral episomes it appears that the pathway leading to an ordered chromatin organization (replication as part of the genome of the host cell or as an independent unit) is of secondary importance; S/MARs per se do not enhance transcriptional levels but rather prevent silencing. This is supported by our observation that highly expressed genomic sites are no further improved by the presence of these elements [13]. Stringent selection procedures have even led us to conclude that highly expressed loci are governed by pre-existent genomic S/MARs [14]
5
Under these circumstances S/MARs clearly reveal “insulator functions” the effect (but not the molecular basis) of which is comparable to the classical insulator cHS4 (a prototype insulator from the chicken beta-like globin gene cluster) at some but not at all genomic sites [13]. If subjected to the classical tests underlying the definition of S/MARs, cHS4 is clearly different, which can be explained by the fact that it associates with a particular protein, the CCCTC-binding factor CTCF that forming bridges to the nucleolar surface, which is mediated by nucleophosmin ]13]. Whereas S/MARs shield a gene from silencing, their insulator functions do not necessarily share enhancer blocking activity with cHS4. Although extended boundaries consisting of “constitutive S/MARs” clearly prevent interactions across domain borders, this is not the case for “facultative S/MARs” that are much shorter and depend on the simultaneous presence of an additional associating factor such as YY1 (otherwise called NMP1 [6], i.e. nuclear matrix protein 1 or SATB1; [7,8,15]). At promoter-upstream positions or as part of an early intron they may even be required for enhancer actions, for instance by introducing loops that enable the apposition of a promoter with its coordinated enhancer. Prominent examples are again the huIFN-ß gene [7,8] or the mouse immunoglobulin κ- and µ- chain genes [16,17]. By necessity, intronic S/MARs have to be transcribed. Since they do not impede passage of Pol II, their occupation must be regulated. In yet another scenario transcribed S/MARs occur in intergenic regions. An element of this type coincides with the replication origin of the chicken alpha-globin domain, which, in normal and transformed erythroblasts, becomes part of a full-domain transcript [18]. After the transcription process has led to opening of the domain in dedicated cells, the element re-attaches to the matrix separating the individual transcription units. Finally, extended S/MARs coinciding with the domain borders usually define the termini of a replicon [19], whereas the function of short S/MARs with a role in S phase is modulated by transcription. The rules underlying such an event could be studied on retroviral integrates, which have the particular advantage that, at low MOIs (multiplicities of infection), they cleanly integrate as single copies. Therefore this provirus model enables the study of single-copy inserts with defined ends (LTRs) at otherwise unperturbed genomic integration sites. Except from the basal vector carrying a 4.3 kb transcription unit, derivatives were transduced, each containing an 800 bp huIFN-ß sub-
6
S/MAR insert (element “IV” in Fig. 1A) at a different position. Whereas the S/MAR-IV insert impeded transcription at distances below 2.5 kb downstream from the promoter, it strongly supported transcriptional initiation in case the distance exceeded 4.5 kb, i.e. at localizations within the LTRs or ahead from the 3´-LTR [20]. These findings could be accommodated in the classical twin supercoiled domain model of transcription, which comprises a over-wound domain in front of and an under-wound (negatively supercoiled) one behind RNA polymerase [5].
S/MAR-actions are multifold and context-dependent Our findings that an S/MAR fragment supports transcriptional initiation when placed at a certain distance downstream from a promoter has since been exploited for a variety retroviruses and cell types (compiled in Tab. 1). The transcription of these proviruses is known to become down-regulated by negative regulatory factors associating with silencer elements within the LTRs or the tRNA primer binding site. Initial experiments relied on Mo-MuLV vectors for which silencing of a reporter gene is accompanied by 3´LTR methylation. In a pilot study ([21]; Tab. 1) the 800bp S/MAR-IV was placed, in both orientations, either into the LTR (generating a proviral double-S/MAR status resembling a chromatin domain) or next to the 3´LTR upstream end. While the experiments revealed an unanticipated orientation effect (activity in the “+”, but not the “-“ direction; [21,26,27]), the location of the S/MAR at or within the 3´LTR (plus the 5´LTR) was of minor relevance as anticipated by the above pilot studies: in both cases the expression remained stable for more than four months, and no LTR methylation was observed. This fact directly supports observations that S/MARs prevent methylation in transcriptionally active loci [22]. Since the single-S/MAR setup with element IV next to the 3´LTR enabled higher virus titers, all subsequent studies relied on this situation. These experiments were extended to various other cell types and retroviruses with S/MARIV alone or in direct contact to a double-copy core sequence from the prototype cHS4 insulator. While some combination of the two elements seemed beneficial in one system [28] S/MAR-IV alone seemed largely superior in another [29]. Studies on a non-S/MAR reference revealed further mechanistic details: by assessing the acetylation status of histone H3 (i.e. a prototype euchromatin marker, cf. Fig. 2) a significant provirus deacetylation occurred with time indicating silencing within
7
the stem cell. At early stages this effect could be reversed by a histone-deacetylase inhibitor (HDACi), i.e. Trichostatin A (TSA). Contrary to HDACi actions, increased CpG methylation became evident only at a later stage at which reactivation attempts using either TSA or the methyltransferase inhibitor 5´-azactidine (5´-azaC) remained inefficient. These observations confirm a current model implying that, while silencing is initiated by histone-deacetylation, the silenced state may become locked, by DNA methylation, only at later time points (Fig. 2). An observation deserving further attention is the fact that, while S/MAR IV acts in an orientation-dependent fashion in three reports, in the latter example [29] the same element is effective regardless of its orientation. While the molecular basis for these particular differences remains undetermined, they nevertheless confirm the context-dependent action of facultative S/MARs. At a later point examples will illuminate the way S/MARs can modulate the superhelical status of neighboring regulatory elements, depending both on their sequence and associated structural features.
Stress-induced duplex destabilization (SIDD), a unifying property of S/MARs S/MARs have been operationally defined according to the protocols that led to their detection [13,30-32]. The respective elements have been implicated in a variety of biological activities, all of which are compatible with an affinity for the nuclear matrix. Besides insulator-, augmentation- and enhancersupport functions these include the long-term maintenance of high transcription levels by counteracting histone- and DNA methylation steps, the support of histone acetylation, and accessory origin-of-replication functions. In spite of this wide spectrum of activities, all S/MARs have one property in common: they consist of a more or less regular succession of DNA-unpairing elements (UEs) which initiate double strand separation under negative superhelical tension (Fig. 1A and [32]). These UEs together constitute a base-unpairing region (BUR) with an architecture enabling the accommodation of prototype nuclear matrix proteins [33]. UE properties were first analyzed for the standard pBr322 plasmid [34] for which the SIDD profile reflected preferential opening of the intrinsic Ori. Subsequent analyses on pro- and
8
eukaryotic DNA were performed at a standard superhelical density of σ=−0.05 as first determined for the bacterial plasmid. It is of note that site-specific nucleases have opened the possibility to excise pieces of genomic DNA between integrase target sites, which have been strategically positioned within a eukaryotic chromatin domain [35]. Since an integrase-mediated excision process preserves the preexisting superhelicity within the resulting circle, σ- values for eukaryotic genomic loci can be determined with precision. Results so far demonstrate a similar range for active eukaryotic loci. Our first studies on the structure/function relationships of S/MARs concerned the domain organization of the human interferon ß (huIFN-ß) gene located at position 9p22 on the short arm of chromosome 9 (Fig. 1) . Apparently, the 14 bp domain is flanked two ~5kb constitutive S/MARs comprising the 2.2 kb EcoR1 fragment “E” (upstream border) and most of the ~ 4.5 kb Hind III fragment that had been localized before by the classical scaffold-reassociation assays [36]. Other marks are certain intense and widely-spaced individual peaks, which triggered in-depth investigations by Klar et al. [7,8]. They showed an association of these sequences that could later be associated with DNAseI hypersensitive sites with regulatory potential (the mentioned “facultative S/MARs”). SIDD analyses and functional tests on S/MARs from mammals and plants explain the cross-species activity of these elements: without exception, active S/MARs are BURs with a related architecture: all of these comprise a register of UEs that obey certain rules regarding the minimum number, spacing and threshold destabilization. Together these features mediate the association of a multifunctional protein called SAF-A, SP120 or hnRNP U [37,38]. Assembly as a multimeric complex results from cooperative interactions with the S/MAR (“mass binding”, cf. Fig. 1A). These properties could be reproduced in an in vitro assay where SAF-A - S/MAR association occurred in the presence of nonspecific competitor DNA and were assigned to a short stretch of amino acids in the N-terminal region, designated SAF-box/SAP domain. SAF-A recognizes AT-rich sequences (“AT patches”) that are common for S/MARs. Apart from this the co-purification of SAFA with proteins such as histone acetyltransferase (HAT p300/KAT3B, introduced in Fig. 2 as an enzyme modulating histone H3-structure and function) indicates that SAF-A serves as a platform
9
for the assembly of factors modulating S/MAR functions. The presence of an RNA binding motif (RGG box) in its C-terminal domain is in accord with its designation as a member of the hnRNP family of proteins involved in the processing of pre-mRNAs .
Chromosome-based expression strategies: Episomes and/or predetermined integration sites (RMCE) Two of our central approaches addressing the design of chromosome-based vectors are outlined in Fig. 3. Both concepts comply with a set of rules that have been covered extensively in a recent review [1] : -
The incorporation of S/MARs which, due to their strand-separation potential support
transcription, provides accessory functions to origins of replication and enhances the efficiency of recombinases [14] ; -
Flp-recombinase target sites (FRTs) are recombined in the presence of the “flippase” (Flp),
provided that they are identical and thereby able to cross-interact [1,39]. Site-specific recombinases (SSRs) have opened new options for the systematic modification of eukaryotic genomes. In case two identical, equally oriented 48 bp target-sites are parts of a given DNA segment, the intervening sequence will be quantitatively excised. If applied to the S/MARplasmid (the so called “parental plasmid”, PP) in Fig. 3B the procedure generates two daughter molecules, a miniplasmid (MP) accommodating prokaryotic vector parts and accessory sequences, and a minicircle (MC), which exclusively consists of the desired functional eukaryotic sequences. According to the above definitions the MC represents a minimal model for a functional chromatin domain, though at an extrachromosomal location. The formal reversion of this excision process would be the addition of MC and MP entities (i.e. re-formation of the PP) but also of oligomeric derivatives containing products arising from MC x MC or MP x MP recombination. Since these “reverse-type” reactions are bimolecular processes that have to occur against kinetic and entropic barriers [40] they would have to be enforced by extreme educt concentrations in the presence of Flp activity. This is the likely reason that complications of this type have not been encountered.
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B – REPLICATING NON-VIRAL EPISOMES
Nonviral gene delivery strategies are usually based on bacterial plasmid-DNA (pDNA) carrying the gene of interest. Already in 2007 pDNA contributed to 26 % of all clinical trials. Due to its relative safety, simplicity, and reliability, naked DNA received particular attention for transfer into muscle tissue. Efforts to improve the efficiency of non-viral gene vehicles require a better understanding of delivery kinetics for different types of DNA into clinically relevant cells. Three DNA species have been compared: linearized plasmid DNA (l-DNA) formulated by single-site digestion of c-DNA, reduced-size linear gene cassettes generated by PCR (pcr-DNA) and a covalently closed circular (ccc-) vector with a certain superhelical status. The latter specimen deserves particular attention as it surpasses linear DNA regarding transcriptional potential [41], resists integration in diploid cell genomes [42] and facilitates the transfer across cellular membranes Another step forward concerns nonviral circular episomes that could be converted into minicircles following the general scheme depicted in Fig. 2B (and detailed in Chapter C). To this end site specific recombinases (the Tyr-dependent recombinases Cre and Flp or Ser-dependent variants, such as ΦC31 integrase and ParA resolvase) could successfully be applied [4]. Resolvases are sometimes preferred since absence of accessory factors leads them to operate in an irreversible fashion. Before addressing the advantages of nonviral, replicating S/MARminicircles we will briefly describe the properties of S/MAR plasmids which enable the generation of ARS-type vectors.
• Can the yeast-ARS principle be verified for mammalian cells? 5µm
In yeast an origin of replication is specified by ~125 base pair DNA-segments called autonomously replicating sequences (ARS). ARS elements are putative origins of replication, which cause plasmids including an ARS to be maintained autonomously in the absence of integration or other sequence rearrangements. A closer inspection revealed an 11-bp core sequence (ACS, ARS consensus sequence), which is part of the recognition site for the origin recognition complex
11
(ORC). However, while central properties of the ORC are evolutionarily conserved, the replication promoting sequences are not. Thus, the nature of replication origins in metazoan genomes has remained largely elusive. A more direct access to mammalian Oris was expected from screening chromosomal DNA for sequences which might confer the ability of autonomous replication in homologous mammalian cells. For mouse genomic DNA this approach led to several, apparently functional DNA segments, which later turned out to have mere plasmid-DNA amplification capacity. A variable subpopulation of episomes could subsequently be ascribed to concatemeric integrates which recombined yielding extrachromosomal circles with limited persistence.
•
ARS and S/MARs: common (SIDD-) properties
There are definite relationships between ARS elements and S/MARs. An early report goes back to Amati and Gasser [43] who demonstrated specific sequences bound to the yeast nuclear scaffold to provide ARS functions. Certain regions with scaffold association potential could be shown to include the 11 bp ARS consensus, suggesting that scaffold binding is related to ARS activity. A later report by Ak and Benham [44] confirms that highly conserved properties of yeast origins concern S/MAR-like characteristics, in particular. a definite susceptibility to superhelically driven DNA duplex destabilization. It is suggested that these features, in conjunction with other characteristics, might be exploited for the localization of Oris in the yeast genome. These ideas gained support by Li et al. [45] investigating the ARS properties of S/MARs from tobacco in the yeast system. In fact, two out of six elements complied with the relevant criteria. This confirms relationships between scaffold attachment and replication potential also for higher eukaryotes. Other replication minimal models go back to viruses, such as SV40, BPV or EBV that replicate episomally in mammalian cells. Also there replication initiation is supported by an easily melting DNA tract, i.e. a base-unpairing region (BUR). Conformational coupling permits the energy absorbed by base-unpairing to be delivered to a DNA unwinding element (DUE) where it serves to establish secondary structures such as hairpins or stem-loops. Once more these are prerequisites for an ORC initiating replication at the origin recognition element (ORE; [46]).
12
For more than a decade vectors sharing functions with natural chromosomes were thought to solve problems related to safety and reproducibility. These vehicles do not require viral factors for their function, and should be stably maintained in the cell for many generations in the absence of continued selection. In case of linear minichromosomes three functional elements are required: telomeres, centromeres and an Ori. While functional telomeres and centromeres could be provided, the megabase-size of these entities per se granted the occurrence of Ori-characteristics, although these features had to remain largely undefined owing to Ori-extension. Most of these approaches suffered from the long-term instability of artificial chromosomes (ACs), however, which motivated the systematic exploitation of ARS-principles for mammalian cells.
•
S/MAR plasmids: verification of the concept
The established strand-separation potential of S/MARs lends support to the idea that there is a regular association of these elements with origins of replication as exemplified by the dihydrofolate reductase domain [47]. This assumption led to the generation of an S/MAR plasmid with replication potential in a variety of eukaryotic cell systems [48]. Available evidence indicates that it is the 2 kb fragment of the huIFN-β 5´ S/MAR (Fig. 1A) that recruits components of the cellular replication apparatus to support authentic replication and segregation [49]. After its establishment in the nuclear architecture depending on an initial phase under selection pressure, the replication apparatus of the host cell is utilized in a way that S/MAR episomes replicate once during the early S phase of the cell cycle in synchrony with the cellular genome. Quite unexpected at first, this vector does not require specific DNA sequences to accommodate the origin recognition complex in vivo. This indicates that the site on the episome where replication initiates is determined by epigenetic principles [50].
Transcription into the S/MAR: directionality and rate A stringent prerequisite for an S/MAR taking over Ori functions is its combination with an active transcription unit to enforce strand separation. Fig. 3B indicates that transcription has to run into the S/MAR causing its over-winding within the positive superhelical part of the classical twin-domain model. In this situation histones
13
will be driven off by the tracking protein but will re-associate and reform nucleosomes within the under-wound (negatively supercoiled) domain [51]. At what time point the underwound and overwound parts of the plasmid will compensate each other is hard to decide due to the dynamic (binding-)properties of the interposed S/MAR region. The Fig. 4 experiment [52] demonstrates that the direction of transcription is the prerequisite for efficient episomal persistence, at least for the prototype vector pEpi. Here we made use of our toolbox, i.e. Flp- or Cre- recombinase in conjunction with two oppositely oriented recognition sites at the flanking the transcription cassette to invert this unit in a remarkably slow [1] recombination reaction. After terminating the process 10 out of 15 clones were found with an inversely oriented unit (“i”), i.e. a transcriptional direction that poses the S/MAR in the negative superhelical domain. Four constructs maintained the original orientation (“o”) and one harbored both orientations (“i/o”). It is of note that within this collection the episomal state was apparent only for the original S/MAR plasmids, while constructs with an inversely-oriented transcription unit yielded Southern blots with a considerable background in the absence of a clear-cut signal. Concerns that mechanistic particularities of the recombinase-mediated inversion process might have triggered integration were invalidated by the observation that corresponding results were obtained in case plasmids with either the “o”- or the “i”- orientation were applied in separate experiments [53]. So far all studies agree in that the “o”-orientation is more efficient. This simplistic statement is refined by a recent report on a sophisticated pEpi derivative that yielded fewer, but obviously episomal copies in the “i”-orientation [54].
Cell and nuclear permeation Only part of naked DNA that gets in contact with the outer cellular membrane can actually enter the cell. In order to improve gene transfer efficiency and to obtain adequate expression, transfection agents and electroporation/nucleofection procedures are now in common use. The apparently higher efficiency of the second class of methods is, at least in part, due to introduction of strand breaks into both the circular vector and the genomic DNA, which trigger non-homologous end-joining (NHJE) and thereby integration [55,56]. For obvious reasons
14
these approaches should be avoided when it comes to vectors for which performance depends on an authentic ccc- status (see the PP and MC species in Fig. 3B). This may turn out to be different for femto-second laser pulse transfer techniques which are under intense present development [57]. The following section still has to rely on nonviral carriers such as cationic lipids and polymers that interact with the anionic DNA via charged moieties, thereby forming compact, nano-sized particles suitable for cellular uptake. -
Transduction
principles
polyethyleneimine (PEI),
Hsu
and
Uludağ
poly-L-Lysine (PLL),
[58] palmitic
have
applied
four
gene
carriers,
acid-grafted PLL (PLL-PA),
and
Lipofectamine-2000 to test the delivery and expression for each of three DNAs, a ccc-plasmid, a linearized version thereof (l-DNA) and a shorter l-DNA variant, obtained by PCR amplification of its center section (pcr-DNA). ccc-DNA exhibits a higher intracellular diffusion capacity facilitating nuclear targeting and/or expression compared to its linearized forms. On balance, pcr-DNA bears only the promoter-GOI unit in the absence of prokaryotic vector sequences and may have an improved potential to traverse the nuclear membrane. Unfortunately, all forms of l-DNA are prone to intracellular nuclease attacks unless they are capped, i.e. provided with hairpin structures at both ends to comply with the Minimalistic Immunogenically Defined Gene Expression (“MIDGE”-) principle [3]. The results show no obvious difference in the morphology of particles regarding interaction, binding kinetics, dissociation characteristics and DNA uptake depending on either DNA structure or transfection reagent. Using PEI, however, the best expression was observed for ccc-DNA followed by l-DNA and pcrDNA. Although the latter specimen was delivered to the same extent, exonucleolytic actions may have invaded its functional core invalidating the (otherwise desired) absence of prokaryotic vector parts. These results are in accord with a superior expression of ccc-DNA. Recent indications from yet another system let it seem likely that this status also facilitates passage of the nuclear membrane: While the technically demanding injection of linear expression constructs into the pronuclei of fertilized mammalian eggs is the traditional method for generating transgenic embryos, an effective nuclear transfer can also be achieved upon cytoplasmic injection of ccc-specimens. This
15
originally unexpected phenomenon indicates that the circular superhelical status enables a specific nuclear transfer route of yet undetermined nature [59]. Still another factor has been associated with the kind of promoter(s) on the vector. Exemplified by the SV40 unit, association of ubiquitous transcription factors and the subsequent exposure of their NLS signals were found to facilitate passage of the nuclear membrane [60]. The specificity of this effect could best demonstrated by controls (such as the CMV promoter) that are devoid of such an activity.
Nuclear association sites Nonviral episomes are able to recruit the replication apparatus of the host cell. Contrary to their viral counterparts, they do not need external accessory factors. Again, it is the S/MAR providing the link to the nuclear matrix. In this position it does not only enable use of the endogenous transcription factories, but it also counteracts silencing. Transcriptionally active genes replicate early in S phase, possibly supported by certain transcription factors [61]. Chromatin-DNA interactions obey a “histone code”, i.e. particular patterns of covalent histone tail modifications, which, together with DNA methylation patterns, is part of the epigenetic code. While it is accepted that histone tails are modified by processes like methylation, acetylation, ADP-ribosylation, ubiquitination, sumoylation and phosphorylation, functional details have only been worked out in specific cases (Fig. 2). Of primary diagnostic value are acetylation and methylation processes concerning the core histones, H3 and H4. A number of diagnostic immunoprecipitation kits have become available to this end. Lysine N-ε-acetylation is a dynamic, reversible and tightly regulated modification with a major role in chromatin remodeling and in the regulation of gene expression, especially at the level of transcription. For H3 the process occurs at several different lysine positions in the N-terminal domain
where
it
is
performed
by
histone
acetyltransferases
(HATs/KATs)
such
as
CBP/p300/KAT3B (Fig. 2). For the non-viral episome pEpi histone H3 acetylation was found to be enriched on the expression unit while the same gene residing on an integrated control underwent histone H3K9 trimethylation (K9me3), and thereby silencing [62]. This study showed S/MAR episomes to preferentially interact with early replication sites that are spread throughout the nucleoplasm.
16
Immobilization of an episome at these sites is the likely consequence of S/MAR-mediated binding to nucleoskeletal structures [63]. Later work extended these findings by a systematic exploitation of alterations in the H3 methylation status at lysines 4 (K4me, K4me3) and -36 (K36me3) for both the pEpi vector and its S/MAR-free, integrating precursor construct, pGFP-C1: - whereas pGFP-C1 is mostly decorated with K9me3 as mentioned, pEpi-eGFP is preferentially associated with modifications typical of active chromatin. For K4 the modifications are enriched on the S/MAR, but K36me3 was uniformly distributed over the entire vector; -
for pEpi the pattern remained stable throughout the G1-, and G2-phases in accord with a
persistent association with early replicating (perichromatic) domains accommodating replication and transcription machineries and RNA processing factors; -
activating histone modifications are removed during mitosis, at a time when the association
with the host chromosomes is initiated. To enable tracing the episome and its chromatin status in vivo Tessadori et al. [64] have prepared a pEpi-derivative, “pELO64”, with a tandem array of 64 LacO sites. The lacO/lacR technology of Belmont [65], i.e. the transient expression of a mCherry-lacR fusion served the visualization of constructs in the living cell. After establishment (3 weeks of continuous culture in selection medium) immobility of episomes could be confirmed and shown to last tens of minutes. The absence of a “corralled” local low amplitude movement, which is otherwise typical for clustered vectors, suggests that episomes become individually and firmly bound to host chromatin. Despite their immobility, episomes re-locate to positions closer to the nuclear center if their gene expression is stimulated by the addition of a histone-deacetylase inhibitor (TSA) and the inhibition of DNA de-methylation (5-aza-dC) or, even more convincingly, by targeting a VP16 domain to pELO64. The latter treatment showed that transcriptional activation mediates a relocation of the signals towards the nuclear center (64). Together, these results prove that the regulatory mechanisms for episomal genes comply with those of the host genes.
17
RMCE- based elaboration following establishment Establishment of nonviral episomes and their viral equivalents in the nuclear architecture usually occurs at low copy numbers (4-8 [46]) and rates (usually 20 weeks of continuous cultivation. We used the incidental observation of independent, but identical S/MARinternal deletion events within the 4.1 kb minicircle as they occurred during the long-term cultivation of CHO-strains (cf. clone M18 in Fig. 11B). The size-reduced S/MAR was recovered by PCR and used to construct an S/MAR-minimized parental plasmid analogue (cf. Fig. 3B). Processing this PP by Flp-mediated excision led to a 2.9 kb minicircle with a largely reduced 733 bp S/MAR-insert. Relative to the 4.1 kb precursor this step again caused a dramatic improvement of expression characteristics, both regarding its level and the stable persistence of the “M18” minicircle [46]. The fact that the parental plasmid precursor of M18 outperformed pMARS regarding its long-term stability led us to abandon the idea to generate minicircles from artificial S/MARs with internal sequence repeats. While vector stability per se may contribute to high level expression, the relevance of authentic transcriptional termination/polyadenylation has already emerged before. Northern blots in Fig. 11C demonstrate prematurely-terminated transcripts within the extended, 2kb S/MAR for both pEpi and its 4.1 kb minicircle derivative, but an authentic usage of the SV40poly(A) signal for the short-S/MAR versions pMARs and “M18” (Fig. 11C). Corresponding SIDD profiles in Fig. 11D
31
provide evidence that the deletion that gave raise to “M18” has inactivated (but not removed) the cryptic internal polyadenylation signal and, at the same time, re-activated the genuine SV40 poly(A) sequence. Obviously, the same poly(A) signal is less destabilized if it is part of the extended 2 kb S/MAR (see the respective UEs in the Fig. 11D SIDD profiles). This difference is ascribed to a competition of the SV40 derived poly(A ) signal with the large number of UEs in the 2 kb S/MAR, which reduces its strand-separation- together with its secondary structure forming potential. Benham [34,84] has shown that for higher eukaryotes poly(A) consensus sequences are only used if they coincide with a region of significant strand separation potential, whereas in yeast the requirements are restricted to the strand-separation requirements. Only the absence of an extended, competing BUR will therefore permit the SV40poly(A) signal to adopt the secondary structure enabling its recognition by the polyadenylation machinery [85].
Transcriptional termination and polyadenylation: an intricate interplay To date all nonviral replicating episomes rely on an S/MAR element that is transcribed over at least part of its length. In any case this process extends the primary transcript in a way that may affect mRNA stability and gene expression. An evaluation and optimization of these facts is stringently required, unless the desired GOI is accommodated in a separate transcription unit, cf. Fig. 10, where the L- or the Hencoding “gene of interest” (GOI) performs functions apart from the “gene on duty” (GOD, here a fluorescent marker). Meanwhile this configuration has proven its value by consistent results in various experimental setups. The performance of different GOI-S/MAR combinations on the other hand is hard to predict. An example is the first version of pMARS (Fig. 7) that had to undergo rectifications before its fluorescence could be evaluated [46]. Polyadenylated mRNAs contain variable extensions between the coding region and the poly(A) tail. The length of both the 3′ untranslated region and the poly(A) tail relates to the translational efficiency and the stability of mRNA. Besides, polyadenylation is intimately linked to transcription termination in a mutual, reciprocating way: co-transcriptional cleavage within the downstream RNA Pol II transcription termination region depends on the presence of an upstream AATAAA-type element. Termination, in turn, is the prerequisite for subsequent pre-mRNA 3′ end
32
processing/polyadenylation guided by the AATAAA tract. This interdependence has opened the chance to preserve the essential S/MAR passage by the transcription complex while polyadenylation can be directed to a location close to the end of the coding region. The concept was verified for a β-globin transcription (HBB) unit for which the proximal termination region was relocated to a position behind the S/MAR while preserving the polyadenylation signal. In fact, the resulting construct enabled replication and retention of the episome as anticipated in the absence of any unwanted mRNA extension [54,71]. Whereas a low-rate transcription through the S/MAR suffices for the establishment and maintenance of plasmid episomes, the same study indicates that higher rates are associated with elevated episome copy numbers opening yet another regulatory option.
Episomal status: Proof and persistence The criteria that are sometimes used to establish the episomal status are subject to considerable contention [83]. Among these are -
plasmid-rescue, i.e. a re-transfer of circular episomes from mammalian cells to E.coli. This procedure is not feasible for minicircles, which, according to the pFAR- concept do not comprise the necessary bacterial DNA components. Although plasmid rescue can verify the principal presence of plasmid derivatives, it does in no way prove an episomal status for all transgenes in the recipient cell. Finally, circular plasmid entities may originate from multimeric integrated concatemers in conjunction with intramolecular recombination events [86].
-
Full-length PCR amplification, which can only serve as a preliminary indication for the presence of episomes. Again, an identical effect may go back to multiple transgenes that have integrated in a concatemeric head-to-tail fashion. This status is a typical concomitant of the classical Ca++phosphate transfection procedure.
-
Linear amplification-mediated (LAM-) PCR [87], a technique originally developed to characterize retroviral insertion sites. The sensitivity of the method results from preamplification of vector-genome junctions and its efficiency is boosted by magnetic capture, dsDNA synthesis, restriction, linker ligation and nested PCR steps. A subsequent study addressed the episomal status of integration-deficient lentiviral (IDLV-) vectors based on the
33
fact that LAM-PCR would detect LTR- and non-LTR mediated integration. While such an event could be traced [88], it is a rare exception. At present these reports are leading to an increasing number of LAM-PCR applications in order to verify the performance of LTR-based episomes (so-called “LTR-circles”; (ESGCT 2011). -
A clear-cut Southern-blot signal is a more stringent criterion as additional bordering
fragments would arise in case of integration. But there are typical and frequently neglected shortcomings as demonstrated in Fig. 12. These examples clearly show that analyses on clonal mixtures can be meaningless. On the other hand the episomal state becomes obvious for single clones such as M23 and H11 (cf. also insert to Fig. 9). -
The traditional extraction procedure according to Hirt leads to the enrichment of non-
integrated DNA - at least at early passages. The efficiency of this protocol may decrease with time since repeated rounds of replication can give raise to extrachromosomal chains (concatenates) even in case of the viral systems [89]. -
The conclusions from Southern blot- and Hirt- extraction procedures can be reinforced by
ATP-dependent nuclease treatment. More commonly known as “Plasmid-Safe”, this system relies on a selective DNase that is mostly used for the removal of contaminating bacterial chromosomal DNA from plasmid preparations [90]. The enzyme rapidly degrades linear DNA under conditions that leave duplex circular DNA intact, justifying its classification as an exonuclease. Paradoxically, however, it acts as an endonuclease on single-stranded DNA. The latter activity is inhibited by the presence of linear duplex-DNA explaining the order of preferred DNA substrates: linear dsDNA > linear ssDNA > circular ssDNA >> nicked circular DNA > circular dsDNA
Consequently, an adequate first step prior to Southern blots would be digestion of genomic DNA by a MC-non-cutting restriction enzyme. Post-treatment of Hirt extracts with the nuclease would indicate the presence of circular dsDNA and thereby enhance the stringency of the assay. Apart from these approaches the ultimate criterion appears to be -
FISH-visualization of transgenes on metaphase spreads, which has proven its potential
before [49,52]. The approach generates multiple focused fluorescent spots in association with
34
the chromosomes when we have to deal with intact episomes. Such an association is lost if the preparation involves shear forces [52]. Alternatively, we find a single intense doublet indicating the typical co-integration of multiple copies subsequent to DNA transfer [49]. For the maxicircle (and the respective parental plasmids) there are obvious examples where intense doublets, one signal on each chromatid, indicate integration events that occurred during continued cultivation and replication (Fig. 13). In conclusion, while full-length PCR may serve as a preliminary hint, the Southern-blot and Hirt extraction protocols may strengthen the evidence for the episomal status in case they are combined with ATP-dependent nuclease treatment. Though more demanding, the most comprehensive information on copy number and -status is enabled by metaphase-FISH. A final comment addresses the apparent discrepancy between the results of Southern-blots and metaphase-FISH data in Figs. 12 and 13, since the blots reveal a considerable contribution of non-episomal minicircles in contrast to the fluorescence microscope data that are compatible with an exclusively episomal status. Non-episomal specimens can arise from rigid treatments at stages, prior to establishment. These treatments may involve FACS sorting – [91] as well as clone pickingroutines. Following these considerations it is recommended to allow an extended period of time for establishment and/or to adapt the relevant parameters. Results in Fig. 9 strongly suggest that clones that have been established after reaching their ultimate chromatin structure resist even vigorous treatments.
•
Emerging extensions and refinements
While the history of authentic nonviral replicating episomes (S/MAR-plasmids) goes back to 1999 [48] and while its purification can rely on standard procedures, the generation, identification and recovery of minicircle derivatives is much more demanding as it has to rely on several dedicated routines. These routines may even enhance the danger of secondary rearrangements, due to S/MAR-intrinsic instabilities. Common protocols involve the action of site-specific nucleases of either the threonine(lambda- Cre-, Flp-) or the serine (ΦC31-, and ParA-) family. These activities can be provided
35
-
either from inducible, genomically anchored genes of modified E.coli strains; induction is
either performed by a temperature shift [83] or a metabolite, typically arabinose [82]. -
alternatively, the recombinase is located in the (MP-)part of the parental plasmid, which
accommodates the auxiliary functions (miniplasmid-section). Whereas our initial experiments firmly relied on the first concept (E.coli strain MM294 with a genomic copy of heat-inducible Flp [83]), our efforts have recently shifted to the second option. Due to the fact that for bacteria mRNA is translated into protein as soon as it is transcribed, this altered concept led to vast improvements regarding yield and also purity (S. Binius, Dissertation TU Braunschweig, 2011). Another important consideration motivating this change is the possibility to select the bacterial strain according to the requirements, especially regarding its propensity to generate unwanted concatemeric side products, which (among others) depends on RecA+ and RecF+ functions. A reverse switch of strategies (transfer of auxiliary genes from the parental plasmid to genomic locations) was taken by Kay et al [82]. Their refined protocol has to accommodate three auxiliary genes (two for recombinases, and one encoding the homing endonucleases I-SceI), excluding alternative options. A more recent approach addresses the creation of minicircles from plasmid precursors in vitro. The procedure involves the simple excision of unwanted sequences by restriction enzymes and subsequent end-ligation. While this protocol per se would fail to produce active minicircles due to the absence of a superhelical structure, the appropriate topological state can be adjusted by in vitro treatments, either using histone-like proteins followed by topoisomerase [92] or by gyrase ([93] Dissertation M. Heine, TU Braunschweig 2012). Such a procedure may profit from the fact that superhelicity can be controlled. After optimization, this may positively influence early transcription and replication steps and thereby establishment in the host.
Combination of excision- and RMCE strategies Following a long standing tradition our focus is on Flp-dependent recombinase protocols as these open a variety of simultaneous or successive modification steps (see, for instance, the RMCE example in Fig. 5 and the toolboxes described in ref. [39, 67]). Concerns that the Flp-mediated generation would suffer from the principal
36
reversibility of Flp reactions did not hold in this case, since excision is strongly preferred over addition due to kinetic and thermodynamic principles 40]. As an intermediary solution we have developed a protocol that allows minicircle generation directly in the recipient eukaryotic cell. In contrast to the replicating MC, the MP product will be lost in dividing tissues by dilution. Currently, this procedure enables a wide range of pilot studies. In case of success the relevant experiment can be repeated using a parental plasmid derivative that enables MC generation in E.coli followed by purification routines (S. Binius, Dissertation TU Braunschweig, 2011). Work is underway to recover the MC-sequences from an auto-processing PP system which proved effective in vivo. These sequences will then be incorporated into the standard backbone permitting large-scale MC production in E.coli (ref. [4] and contributions of these authors in the present book edition). Central features of our processing systems for eukaryotic cells are summarized in Fig. 14. A PSV40-Flp-expression unit becomes active as soon as the parental plasmid is transferred to the recipient cell. The educt is then processed by Flp-catalyzed crossover between two FRT wild type sites (red half arrows). This excision separates Flp from its promoter, which now serves to drive a positive/negative selection marker and/or the egfp reporter gene to provide the following functions: -
an auto-limiting feature (no additional Flp-activity is generated after this step);
-
if placed appropriately, the reporter gene becomes activated and permits quantification of
the excision reaction; controls have proven that this process proceeds to completion (A. Oumard, N. Heinz et al., in progress). To add RMCE options comparable to Fig. 5, we will provide the PP with a third, heterospecific FRT site (yellow half arrow in Fig. 14). This site does not interfere with the excision reaction but enables subsequent exchange of a cassette and thereby the introduction of new functions into an established episome (cf. Fig. 5). In the given example, a positive-negative selection marker (for instance the hygtk fusion gene) may have served to support establishment of the minicircle in the presence of Hygromycin and to enrich successful RMCE events by counter-selection (here in the presence of GANC). Advantages of this process arise from the facts that (i) no potentially
37
debilitating, sorting steps are required at early time points and (ii) RMCE will establish a pFAR situation. Due to the chronological sequence of events that led to the development of minicircles from the original S/MAR-plasmids, most accessory options have originally been explored at the level of (parental-) plasmids before being transferred to the ultimate vectors. In this context the exploitation of selection procedures for episomal establishment and supplementation of S/MAR activities by initiator-of-replication (IR-) are of prime importance (see chapter “Establishment and maintenance: The EBV paradigm”). On balance, certain intrinsic differences between the PP- and MC systems have to be taken in consideration, exemplified by the following paragraph.
MC withdrawal at will For several applications it would be valuable to have a tool available for withdrawing episomal vectors at will. Since the Yamanaka group [94] could apply the combined expression of reprogramming factors to establish induced pluripotent stem (iPS-) cells the development of novel approaches for their delivery in a reversible, dose-controlled fashion has become an active area of research. Current refinements concern the targeted differentiation of iPSCs into functional somatic cells, and the approaches for a conditional elimination of certain iPSC-progenitors that might otherwise raise to teratoma-intiating cells (TICs),. Due to the risk of insertional mutagenesis, viral transduction routes become increasingly replaced by nonviral methods in order to induce the iPSC status. Among the alternatives is a recent report building on a standard minicircle, which, in contrast to a regular plasmids, has enabled the generation of iPSC clones from human adipose stem cells [95]. The superior performance of the MC is ascribed to higher transfer efficiency together with stronger and more persistent expression characteristics. Current extensions of this concept rely on replicationcompetent S/MAR minicircles as these permit a prolonged expression in diving cells. We might be able to terminate this phase at will, given the availability of strategies that permit withdrawal of the expression unit. A candidate approach might exploit the role of active transcription into the S/MAR but so far there are indications that traversal of the S/MAR is only required for episomal establishment rather than long-term maintenance. Two recent reports address this question, but lead to opposite conclusions.
38
The first study [96] concerns persistence of episomes in CHO-K1 cells in case transcription of the egfp gene is regulated using the Tet-On system, which permits transcription only in the presence of doxycycline. Removal of the antibiotic is shown to cause three-fold reduction of expressing cells and an about twelve-fold reduction in fluorescence activity. Although the data indicate some leakiness, these observations led to the conclusion that these phenomena were governed by vector loss. In the second example [64] pEpi derivatives carrying a tandem array of lac operator sequences are used to enable visualization and modulation of the episome´s chromatin status. For CHO-K1 cells carrying established episomes, only 5% express the reporter gene at a detectable level. Treatment with inhibitors of DNA-methyltransferases (5-aza-dC) and/or histone-deacetylases (TSA) resulted in an almost 4-fold increase in the percentage of cells with detectable eGFP expression. This indicated that decreasing fluorescence had to be ascribed to silencing rather than vector loss. Our lab has applied the capacity of retroviral particles to transfer mRNA (RMT), episomal DNA (RET) and membrane- as well as intracellular proteins (RPT). These processes rely on systematic blocks within the regular RV life cycle, and are covered by the term retroviral “pseudotransduction” [97]. In the present context RET is of particular interest, since episomal intermediates persist due to inactivated integrase functions (cf. the retroviral delivery of LTR-circles indicated in Fig. 1). Episomes of this origin have been successfully applied for gene expression over a limited period of time. [88,98]. Although we anticipated that these entities would gain replication capacity by the introduction of an S/MAR we had to find a rapid disappearance of reporter gene fluorescence. This phenomenon could be reproduced using a fully synthetic 2-LTR minicircleanalogue that could be transferred, by lipofection, at elevated copy numbers. Induction of histone hyperacetylation by butyrate resulted in a transient recovery of fluorescence for a cell population with the 2-LTR circle in an episomal state. Since these observations could be repeated in several subsequent inactivation/activation cycles, they are consistent with the idea that, once established, lack of transcription does not lead to vector-loss (A. Oumard, unpublished). It is of note that LTR-
39
dependent silencing is a well known phenomenon that has been ascribed to negative regulatory factors associating with its 5´end. A similar conclusion goes back to minicircles encoding a GFP reporter and a separate milkspecific expression cassette. After establishment these MCs were stably transmitted for more than three month in monoclonal primary bovine fibroblast lines even in the absence of continued selection. Serum starvation greatly reduced GFP fluorescence, which, however, could be fully restored after serum was re-added to the medium. These data confirm that established minicircles are not lost during periods without transcription ([99] and in preparation), which is the prerequisite for cell modifications to survive early embryonic development phases in the absence of gene activity. Only further studies can show whether or not early stages of establishment are different for minicircles and S/MAR-plasmids, in that the latter group meets more stringent requirements for its persistence as indicated by differences under the conditions of the Fig. 8, 11 and 13. To overcome barriers of this type, methods are being developed that will disable minicircle persistence by the recombinase-mediated excision of “floxed” or “flirted” (loxP- or FRT-flanked) S/MAR inserts [1].
Pronuclear injection and somatic cell nuclear transfer Very little has been reported on attempts to produce animals that ubiquitously express episomes. Manzini et al. [100] generated transgenic pig fetuses by sperm mediated gene transfer (SMGT) and showed the episome to confer expression of the transgene marker GFP in most cells and tissues. To our knowledge, though, no live animals with episomes have been generated. Recent experiments try to fill this gap, and will allow addressing the question as to whether episomes are stably and ubiquitously expressed and passed on to the next generation through the germ line. Here two approaches are of prime relevance. Somatic cell nuclear transfer (SCNT) is a twostep process in which a gene construct is first introduced into somatic cells, followed by transfer into enucleated oocytes. Since the birth of “Dolly”, the first animal to be cloned from an adult cell in 1997, it is established that physiologically normal beings can be generated by SCNT. Since then the procedure has been verified for the major livestock species including cattle, goats, pigs and
40
deer in addition to laboratory rodents. Ongoing studies use minicircles for the generation of cows expressing transgenic proteins in milk as mentioned above [99]. To this end MCs with a GFP reporter gene and a lactation-specific expression cassette under the control of the murine whey acidic protein (WAP) are transfected into bovine fibroblasts. Following molecular characterization, these cells are applied as donors for SCNT to generate transgenic offspring. Another approach starts with the pronuclear transfer of minicircles and other supercoiled DNAs enabling ectopic gene expression in embryos, used to study reprogramming events during early ontogenesis. While these expectations can be met by the classical, demanding pronuclear injection technology, identical results were obtained by simple cytoplasmic injection of vectors with ccc-status [59]. This reveals a nuclear transfer mechanism of yet unknown nature. While the conventional technology had to live with random aspects as it resulted in the integration of one or multiple copies of a gene into one or several unspecified genomic loci these can, at least in principle, be overcome by minicircle transfer. Present emphasis is posed on a better understanding of the molecular interactions by which episomes become productively established such that minicircles gain replication potential in early embryos.
From cells to organs S/MAR vectors devoid of extraneous bacterial sequences could be applied to provide high and sustained transgene expression in the recipient cells. A common in vivo model relies on liver into which either S/MAR plasmids or S/MAR minicircles can be transferred by hydrodynamic injection. While the expression from a prototype S-MAR plasmid dropped to 10% of its initial level within 25 days, in case of the minicircle its luciferase expression remained for the entire three-month period of the experiment. At this time it was approximately two orders of magnitude higher than for both the S/MAR-free minicircle control and the S/MAR parental plasmid [101]. Partial hepatectomy on S/MAR minicircle treated mice caused a rapid drop of expression due to the lack of vector replication but there are present approaches to overcome this phenomenon by providing a survival advantage associated with the MC. Already now the ongoing studies underline the utility of minicircles for persistent, atoxic gene expression in the liver. They
41
clearly demonstrate the benefit of an intrinsic S/MAR also for expression parameters not directly related to active replication [81].
SUMMARY AND OUTLOOK
Recombinant viruses are widely utilized as vectors for gene transfer. They have, however, certain intrinsic drawbacks including a limited opportunity for repeated administrations due to acute inflammatory and delayed immune responses. For vectors that integrate foreign DNA into the genome insertional mutagenesis has become a major issue, which has directed attention to viral episomes such as OriP vectors that rely on the replication machinery of the Epstein-Barr virus. In this and other cases viral proteins (here: EBNA1) are able to mimic the function of chromosomal proteins in order to exploit the replication functions of the host cell. Since each viral vector carries a potential risk, intense efforts have been launched to create artificial chromosomes using telomeres, centromeres and intrinsic Ori functions. These systems have suffered from considerable instability in addition to the fact that, with a few exceptions, functional mammalian Oris have remained barely defined due to their extended and multifaceted structure. This is definitely different for yeast where Autonomously Replicating Sequences (ARS) have been defined permitting the facile construction of episomes with a function that is largely restricted to this species. A property common to yeast and mammalian Oris is the association with a Scaffold/Matrix Attachment Region (S/MAR), an element than can confer a multitude of activities to vectors provided that these obey chromosomal organization principles. A parameter of particular relevance is the topological status, which provides structural imprints adapting the recognition potential and function of S/MARs to a given situation. The observation that an S/MAR, tuned by an adjacent transcription unit, can be used to provide plasmids with replication potential led to the first nonviral plasmid episome (pEpi) in 1999. pEpi, in fact, was the first example of a vector with ARSlike functions in mammalians. Regarding the associated properties, an S/MAR episome resembles EBV vectors in that it provides both “molecular glue” (MG-) and “initiator of replication (IR-) functions. EBV builds on
42
interaction of the viral EBNA1 protein with the repetitive EBV-derived “family of repeats” (FR) to address chromosomes and replication machineries of the host cell. For the nonviral homologue the respective functions go back to the S/MAR in association with the cellular SAF-A/hnRNP-U protein. In both cases complex formation is governed by the repetitive structure of the DNA site, which leads the partner protein to oligomerize and to enter strong, but reversible “mass-binding” interactions. The IR-function on the other hand is well characterized for EBV, where it localizes to a dyad-symmetry (DS) element accommodating the EBNA-dimer. Unexpected at first, S/MARs do not harbor a defined initiator of bidirectional replication element but rather induce structural changes permitting replication initiation over the plasmid’s entire length. Present efforts are directed to combining S/MAR- and IR- functions on a single, nonviral episome, which however is only one among a variety of approaches described in this review to optimize this vector class. A relevant improvement that is well underway is the conversion of S/MAR plasmids into “minicircles”. Minicircles per se are already well established vectors, which, due to the deletion of prokaryotic plasmid parts, resist host-defense actions that would otherwise lead to silencing. Minicircles have proven considerable therapeutic potential after hydrodynamic gene transfer or jet injection into barely dividing tissues such as liver and muscle. This review demonstrates that these entities can be supplemented by an S/MAR, which, per se, largely improves the expression properties of this novel vehicle. In conjunction with a transcription unit it provides replication functions and nuclear establishment, which, although largely superior to S/MAR plasmids, remains a target for further optimization. The term “establishment” covers the interval between vector transduction and its functional association with the nuclear substructures providing replication potential. After this stage the vector has reached its ultimate chromatin structure, which permits its long term maintenance with an efficiency that is unprecedented by other types of episomes. In the present article we have shown that this stable association permits the isolation of clonal cell lines with predictable properties going back to one or even several distinct minicircles that can be accommodated in parallel and lend themselves to further modification in situ.
43
In conclusion, S/MAR-minicircles combine the properties of efficient transient expression systems (facilitated membrane transfer and physical stability leading to an extended transcriptional burst) with those of stable expression systems. The transition between both phases is smooth such that comprehensive procedures for a wide variety of purposes can be envisaged. Current bottlenecks on the way to S/MAR-MCs with adequate purity and an established ccc-status have been identified and enable industrial scale routine preparation (102).
Acknowledgements Our particular thanks go to all colleagues who communicated ideas and contributed to this project [44, 49, 54, 59, 64, 71]. We gratefully acknowledge support by Martin Schleef (PlasmidFactory Bielefeld) who provided a replicating S/MAR minicircle (corresponding to our vector M18) produced from our components and ideas for upcoming joint projects. Work in the authors lab at Hannover Med.
School
has
been
supported
by
the
the
CliniGene
Network
of
Excellence
(EuropeanCommission FP6 Research Program, contract LSHBCT-2006-018933), the Excellence Initiative “REBIRTH” (From Regenerative BIology to Reconstructive THerapy, the SFB 738 (Optimierung konventioneller und innovativer Transplantate) and ReGene (Regenerative Medizin und Biologie) grants, provided to the consortium by the BMBF.
44
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FIGURE LEGENDS Fig. 1: Chromatin domains, the smallest autoregulatory expression units A. Characterization of boundary elements (“constitutive S/MARs”/CSs) and of domain-internal context-dependent elements (“facultative S/MARs”). CSs are characterized by their attachment functions (hooked symbols) whereas facultative S/MARs mostly coincide with DNAse I hypersensitive sites (HS). Both types can be identified in SIDD profiles indicating the energy required for DNA strand separation at any location along the X-axis [kb]. Negative peaks (so called “unpairing elements”, UEs) that reach a G value of zero (0 kcal/mol) are expected to open at a given superhelical tension (usually σ = -0.05). Exemplified by the human interferon-ß (huIFN-ß-) domain the structure of constitutive S/MARs (base-unpairing regions/BURs at the domain borders) is associated with an oligomeric complex of scaffold-attachment factor A (SAF-A) and accessory proteins (here: the histone acetyltransferase p300, green labels). Other imprints in the SIDD profile mark the polyadenylation site (T), which is involved in transcriptional termination, and a breakpoint junction (A1235), i.e. a site involved in genomic deletions [5]. B. Interpretation of these data in the framework of a popular chromatin domain model. Each domain is delimited by extended, constitutive S/MAR elements, which, in case of the huIFN-ß domain, extend over ~ 4 kb each, covering the ~2.2 kb EcoRI fragment “S/MAR E” (upstream) and the ~ 3kb Hind III fragment “b” (downstream). The minicircle (MC), which is in the focus of this review, can be considered as a simplistic domain model in which a single constitutive S/MAR delimits a chromatin loop. Whereas these extended elements remain scaffold-associated independent of the organism’s cell type, the association of restricted, “facultative” S/MARs is governed by the cell- and stage-specific association of specialized factors, exemplified by YY1 (originally termed “nuclear matrix protein 1” / NMP-1) [6-8]. Facultative S/MARs can serve enhancer-accessory functions rather than acting as an insulatos. Common to both classes is the ease of strand separation, which causes spots of DNAse I hypersensitivity at the transition points to regular B-type DNA. These “HS sites” proved to be preferred integration targets for retroviruses [9] that are released from a pre-integration complex (PIC; see right-hand symbols characterizing the delivery of a circular provirus-precursor).
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Fig. 2: Components of the epigenetic code exemplified by histone H3 H3 lysines (residues K4/-9/-14/-18/-27/-36 and -79), indicating the transcriptional status of chromatin sites are predominantly located in its N-terminal tail domain. They are modified by either methyltransferases (HMT, now named “KMT”*)) or by acetyl transferases (HAT/KAT) and – deacetylases (HDACs). Activating modifications are indicated in green, italicized letters whereas inactivating modifications are symbolized by regular letters in red. It is of note that the degree of modification may induce different responses (K9- and K27 methylation reactions) whereas in other cases activation or inactivation depends on the chromatin context. H3-modifications inducing an inactive state occur in a stepwise fashion: Lysine residues K9 or K27 have to be de-acetylated in order to permit mono-methylation, which is still compatible with the active states. Subsequently, a methyltransferase induces the di- or even tri-methylated state. By recruiting the heterochromatin protein HP1, gene silencing is initiated at H3K9me where it is caused by DNA methyltransferases (KDM3/-6) to yield the triacetylated forms K9/-27. What follows is a complex, barely understood multi-component interplay involving, for instance, H3K9me3, Suv39h and DNA methyltransferases. *) The rationalized “KMT” nomenclature follows doi:10.1016/j.cell.2007.10.039.
Fig. 3: Chromosome-based expression principles: two current approaches and their combination In both branches of this representation, chromosome-based vectors comply with guidelines 1-3 (top). Both approaches (RMCE, left branch and minicircles/MCs, right branch) share the following principles: -
eukaryotic expression units are delivered in the form of an autonomous chromatin domain,
characterized by either two bordering S/MAR elements (hooked boxes, cf. Fig. 1) or, in case of a circular entity, by a single one (Fig. 1B); -
the application of a site-specific recombinase (Flp) inducing the recombination between two
identical Flp recombinase target (FRT-) sites (red half-arrows). If these sites are located on a single DNA molecule, as in branch B, the result is excision - here cleavage of a “parental plasmid”
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(PP) to yield two circular derivatives, the “miniplasmid” (MP, not shown) and the minicircle (MC); further details will be explained in Fig 14. While the latter contains the eukaryotic vector parts, the MP comprises prokaryotic and auxiliary sequences, which are lost as the cell divides; -
if two identical sets of different (“heterospecific”) FRTs are part of a genomic target and of a
donor plasmid as in branch A, the genomic cassette (Fmut-hygtk-Fwt, here the classical setup F3hygtk-F) is cleanly replaced by a matching donor cassette (F3-GOI-F). This Flp-recombinase mediated cassette exchange (Flp-RMCE) process avoids co-introduction of the prokaryotic vector parts (dashed line), i.e. the cassette on the donor plasmid takes the place of the selection marker cassette in a so called “flp-in” process. “+/-“ symbolizes a fused selection gene, typically composed of a positive (hyg) and a negative (HSV-tk) selection marker.
Fig. 4: Episomal persistence depends on the relative orientation between a transcription unit and the S/MAR A derivative of the parental S/MAR plasmid (PP) depicted in Fig. 3B was prepared such that the GFP transcription unit was flanked by two recombination target sites in inverse orientation. During a pulse of recombinase activity the transcription unit became inverted for 9 out of 15 clones; one clone (EG78.1) contained constructs with both orientations (compare PCR analyses in the top section with scheme in the insert). Subsequent Southern analyses, performed after vector linearization by HindIII, indicated full length episomes only in case of the functional original orientation (“o”) for which the transcript extended into the S/MAR; the signal at 7.3kb reflects the size of an intact episome. In cases where PCR analyses revealed inversion (“i”) of the transcription unit the blots were dominated by plasmid loss and/or multiple integration events.
Fig. 5: An established episomal vector permits RMCE-based modifications in situ
56
Following the principles in branch “A” of Fig. 3, a cassette (here a luciferase expression unit flanked by two different, “heterospecific” FRTs) can be exchanged by a cassette obeying the same architectural principles. An authentic modification requires the prior establishment of the target vector (here: a pEpi-type episome with a luciferase expression cassette), which is replaced by a secondary cassette (here: egfp) provided by a donor plasmid. RMCE is driven by molar excess of both the donor and the recombinase [1]. In the given example the exchange reaches 7%. RMCE events can be enriched by FAC-sorting, which results in a collection of mostly stable clones (here: 70%). Decreased expression levels can be ascribed to the silencing effects of plasmid episomes since the reaction is not reversed in the absence of Flp recombinase.
Fig. 6: S/MAR activities of eukaryotic base-unpairing regions (BURs) can be deduced from SIDD profiles DNA duplex destabilization properties of a DNA segment can be visualized after virtual cloning of the element in question into a standard plasmid (here the PTZ-18R vector, a pUC derivative). This corresponds to the context in which the experimental strengths of scaffold binding of many of the S/MAR fragments have been assessed. A - Sequence of pTZ_18R, which per se provides three unpairing elements (UEs) at the promoter and the terminator of the ampicllin resistance gene, as well as at the phage f1 Ori; these signals serve as internal standards. Besides the prototype SIDD profile [map position vs. G(x)], the respective probability profile (map position versus p) is indicated. This representation is usually of lower complexity (here: a single peak at the position of AmpT) indicating the site where strand unpairing initiates. B - The ~2 kb S/MAR insert providing the pEpi vector with replication potential, inserted into the pTZ_18R backbone. Note the repetitive pattern of UEs, which obeys certain spacing criteria. Obviously, the strongest S/MAR-UE (the “CUE” at position 800) surpasses the AmpT-associated peak regarding its strand separation potential C - S/MAR activity of the most strongly destabilized UE within the 2kb BUR in part B, determined by an in vitro assay for the oligomer series M1 (monomer) to M4 (tetramer)
57
D - SIDD profile for the tetramer (M4) in the common pTZ_18R context. Note that the insert competes with the AmpP- and f1-Ori-associated UEs but barely with the otherwise dominant “coreunpairing element” (CUE ) derived from AmpT.
Fig. 7: Minimalization approaches - pMARS and Minicircles pMARS is a derivative with a functional, minimal S/MAR composed from four units of the most effective UE (element M4 in Fig. 6D A – Upper scheme anticipates the conversion of a pEpi-derived parental plasmid (circle) into two derivatives, the minicircle (derived from the lower half) and the miniplasmid (from the upper half, containing the selection gene and the prokaryotic Ori) A – Bottom: FACS profile (eGFP-fluorescence versus size) for the pEpi-type vector containing the full 2 kb S/MAR and a corresponding profile for the pMARS derivative, i.e. the tetrameric synthetic sub-S/MAR “M4”. B, C – FACsorting for the pEpi derivative (~2 kb S/MAR insert) and the minimized pMARs version (4 x 155 bp “M4-“ insert)
Fig. 8: Establishment process of a 6.4 kb S/MAR plasmid and its 4.1 kb minicircle derivative, both containing the same ~2 kb S/MAR sequence Long-term expression of replicating episomes in CHO-K1 cells after 5-days (5 population doublings, PDs) of establishment. After this period fluorescent cells were recovered by FACsorting. 10 days (~10 PDs); after sorting 70% of fluorescent cells remained for the minicircle (MC), but less than 10% for the parental plasmid (here: the pEpi-vector shown in Figure 7A). If, at this time, the latter population is subjected to G418 selection, the remaining fluorescent subpopulation becomes dominant reaching 60% after 50 PDs in which case, however, FISH analyses proved integration events for 40 % of the cells (cf. Fig. 13). It is of note that presence of the HDACi butyrate (“+butyrate”) for a 24 h period - followed by release from this treatment 18 h prior to transfection of the S/MAR plasmid - significantly slows down the decay of the fluorescent cell
58
population. Due to the pFAR principle selection is neither possible nor required in case of the minicircle as a substantial proportion (65% in this experiment) of cells maintains fluorescence during the entire time interval. Insert: Butyrate pre-treatment (“+B”) improves establishment rates also for the MC. Under the given conditions [46] the population of fluorescent cells stabilized at 20% of eGFP-expressing cells if transfection was performed without prior butyrate treatment (“-B”) but at 33-40% if transfection followed a period in the presence of the HDACi. Controls demonstrated that the butyrate effect was not due to synchronizing cells at the G0/G1 case (the value determined after 24-36 h in 5 mM butyrate) but rater the consequence of a persistently altered epigenetic status initiated by, for instance, histone hyperacetylation.
Fig. 9: Individual MC-clones established in the nuclear architecture MCs of 4.1 kb have been transferred, by lipofection, into CHO recipient cells. FACScan (eGFP expression profiles) are shown for a cell population containing clones with 4–8 (average 5) copies of the MC (“mixture”). Authentic single clones (M23, L2 and H11) were analyzed correspondingly 37 days post-transfection. Clones M23 and L2 are associated with symmetrical FACS profiles. The Gaussian distribution found here is compatible with a unique (class of) association site(s). Interestingly, this pattern resists freeze–thawing cycles (freezing at day 37 post-lipofection, storage for 14 weeks, and FACScan 28 days after renewed continuous culture) as shown in the bottom row. KWT, control from non-transfected CHOK1 cells. The time course indicated on top (arrow) symbolizes the relevant stages during the MC life cycle between preparation/transfer and withdrawal. The present study comprises the maintenanceand expression stages following establishment.
Fig. 10: Co-transfer of minicircles accommodating heavy-chain (H) and light-chain (L) antibody genes supports survival of cells with an optimum expression ratio Left: L- and H- genes are provided as transcription cassettes that are incorporated separate from the fluorescence marker genes (rfp or egfp, respectively).
59
Right: FACS analysis after 20 generations – if red fluorescence is plotted versus green fluorescence, surviving cells accumulate along a diagonal representing a constant expression ratio. The upper-right quadrant contains 83% of all cells and served to recover high-producer strains (R. K. Masters thesis, TU Braunschweig, 2008).
Fig. 11: AN S/MAR-internal auto-deletion process and its functional relevance A, B - After long-term cultivation, PCR revealed a reduced size (2.9 instead of 4.1 kb) for 2 out of 13 clones (exemplified by “M18” in B). According to sequence analyses these clones underwent identical deletions (dashed red lines in A). The yellow star marks a premature transcription termination site functioning in the context of the extended, 2kb S/MAR element but not after the S/MAR-internal deletion as shown in section C. C - Northern blot analyses were performed on authentic polyadenylated mRNA and hybridized with a labeled eGFP-DNA probe. It is seen that transcript length depends exclusively on the extension of the S/MAR, which is reduced (prematurely terminated) in case of the full 2 kb fragment (4.1 kb MC and pEpi, lanes 1 and 3). In contrast, it is full-size for MC-derivative M18 (lane 2), its PPprecursor (lane 4) and the plasmid-vector pMARS (lane 5). D - SIDD profiles provide an explanation for premature termination since in these cases the destabilization of the SV40 poly(A) sequence (marked by the left-hand circle) is reduced by competition with the extended ~2kb BUR. This is depicted for pEPI (top SIDD profile) relative to the M18 minicircle derivative (bottom profile [46]). E - S/MAR size-reduction leads to improved expression (and long-term stability; [46]), a plausible consequence of authentic transcript termination and –polyadenylation (red star-symbol in A).
Fig. 12: Southern blots for a number of expressing CHO-K1 clones Clones have been pre-sorted, by FACS, 7 PD after DNA transfer. An aliquot of the clone mixture (T) and single clones were cultured before high molecular weight DNA was harvested from 1×106 cells. Genomic DNA was cut with the MC-single cutter BstZ17I, subjected to Southern blot
60
analyses and visualized by a radioactive egfp-probe. The selection of single clones comprises clones M23 and H11, which have been analyzed for expression and long-term persistence as shown in Fig.11.
Fig. 13: Metaphase-FISH analyses FISH-analyses were performed 55 PDs after transfection. Sections MC(1) and MC(2) demonstrate the presence of individual minicircles while sections pEpi(1) and pEpi(2) give evidence of integration events (intense doublets across the chromosome arms are found in 40% of the cells). Note that parental plasmids and minicircles are presented at different magnification while the overview (“wt”) corresponds to ~half the magnification chosen for the pEpi slides. See ref. [83] for technical details.
Fig. 14: Multiple options for generating and elaborating Minicircles (MCs) in situ, i.e. in the recipient eukaryotic cell The replication potential of this vector class is due to the properties of a scaffold/matrix attachment region (S/MAR), which enables non-covalent anchoring the MC to chromosomes in the host cell. MCs and active precursors (PPs) are the first true mammalian equivalents to yeast ARS plasmids. -
MCs can be generated from parental plasmids in situ using the Flp recombinase encoded
on the vector backbone (blue arrow). After recombining two identical target sites (FRTs, red halfarrows) it is separated from the promoter becoming part of the MP. -
The MP has no replication capacity and, in contrast to the MC, is lost as the cell divides.
MCs consists only of eukaryotic sequences, among these the gene-of-interest (here: eGFP, green arrow) and a 730 bp S/MAR (derivative “M18”). -
The additional inclusion of a heterospecific FRT (yellow half-arrow) permits subsequent
elaboration of the MC after its establishment in the nuclear architecture (principle in Fig. 5). In the given example RMCE is applied to introduce the GOI, which removes and takes the place of a positive/negative selection marker. Note that the functions of the respective coding regions (+/-
61
selection marker or GOI), individual promoters, IRES elements or fusion genes have not been specified as these may vary from case to case. Optimal versions are about to enter large-scale minicircle production routines as described in the text.
62
Table 1: Context-dependent and anti-silencing actions of an 800 bp S/MAR element (origin: 5´boundary of the huIFNβ gene domain). The element is used alone or in combination with the cHS4 element (an insulator from the chicken - beta globin gene cluster) Insulator [S/MAR IV / cHS4]
RV vector system
Cell type
Activity
Orientation dep.
Ref.
IV
MoMuLV
huPBL; CD4+/8+ T (resting!)
Similar function within or ahead from 3´LTR. Prevents silencing, not: IS-dependence; HIV replication in CD4+ 100x more inhibited
++1
[21]
IV
MoMuLV
human CD4+/CD8+ T; primary MΦ
HIV replication inhibited in CD4+ T but not monocytes. Effect due to 2-10x expression of RevM10 in T . No of expr. Cells and level ↑
[22]
IV
MoMuLV
human T-cells (CEMSS)
Prevents 5´LTR de-novo methylation (100%); continued stable expression; level parallels copy No.
[23]
IV
MoMuLV/MSCV
mobilized CD34+ HSCs
4x long-term increase of transgene express./cell
[24]
IV
Onco-RV vector (Phoenix GALV)
Baboon marrow cells prestimulated/
Retransplant mixed (+/- S/MAR) population. S/MAR increases expression in all hematopoietic lineages 2-9x for 6-12 mo
[25]
transplanted IV (+/-cHS4)
Lentiviral VSV-G
human hematop. stem cell line KG1a and progenitor cells
Optimal expression in presence of SAR-cHS4-3´LTR
IV(+/-cHS4)
pSFb91 (SFFVxMESV)
K562, HEK293, KB3.1 lines
S/MAR-[cHS4]2 -3´LTR most efficient (elements “oppositely oriented”)
IV(+/-c)HS4
VSV-G
human mesench. stem cells (ASCs)
S/MAR alone superior. No 5´LTR CpG-meth. at day 2 180.
++
[26, 27] [28]
-- (!)
[29]
1
directional effect argues against enhancer mechanism; 2 H3 deacetylation precedes methylation in control. CpG methylation, does not establish but rather fixes inact. state
63
Nehlsen 2011, Fig. 1 huIFN-β
A1235 T
HS-sites Mass-
Binding
P enh MC I Nuclear Scaffold/Matrix:
I
I
- Lamins - Matrins (ARBP/meCP2, Calmodulin, DNA-Polß, HAT, HDA, HMG, HAT, HDA, HMG 1/2, Nucleolin, NuMa, PARP, SAF-A/hnRNP-U , SAF-B, Topo II, ssDNA-Binders); - Transcriptional modulators (SATB1) and specific transcription factors (YY1/2).
64
Nehlsen 2011, Fig. 2
N-terminal tail M1
K4
K9
K14 / K18
me1)
me2)
me3)
me2
me2
me
me2
me2
me36)
me37)
me38)
me34)9)
me3, me3
ac10)
ac11)
ac12)
• de-acetylation →→ • tri-methylation → • DNA-methylation
K27
K36
K79
A136
me5)
ac13)
1)
KMT/Set1; 2) KMT1/KMT8/G9a; 3 ) KMT6; 4) KMT3; 5) KMT4; 6) KDM5; 7) KDM3; 8) KDM6; 9) KDM4; 10) KAT13; 11) KAT2/12; 12) KAT3/12; 13) KAT3A/B
65
Nehlsen 2011, Fig. 3
Guidelines and Strategies: 1 – No random integration (→ RMCE or episomal DNA) 2 – Never co-introduce prokaryotic (plasmid-) DNA 3 – No co-expressed gene / sel. marker besides the GOI ampr
Flp
*
off
II Nonviral Episomes Flp - excision
A
*
„Minicircle“
*
Epi-retroviral transfer
eGFP
B
A - Establishment B - Maintenance 66
67
68
Nehlsen 2011, Fig. 6
A
B
C
D
69
Nehlsen 2011, Fig. 7
A pEpi-FGSARF
B
31 %
C
pMARS
64 %
70
Nehlsen 2011, Fig. 8
parental pl. + butyrate. (minicircle)
PDs
71
Nehlsen 2011, Fig. 9
Preparation → Transfer → Establishment → Maintenance → Expression → Withdrawal
72
Nehlsen 2011, Fig. 10
2-Minicircle System Supertransfection of egfp expressing cells with an rfp vector
RFP GFP
73
Nehlsen 2011, Fig. 11
A
B
PSV4 0
polyA
eGF
* *
S/MAR R
C
D
E
∆1.3 kb
pEPI
pMARS
74
Nehlsen 2011, Fig. 12
75
Nehlsen 2011, Fig. 13
5µm
5µm
MC (2) 76
Nehlsen 2011, Fig. 14
ampr
Oripro
hygtk
Flp
parental plasmid (PP)
off PSV40
eGFP
S/MAR
donor plasmid
1 - Excision ÷ Flp termination
2 - Hyg-selection
on
÷ ESTABLISHMENT miniplasmid (MP)
3 - RMCE 4 - Ganc-countersel.
minicircle (MC) 77
