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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 / Nationale Forschungsförderung.

3) Department of Pathology, Josephine Nefkens Institute Erasmus MC 3000 CA, Dr. Molewaterplein 50 Rotterdam, The Netherlands Germany

4) Hannover Medical School (MHH), Carl-Neuberg-Strasse 1, D-30625 Hannover, Institute for Experimental Haematology OE 6960, Room J11 01 6530; Tel.: +49 511-532-5136; Fax: +49 3212 106 7542; bode.juergen@mh-hannover.de;

*) Corresponding Author

5) Rentschler Biotechnologie GmbH Erwin-Rentschler-Straße 21, 88471 Laupheim

Keywords: minicircles; nonviral episomes; ARS assay; oriP; S/MAR

Abbreviations used: BPV, bovine papillomavirus; BUR, DNA base-unpairing region; CHO, Chinese hamster ovary; CUE Core-Unpairing Element; CS, constitutive S/MAR; DS, dyad symmetry element; EBV, Epstein–Barr virus; eGFP, enhanced green fluorescent protein; egfp, the corresponding coding region; FACS, fluorescence-activated cell sorting; FISH, fluorescence in situ hybridization; Flp, flippase (site specific recombinase); FR, Family of Repeats (OriP); FRT, Flp-recognition target; GANC, ganciclovir; GOI, gene of interest; GOD, gene on duty; HDACi, histone deacetylase inhibitor; HMT, histone-methyltransferase; IR, initiator of replication; IRE, inverted repeat, LRT, long terminal repeat; LUC, luciferase; MC, minicircle; MP, miniplasmid; Ori, origin of replication; MRE, mirror-repeat; Ori, origin of replication; OriP, origin of plasmid replication; ORC, origin-recognition complex; PD, population doubling; pEpi, plasmid-episomal; pFAR, plasmid free of antibiotic resistance genes; PP, parental plasmid / educt for MC preparation; RMCE, (Flp-)recombinase-mediated cassette exchange; S/MAR, scaffold/matrix attachment region; SIDD, stress-induced duplex destabilization; SV40, simian virus 40; UE, DNA Unpairing Element; TIC, teratoma-initiating cell.

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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

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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

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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]

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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-

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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/MAR-

IV 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

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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 methyl-

transferase 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 uni fying 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 enhancer-

support 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

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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 SAF-

A 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

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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 an d/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/MAR-

plasmid (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/MAR-

minicircles 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 mamm alian cells?

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

5µm

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(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]).

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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 ra te 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

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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 over-

wound 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

15

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 Hsu and Uludağ [58] have applied four gene carriers,

polyethyleneimine (PEI), poly-L-Lysine (PLL), palmitic 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 pro-

nuclei 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

16

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 tri-

methylation (K9me3), and thereby silencing [62]. This study showed S/MAR episomes to

preferentially interact with early replication sites that are spread throughout the nucleoplasm.

17

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.

18

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 <5%). After establishment the episomes are stably maintained for hundreds of

generations. As mentioned, major efforts have been invested to overcome these limitations, among

which the excision of prokaryotic vector parts, i.e. the generation of minicircles (MCs) appears as

the most promising one [66]. Since a time-efficient MC preparation proved to be a major

bottleneck, certain exploratory studies still rely on SMAR-plasmid precursors, such as the pilot

study in Fig. 5. This experiment explores the potential RMCE- (recombinase-mediated cassette

exchange) for replacing, within an established nonviral episome, a marker gene expression

cassette (here: luciferase) for a cassette bearing a gene of interest (exemplified by GFP).

RMCE (Fig. 3A) is a relevant extension of the Flp/FRT-methodology [67], which is

thoroughly addressed in a parallel review [1]. RMCE is based on sets of heterospecific, 48 bp FRT

sites differing in the sequence of the 8 bp spacer, which separates two inverted 13 bp Flp-binding

elements. The term “heterospecific” implies negligible or no cross-interactions between two spacer

variants (FRTmut x FRTwt) but maximal recombination between identical sites (FRTmut x FRTmut =

FRTwt x FRTwt). Under these conditions, a cassette FRTmut – GOI1 - FRTwt remains perfectly stable

even in the continued presence of Flp activity [1]. Only in case a second cassette of the same

architecture is provided as part of a donor plasmid and at a molecular excess, this donor cassette

(FRTmut – GOI2 - FRTwt) can be “flipped in”, cleanly replacing the resident one, which is either

genomically anchored (Fig. 3) or part of an established episome (Fig.5)

The feasibility of the latter concept has been shown by exchanging a luciferase gene (here part

of an established S/MAR-plasmid) for an eGFP gene that enters the scene as part of a (non-

replicating) donor plasmid. In the present experiment gain of eGFP fluorescence for 7% of the cells

in the absence of selection reveals a rather high RMCE-rate when compared with integrated

acceptor sites [13,67]. This renders the concept suitable even for successive RMCE-steps. While

these data already indicate a remarkable accessibility of the episomal target, a more uniform and

rather stable population can be obtained after FAC-sorting the fluorescent cells. Of particular note

is the fact that, whereas copy numbers vary by just a factor of two among the clones, expression

19

levels may differ by two orders of magnitude [46,52]. In the case of S/MAR plasmids this may be

ascribed, in part, to silencing with time. In case of minicircles, later experiments will demonstrate,

however, that a similar diversity is governed by clonal properties [52]: while all acceptor sites are

early replicating and rather highly expressed, their occupancy is stable in agreement with their

characterization as described under “Nuclear association sites”.

• Remaining shortcomings and their solution

While the topic “limited establishment of nonviral episomes” has been introduced above, this

parameter has to be judged by comparison with viral paradigms which have the reputation of being

highly efficient.

Establishment and maintenance: the EBV paradigm So far the greatest progress toward the

development of an efficient episomal gene therapy vector goes back to plasmids based on

Epstein-Barr virus (EBV), a member of the gamma subfamily of herpesviruses [68]. Molecular

details of EBV replication are well understood: the EBV latent origin has been identified and named

OriP (origin of plasmid replication). To initiate replication, OriP requires the presence of a trans-

acting factor, EBV nuclear antigen-1 (EBNA-1). OriP extends over 1.7-kb and comprises two

functional elements, the family of repeats (FR) and the dyad symmetry (DS) element. The latter is

a 120-bp region containing two EBNA-1-binding sites separated by 9-bp. The FR element in turn

comprises twenty EBNA-1 binding sites. It has been demonstrated that it enables EBV genome

retention as an episome. EBNA1 complexes on both, DS and FR, interact by a DNA looping

mechanism in a way reminding of nuclear matrix proteins like SAF-A, which has been implicated

in the replication of S/MAR-based episomes (Fig. 1B).

The FR element increases transcription rates depending on the number of EBNA-1-binding

sites. Detailed investigations on Raji cells showed that the reduction of repeat-numbers on an

EBV-derived plasmid decreases the formation of antibiotic-resistant colonies by three orders of

magnitude as the plasmid is lost. For living cells it could be shown that EBNA-1 mediates the

segregation of OriP plasmids and a high mitotic stability by their attachment to metaphase

20

chromosomes. In summary, EBNA-1 mediates replication by binding to two elements, an initiator of

replication (the “DS-“, otherwise called “IR”-element) and to FR, the latter enforcing its retention.

- Complementarity of “molecular glue” and initiato r of replication (IR-) functions “Raji ori”, a

second Ori in the EBV genome permits licensed DNA synthesis over restricted time intervals but

fails in long-term ARS assays [68,69]. While, for oriP, 90–99% of newly introduced plasmids do not

support initial DNA synthesis, for Raji ori, the range is 99.99–99.999%. This situation can be

alleviated, however, if a floxed (i.e. reversibly mounted) DS element is provided in cis. Under these

circumstances, long-term extrachromosomal replication becomes possible and it remains stable,

even in case the DS unit is removed after establishment. These observations confirm that episomal

retention depends on a combination of “molecular glue” (FR-type) and “initiator of replication (DS-

/IR-type) functions and that the initial replication rates (enabled by DS) are of primary relevance.

Again, this observation serves as a guideline for further improvements of the nonviral counterpart.

- Two variants of an L1 transposon system A revealing side-by-side comparison of EBV-type

and S/MAR based episomes was recently reported by Rangasami [70]. Both constructs were

applied to generate L1 transcripts that are spliced and reversely transcribed to generate a

transposon that becomes genomically integrated (retro-transposon principle).

Unexpectedly, chromosomal integrations of the EBNA1-based primary vector were quite

common, explaining a significant loss of copies/expression over 50 days, possibly due to the

repetitive nature of L1 sequences. Using the S/MAR-based counterpart, the L1 cassette proved

increased, prolonged retrotransposition efficiency due to higher expression levels under otherwise

identical conditions. Virtually all cells maintained episomal vector copy numbers for at least 50

days illustrating the stability of the respective L1 transposition construct as part of the nonviral

system for cultured human cell lines.

These differences did not follow the initial transgene copy number per cell, which was about

two for the S/MAR-L1 construct but exceeded 40 in case of EBNA1-L1.

- Can replication-support elements be shuffled bet ween the EBNA1- and S/MAR-vectors?

Considering the beneficial actions of S/MARs in an episomal context with regard to vector

maintenance and transgene expression in mammalian cells, Giannakopoulos et al. [71] introduced

21

the standard 2kb element into an EBNA1 episome in order to improve its maintenance, which

otherwise diminished over time. Unexpectedly, this step completely eliminated the capacity of the

resulting Pcmv-OriP-EBNA1 vector to replicate as an episome. The comparative calculation of SIDD

profiles for a series of constructs suggested that the S/MAR and the DS element are in mutual

competition with DS being on the looser side. As a result, its strongly reduced duplex

destabilization will have affected the vector’s replicative potential. This situation changes if an

alternative replication of Initiation Region (the IR-element from the β-globin gene locus) takes the

position of the EBV-Ori: an Pcmv-eGFP-S/MAR-IR insert not only restores the replicative capacity

but it also enables an enhanced episome retention. These data underline a synergism between the

S/MAR- and the IR-elements with regard to vector-retention and -replication potential. While the

conventional S/MAR vectors have Ori-support (otherwise called “molecular glue-”) functions,

initiation of replication occurs “all over the place” [50]. A strictly localized initiation, however, is

likely to be superior for effective vector propagation.

Recently, these experiences were used to improve episomal maintenance in human

hematopoietic progenitor cells. To enhance the vector’s potential the ß-globin IR element was

mounted as before, while transcription through the eGFP marker was driven by either the

EF1/HTLV or the SFFV promoter. SIDD analyses of the respective S/MAR plasmids anticipated

that these changes would preserve the function of both elements, S/MAR and IR. In fact, after a

single initial sorting step all vectors were quantitatively maintained as stable episomes in mobilized

CD34+ peripheral blood cells (A. Athanassiadou, submitted).

- Selection principles overcoming the need of anti biotics While there is little chance to recover

cells bearing plasmid-derived vectors in the absence of selection, several concepts are underway

to avoid the risk of antibiotic resistance marker dissemination, i.e.

- providing a growth advantage independent of drug selection markers [72];

- using FAC-sorting instead; this approach is necessarily restricted to cells that can be kept in

culture [73];

22

- applying novel approaches to produce plasmids Free of Antibiotic Resistance genes, called

pFARs. The strategy is based on the suppression of a chromosomal nonsense mutation by a

plasmid-borne function [3].

Previous experiments have shown that a prototype S/MAR-plasmid vector encoding the luciferase

reporter gene enabled transgene expression for at least 6 months following hydrodynamic delivery

to mice livers. After partial hepatectomy, however, no detectable vector replication was seen to

persist. To deal with this phenomenon, Wong et al. [72] have developed an in vivo selection

strategy providing liver cells with a survival advantage. Accordingly, the vector was modified to

express the Bcl-2 gene conferring resistance to apoptosis in the presence of a Fas-activating

antibody. In fact, this Bcl-2-luciferase S/MAR plasmid enabled episomal replication and sustained

luciferase expression for more than three month. Quantitative PCR was performed at the end of

this period to compare the copy number of plasmid molecules revealing a tenfold increase for the

S/MAR vector relative to a non-S/MAR control. This confirms, for the first time, the ability of S/MAR

plasmids to replicate and establish mitotic stability at a detectable level after application to an adult

organism, as long as there is a selective advantage.

From a Molecular Biologists point of view, transcription units and promoters of bacterial or

fungal origin are common initiators of inactivating methylation reactions in mammalians. In case

drug resistance genes are applied, their expression next to the GOI contributes to promoter-

interferences and silencing effects [14]. Along these lines, attention is paid to the work by Gossen

and colleagues [73] demonstrating that the method to recover stably transfected cells has a

profound impact on transgene expression patterns. Standard antibiotic selection was directly

compared to FACS methods regarding the establishment of stable cells and proved that only the

second approach could overcome phenomena associated with the spontaneous resistance to drug

selection markers to provide uniform and stabile gene expression patterns. This was therefore the

method of choice for a stringent side-by side comparison of PPs and their MC derivatives (see

below).

Although conventional plasmids encoding antibiotic (aminoside-)inactivating proteins have

been approved for certain clinical applications, pFAR constructs gain increasing attention

23

representing the approach to overcome risks associated with the dissemination of antibiotic

resistance markers (or contaminating antibiotics): whereas luciferase activities decreased within

three weeks after intradermal electrotransfer of conventional plasmids, sustained levels were noted

for a pFAR derivative. Thus, novel strategies have become available for the efficient production of

biosafe plasmids, which has already proven its potential in several organs and tissues.

In this context it should be noted that the chromosomal vector strategies introduced in Fig.

3 are variants of a pFAR design relying either on the clean exchange of a genomic target by an

eukaryotic expression cassette (RMCE) or on the excision of accessory sequences after plasmid

production in bacteria (minicircle concept)

- Targets for DNA methylation: role of CpGs It is an accepted fact that eukaryotes have evolved

elaborate defense systems to fight the expression of ectopic transcription units in order to protect

the integrity of their genomes. In mammals, the insertion of retroviral DNA, the incorporation of

repeat arrays and the co-introduction of prokaryotic vector parts are major triggers of

transcriptional silencing processes. Additional defense strategies go back to the fact that

dinucleotide frequencies in mammals differ from those of other organisms. Of particular relevance

is a relatively low content of CpG dinucleotides, which, in addition, are often methylated (mCpG). In

most bacterial genomes, however, the occurrence of CpGs is in accord with statistical

expectations, and cytosines remain normally unmethylated.

Although the CpG content of mammalian genomes is low, regions exist where these motifs

reach statistical levels. These “CpG islands” are associated with many genes and are protected

from methylation (and thereby from subsequent meC → U transition) by interaction with

transcription factors. While silencing is commonly accompanied by the methylation of CpGs, these

events may depend on a prior methylation of histone H3 at Lys-9 [74]. In the context of Fig. 2 it is

of note that, for ES cells, promoters positive for H3K27me3 are fourfold more likely to acquire DNA

methylation. Such a methylation center can trigger chromatin condensation spreading to a

downstream promoter to provide it with a heterochromatic structure – at least in the cases where

such a process is not blocked by an intervening insulator element [13]. Regarding human

organisms, another level of defense is associated with the innate immune system, which has

24

evolved mechanisms to discriminate bacterial from intrinsic DNA via Toll-like receptor 9 (TLR 9-)

signaling.

- pEPIto Returning to the class of replicating nonviral episomes, consideration of a common

heterochromatization route led to developing a size- and CpG-reduced derivative of the pEpi

S/MAR plasmid, pEPIto [2]. Traditional pEpi-type vectors comprise a pUC-Ori for bacterial

propagation, the S/MAR, a second mammalian SV40-O/P driven transcription unit serving

selection purposes in bacteria (kana) and mammalian cells (G418) in addition to 206 CpGs. In

contrast, in the pEPito backbone there remain 37 CpGs, an R6KOri for bacterial propagation, an

ampicillin selection gene and the common 2 kb S/MAR. A second transcription unit (eGFP-IRES-

BSD) encoding both the fluorescence marker and a drug selection (BSD-) function provides

additional options.

Side-by side controls proved both, increased transgene expression levels and colony-

forming efficiencies, for pEPito in vitro, in addition to a more persistent expression profile in vivo.

While, in the present setup, the establishment efficiency for pEpi-1-replicons was as low as 0.25%

in a colony-forming assay, it was six-fold higher (~ 1,8%) for the pEPito-construct, both controlled

by a CMV promoter. Although the effect of unmodified CpGs is an accepted contribution to

silencing and while it may have determined the outcome of this study, there is a recent report [75]

that CpG content is of minor or no relevance in the context of a minicircle, which mostly consists of

eukaryotic sequences. In particular, CpG islands are clearly exempt from negative actions, which is

particularly obvious in the case of an UCOE. Such a Ubiquitous Chromatin Opening Element

confers resistance to DNA methylation–mediated silencing in line with its origin from two

divergently transcribed promoters that are embedded in an extended methylation-free CpG island

[76].

In summary, to date all findings are compatible with heterochromatization steps initiating at

bacterial vector elements. Apart from CpGs tracts, there seem to be other distinguishing features

between bacterial and mammalian genomes which may trigger structure-based discrimination

steps [77]. Among these is the relative occurrence and secondary structure forming potential of

inverted and mirror repeats. These IREs or MREs can form cruciform- or intramolecular triplexes

25

(H-DNA) in negatively supercoiled domains. Since supercoiling triggers strand separation for B-

DNA, it is one of the prerequisites for these and related structural transitions. Whereas in E.coli

IREs are mostly restricted to transcriptional termination sites, MREs comply with statistical

expectations. Sequences with H-forming potential on the other hand are only typical for humans.

Together these considerations suggest the existence of a multi-facetted structural code that is

recognized in a foreign host to delimit the expression of foreign genes.

Vector-size limitations (?) Over the years evidence for an inverse relation between episome size

and -stability has accumulated. This became particularly evident during cloning, electroporation-

mediated vector transfer, FACsorting routines, persistence of the superhelical state during freeze-

thawing cycles and long-term stability in mammalian cells. For pEpi-type vectors the performance

was best if their size did not exceed 10 kb as it strongly deteriorates above ~15kb.

Regarding these observations the description of a 156 kb iBAC-S/MAR-vector (pEPHZ-LDLR) by

Lufino et al. [78] came as a surprise, the more as the authors demonstrated that such a construct

enables infectious delivery while retaining a 135 kb transcription unit. CHO cells were infected

exploiting the high transgene capacity of herpes simplex virus type 1 (HSV-1). Infected cells were

kept in selective media for two weeks after which 108 early stage single clones could be isolated,

most of which did not survive. Ten growing clonal lines could be maintained and screened, by

plasmid rescue, for their episomal status, which succeeded in three cases. Two clones were kept

in the absence of continued selection and shown to preserve a low-copy number episomal status

for at least 100 cell generations. While these observations indicate that there is no stringent upper

vector size limit impairing episome function, they nevertheless confirm very low rates of

establishment in case a certain size limit is exceeded.

C – MINIMALIZATION APPROACHES

26

Following these tendencies, minimal sizes for nonviral extrachromosomal entities should be of

value. This not only concerns the class of pure minicircles that are derived from so called “parental

plasmids” (PPs) by excision of redundant auxiliary sequences, but also their replicating S/MAR

variants (Fig. 3B). Based on their intrinsic molecular glue- and replication-support activities nonviral

episomes, and minicircles in particular, are in the position to utilize the host´s replication and

segregation machinery.

Another minimalization option was shown to reside in the S/MAR element itself. For

unknown reasons it is so far almost exclusively the 2kb sub-S/MAR sequence from the huIFN-β

domain (Fig. 1) that has been used to provide replication potential – sometimes at the expense of

long-term stability and an increased tendency to integrate. Since the rules determining S/MARs are

known (Figs 1, 6, 7), the stage was set for systematic minimalization efforts at this level. The

relevant principles can be summarized as follows:

- S/MARs occur only in eukaryotic genomes, where they serve a variety functions.

They represent DNA elements, between a few hundred to several thousand base-pairs in

length, which are operationally defined by their affinity for the nuclear scaffold or -matrix. In

the present context the term “scaffold” denotes the common protein network serving

primarily structural support functions, whereas the term “matrix” stands for the entire

complement of proteins resisting a given nuclear extraction routine. A potential overlap of

these functions is reflected by the consensus term “scaffold-matrix attachment region”

(S/MAR). Since S/MARs do not share obvious sequence motifs, an important component

determining their performance is thought to rely on structural particularities;

- a given S/MAR may have predominantly context-dependent (facultative) activities or

constitutive (domain bordering) functions. While the first group can even be shorter than

originally claimed, consisting of a single strongly-destabilized UE of or slightly above 170 bp

plus accessory transcription factor binding motifs [79], the second group comprises an

extended register of moderately-destabilized UEs that have to obey certain structural rules

[5]. This architecture mediates an association with the ubiquitous components of the

27

nuclear scaffold (exemplified by SAF-A), which may acquire secondary nuclear matrix

components.

Finally, several minimalization principles (deletion of prokaryotic vector parts and selection markers

or minimal arrays of UEs) can be combined anticipating that this will enable a second-generation

minicircle with superior establishment and stability, both regarding its physical status and

expression characteristics.

• Oligomerizing S/MAR modules: pMARS and its properti es

The approach by Jenke et al. [80] relies on an oligomerization strategy to investigate the S/MAR

potential of a 155-bp module, i.e. the most destabilized UE in the 2 kb standard element, and of

155n-oligomers. Initial scaffold-reassociation studies in vitro confirmed the continuous increase of

activities when oligomerizing the monomer and showed that the binding strength of the original 2

kb template was approached at the tetramer level (620 bp; Fig. 6C). In this and other examples the

term “activity” is not restricted to affinity parameters but it extends to biological functions such as

transcriptional “augmentation” (shielding a transgene insert from an heterochromatic environment),

the effect of histone hyperacetylation and, in the present case, the capability of episomal

replication in the context of a plasmid. An association of these minimized vector derivatives with

nuclear matrix components could be demonstrated by FISH analyses and by in vivo crosslinking

using cis-diammineplatinum(II)-dichloride (cis-DDP) [63]. FISH analyses per se proved a non-

covalent association with the mitotic chromosome for the S/MAR-tetramer, but not the dimer,

which, using the cisDDP protocol, could be ascribed to SAF-A.

Fig. 7 illustrates a rather dramatic effect of this minimalization during an early time interval.

Transfected cells were sorted out after 5 population doublings (5 days) and kept in continuous

culture for another two weeks before FACScans were recorded. Their comparison shows that after

this interval 31% of pEpi-expressing cells had persisted, while for pMARs 64% of the recipient cells

were still active. In both cases the loss of fluorescence may be due to inadequate establishment

and -maintenance, to silencing or a combination of both. These functions may have been improved

for the size-reduced pMARs derivative, but in case of pEPI (and certain derivatives) there is

28

evidence for yet another contribution: within the standard 2 kb S/MAR polyadenylation of the

growing mRNA occurs at a cryptic signal, whereas the 620 bp tetramer permits the transcription

apparatus to transverse the entire element before being processed at the authentic SV40

polyadenylation signal located at its 3´terminal end (Fig. 11 will deal with these phenomena in a

wider context). Together these findings show a complex interplay of various expression parameters

associated with the extension and stability of the transcript. Recall that this transcript has to cover

S/MAR sequences beyond the translational stop signal.

• Replicating minicircles, a solution with great prom ise

While there is significant progress in the modification, by episomal DNA, of slowly-dividing tissues

like liver, muscle and brain, maintenance problems have so far limited the use of nonviral

episomes for dividing cells, for instance of the hematopoietic system. For liver, the most advanced

vehicles appear to be “minicircles”, small circular vectors that are exclusively composed from

eukaryotic sequences. In contrast to linear DNA, minicircles do not form concatemers and are less

prone to integration. It is also known that, owing to their superhelical status, they are superior

transcriptional templates DNA [41]. Based on this rationale M. A. Kay and co-workers have

demonstrated that transgene expression levels in minicircles can be 45-560- [66,81], or even 10-

1000fold [82] higher and also more persistent than conventional plasmids. These vehicles were

therefore subjected to a critical test to prove their episomal state, i.e. a 2/3 hepatectomy upon

which almost every hepatocyte undergoes one or two cell doublings until the liver mass is

reconstituted. The results show that minicircles per se are not functionally attached to

chromosomal DNA and they anticipate the category of problems that have to be overcome in case

replicating S/MAR variants are used for the modification of proliferating cells [72]. Studies on

S/MAR-plasmids predict that, to be effective, minicircles will not only need replication potential but

also the capacity to become established in the nuclear architecture. To provide these properties

both molecular glue and initiator of replication functions are required, which come to life only in the

appropriate superhelical (ccc-) context.

By subjecting a pEpi-derivative to the machinery required for excising its plasmid parts

while maintaining the ccc-status, we could introduce the first minicircle that met a major part of

29

these requirements [83]. Since we have established an Flp-recombinase based toolbox to permit

the inversion, excision as well as (RMCE-mediated) integration of appropriately flanked expression

cassettes, we preferred the Flp/FRT system over alternatives that have been applied to the same

end.

Establishment and maintenance parameters Following these considerations a side-by-side test

of pEpi-type S/MAR-plasmids and their minicircle derivatives was performed [83]. To enable a

stringent comparison, we applied a single FACsorting step to obtain two populations of 100%

fluorescent cells (Fig. 8), the stability of which could then be followed for extended periods of time

(here: 50 population doublings, i.e. approximately 50 days). If the sorting was performed after an

initial 5 days period, establishment of the minicircle was almost complete, evidenced by the fact

that there was just a slight further decay, i.e. the level of expressing cells remained at about 70%.

Sorting at this point maximized the difference to the S/MAR plasmid, which, as a possible

consequence of continuous silencing, was completely lost during 30 PDs. Although the outcome of

such a comparison is clearly coined by the time point of sorting, major intrinsic differences between

both systems become obvious:

- At first view, the apparent deficiency of the plasmid derivative can be overcome by

drug selection starting as late as at 12 PDs, however:

- analyses on the recovered population, which persists due to the presence of a neor

gene, indicate that ~40% of cells have lost the episome by integration [52] .

Episome establishment and maintenance is a complex process based on epigenetic parameters

and stochastic events of largely unknown nature. A positive effect of histone deacetylase inhibitors

such as Trichostatin A (TSA) and butyrate was provisionally ascribed to arresting cells in G1 and

G2 just ahead nuclear membrane breakdown in early metaphase. This idea had to be abandoned,

however, because this effect did not arise using alternative synchronization approaches. Attention

was therefore paid to the role of open chromatin structures, which persisted after time-limited

histone hyperacetylation [46]. Meanwhile this concept is systematically pursued for improving the

establishment of minicircles. The insert of Fig. 8 shows a corresponding experiment.

30

Clonal behavior It has been noted before that the expression profiles of both, S/MAR-plasmids

and minicircles cover 2-3 orders of magnitude (Fig. 9, top), which has to be interpreted considering

the fact that this range goes back to just a two-fold variation of copy numbers (typically 4-8). For

S/MAR plasmids a partial explanation of this behavior is transcriptional suppression as it occurs at

the episomal state to be increased after its integration. Evidence for such a process comes from

the continuous shift of pEpi-expression profiles to low level positions, which can at least partially be

reversed by the action of a HDACi [83]. Minicircles, in contrast, usually show persistent expression,

which remains unaffected by HDACis. In this case the model suggests that variation simply reflects

the clonal behavior of cells after these entities have been firmly established [46].

Proof of this concept required the isolation of single clones and demonstration of their

persistence. Fig. 9 reports the outcome of these experiments, which gave raise to clones with a

sharp, symmetrical expression profile (M23 and L2 in Fig.9). Besides, there were some clones with

a double-maximum, which retained this property after sub-cloning (clone H11). Additional proof for

stability of M23 and L2 could be provided by an extended freezing - re-thawing cycle during which

both the expression profile and the episomal status were maintained.

Bi-MC systems These data encouraged the use of minicircle clones for expression purposes, the

more as these combine properties of transient and of stable expression systems. Proof-of-principle

comes from two-minicircle transfer experiments in which one entity encodes the light (L-) chain of

antibody and the other its heavy-(H-) chain counterpart. Usually, a certain overexpression of the

(secretable) L-chain proves beneficial because this entity provides chaperone functions when it

comes to correct folding of the H-chain (that would otherwise plug the endoplasmic reticulum

followed by cell death).

Significant conclusion on the system’s properties could first be drawn from the fact that bi-

MC clones can be established either by a synchronous transfer or by successive transfer steps, i.e.

transfection of the H-chain minicircle at a time where the L-construct has already been established.

Comparable efficiencies of both protocols indicate dynamic properties for nuclear substructures

31

exposing a number of sites that are suitable for establishment at any given time-point. This finding

invalidates an alternative explanation, i.e. saturation of binding sites already at low vector copy

numbers.

This model can be expanded by auto-regulatory principles. These emerged from the

observation that, regardless at which ratio H- and L-vectors have been transfected, both entities

are stably maintained at a certain ratio suggesting “survival of the fittest” (Fig. 10). Based on these

concepts technically more demanding setups can be overcome, which enforce certain expression

ratios by the choice of appropriate promoters, integration sites or the use of IRES elements to

create co-expression units with different properties.

MC-size reduction: “ In vivo evolution” The benefit of “survival of the fittest-”, otherwise called “in

vivo evolution-” principles emerged also at yet another level. In spite of their superior persistence

and expression properties minicircles showed instability in the long run, i.e. after >20 weeks of

continuous cultivation. We used the incidental observation of independent, but identical S/MAR-

internal 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

32

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 H-

encoding “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

33

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 pre-

amplification 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

34

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 >> nic ked 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

35

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 picking-

routines. 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

36

- 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

37

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

38

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 replication-

competent 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.

39

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 “pseudo-

transduction” [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 minicircle-

analogue 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-

40

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 milk-

specific 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 trans fer 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 two-

step 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

41

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

42

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 ARS-

like 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

43

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.

44

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.

45

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FIGURE LEGENDS

Fig. 1: Chromatin domains, the smallest autoregulat ory 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).

55

Fig. 2: Components of the epigenetic code exemplifi ed 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”

56

(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-F wt, here the classical setup F3-

hygtk-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 relativ e 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

57

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-unpairi ng 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)

58

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 “core-

unpairing element” (CUE ) derived from AmpT.

Fig. 7: Minimalization approaches - pMARS and Minic ircles

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 pla smid 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 FAC-

sorting. 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

59

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 nuc lear 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 maintenance-

and expression stages following establishment.

Fig. 10: Co-transfer of minicircles accommodating h eavy-chain (H) and light-chain (L)

antibody genes supports survival of cells with an o ptimum 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).

60

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 an d 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 PP-

precursor (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

61

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 elabor ating 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 half-

arrows) 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 (+/-

62

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.

63

Table 1: Context-dependent and anti-silencing actio ns 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)

1 directional effect argues against enhancer mechanism; 2 H3 deacetylation precedes methylation in control. CpG methylation, does not establish but rather fixes inact. state

[29] -- (!) S/MAR alone superior. No 5´LTR CpG-meth. at day

180. 2 human mesench.

stem cells (ASCs) VSV-G IV(+/-c)HS4

[28] S/MAR-[cHS4]2 -3´LTR most efficient (elements

“oppositely oriented”) K562, HEK293, KB3.1

lines pSFb91

(SFFVxMESV) IV(+/-cHS4)

[26,

27] ++ Optimal expression in presence of SAR-cHS4-3´LTR human hematop.

stem cell line KG1a

and progenitor cells

Lentiviral

VSV-G IV (+/-cHS4)

[25] Retransplant mixed (+/- S/MAR) population.

S/MAR increases expression in all hematopoietic

lineages 2-9x for 6-12 mo Baboon marrow cells

prestimulated/

transplanted

Onco-RV vector

(Phoenix GALV) IV

[24] 4x long-term increase of transgene express./cell mobilized CD34+

HSCs MoMuLV/MSCV IV

[23] Prevents 5´LTR de-novo methylation (100%);

continued stable expression; level parallels copy

No. human T-cells

(CEMSS) MoMuLV IV

[22] 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 ↑ human CD4+/CD8+ T;

primary MΦ MoMuLV IV

[21] ++1 Similar function within or ahead from 3´LTR.

Prevents silencing, not: IS-dependence; HIV

replication in CD4+ 100x more inhibited huPBL; CD4+/8+ T

(resting!) MoMuLV IV

Ref. Orien-

tation

dep.

Activity Cell type RV vector

system Insulator

[S/MAR IV /

cHS4]

64

A1235 T

HS-sites

huIFN-β

Mass- Binding

Nuclear Scaffold/Matrix : - 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).

P

enh

MC

I I I

Nehlsen 2011, Fig. 1

65

Nehlsen2011, Fig. 2

me2me2meme2me2

me5)me3)me2)me1)

K79K36K27K14 / K18K9K4

ac12)ac11)ac10)

me3, me3me34)9)me38)me37)me36)

M1 A136

N-terminal tail

• de-acetylation →→

• tri-methylation →

• DNA-methylation

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

66

II - Nonviral Episomes

Flp - excision

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

*

*

amp r

Flp

off eGFP

„Minicircle“ A - Establishment

B - Maintenance

*Epi-retroviral transfer

A B

Nehlsen 2011, Fig. 3

67

68

69

A B

C D

Nehlsen 2011, Fig. 6

70

pEpi-FGSARF

31%

A B

C pMARS

64%

Nehlsen 2011, Fig. 7

71

parental pl.

(minicircle)

PDs

+ butyrate.

Nehlsen 2011, Fig. 8

72

Preparation → Transfer → Establishment → Maintenance → Expression → Withdrawal

Nehlsen 2011, Fig. 9

73

GFP

2-Minicircle System Supertransfection of egfp

expressing cells with an rfp vector

RFP

Nehlsen 2011, Fig. 10

74

PSV4

0

S/MAR

R

eGF

polyA

* *

∆∆∆∆1.3 kb1.3 kb1.3 kb1.3 kb

A

C

B

D E

pMARS pEPI

Nehlsen 2011, Fig. 11

75

Nehlsen 2011, Fig. 12

76

MC (2)

5µm

5µm

Nehlsen 2011, Fig. 13

77

Nehlsen 2011, Fig. 14

Ori pro

amp r

minicircle (MC)miniplasmid (MP)

PSV40S/MAR

Flp

3 - RMCE1 - Excision ÷÷÷÷ Flp termination2 - Hyg-selection ÷÷÷÷ ESTABLISHMENT

eGFP

hygtk

donor plasmid

4 - Ganc-countersel.

off

on

parental plasmid (PP)