Chromosome-based vectors for gene therapy

Gene 304 (2003) 23–33 www.elsevier.com/locate/gene

Review

Chromosome-based vectors for gene therapy H.J. Lippsa,*, A.C.W. Jenkea, K. Nehlsenb, M.F. Scinteiea, I.M. Stehlea, J. Bodeb a

Institut fu¨r Zellbiologie, Universita¨t Witten/Herdecke, Stockumer Strasse 10, D-58448 Witten, Germany b Gesellschaft fu¨r Biotechnologische Forschung, Braunschweig, Germany Received 1 October 2002; received in revised form 22 November 2002; accepted 13 December 2002 Received by A.J. van Wijnen

Abstract Currently used vectors in human gene therapy suffer from a number of limitations with respect to safety and reproducibility. There is increasing agreement that the ideal vector for gene therapy should be completely based on chromosomal elements and behave as an independent functional unit after integration into the genome or when retained as an episome. In this review we will first discuss the chromosomal elements, such as enhancers, locus control regions, boundary elements, insulators and scaffold- or matrix-attachment regions, involved in the hierarchic regulation of mammalian gene expression and replication. These elements have been used to design vectors that behave as artificial domains when integrating into the genome. We then discuss recent progress in the use of mammalian artificial chromosomes and small circular non-viral vectors for their use as expression systems in mammalian cells. q 2003 Elsevier Science B.V. All rights reserved. Keywords: Artificial domains; Episomal vectors; Artificial chromosomes; Non-viral gene transfer

1. Introduction The therapy of human diseases exploiting the powerful tools provided by modern cellular and molecular biology and gene technology is one of the great intellectual and technical challenges of present biology and molecular medicine. It is expected that gene and cell therapy can be Abbreviations: ARBP, attachment region binding protein; ARS, Autonomous replication sequence; BPV, bovine papilloma virus; BUR, base unpairing region; CUE, core unpairing element; DCR, dominant control region; DNA-PK, DNA-activated protein kinase; EBV, Epstein– Barr virus; EBNA, Epstein–Barr nuclear antigen; FISH, fluorescence in situ hybridization; HIV-1, human immunodeficiency virus 1; HPRT 1, hypoxanthin guanine phosphoribosyltransferase 1; HS, DNAse I hypersensitive sites; HS4, chicken beta-globin locus associated insulator; HS5, human beta-globin locus domain border/insulator; HSV, Herpes simplex virus; LCR, locus control region; LTR, long terminal repeat; MAC, mammalian artificial chromosome; MeCP-2, methylated CpG binding factor 2; murine ES cell, murine embryonic stem cell; NF-kB, nuclear factor kappa B; PARP, poly ADP-ribose polymerase; pDNA, plasmid DNA; PE, position effect; ORI, origin of replication initiation; RMCE, recombinase-mediated cassette exchange; SAF-A, scaffold attachment factor A; S/MAR, scaffold matrix attached region; SV40, Simian Virus 40; YAC, yeast artificial chromosome. * Corresponding author. Tel.: þ49-2302-669-144; fax: þ 49-2302-669220. E-mail address: [email protected] (H.J. Lipps).

used for the treatment of many and very common human diseases where conventional medical procedures fail to be successful. While such therapeutic strategies are theoretically possible, their application is still limited by technical problems, among these are the delivery of transgenes into cells and tissues and the lack of efficient and safe vector systems. The final goal of all such strategies is to achieve the authentic expression of transgenes or nucleic acid sequences of therapeutic value in a specific cell type in the absence of undesired interactions with the host’s genome and without the risk of cellular transformation or stimulating the host’s immune system. This is rarely achieved using the conventional vector systems that are currently used for gene therapy. It is apparently possible to transfect muscle cells very efficiently with naked DNA. Using this technique long term expression of a transgene has been observed, although the status of transferred DNA in the cell is yet poorly analyzed. Transfection of other cell types can be very inefficient and therefore appropriate vector systems have to be chosen. Unfortunately, currently used vectors for eukaryotic cells suffer from a number of limitations which makes them only partially useful for gene therapy of human diseases (Prince, 1998). Integration into the host genome by for example retroviral vectors can lead to insertional mutagenesis and in

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all cases profound differences in the pattern and level of transgene expression have been observed. Recently, direct experimental evidence was provided that retroviral vectors can induce leukemia in mice and humans demonstrating the problems related with such integrating and, in particular, retrovirus-derived vectors (Li et al., 2002a,b; Marshall, 2000a,b; Check, 2002). On the other hand, transient expression as achieved for example with adenoviral vectors means that repeated transfections are required which can cause undesired immunological side effects in the host organism. For these reasons multiple transfections are sometimes not possible and in most cases not desirable. A solution to these problems could be the construction of vectors based on chromosomal regulatory elements which either behave in a way that they faithfully mimic gene expression from the natural context following integration or of vectors that are stably maintained and replicated as an episome in the target cell. Gene expression in eukaryotic cells is regulated at many different levels. While DNA-binding proteins and their interaction with the basic synthetic machinery drives transcription, it is now clear that the efficiency and the precision of this process is strongly influenced by higher nuclear organization (Cook, 1999; Cremer et al., 2000; Cremer and Cremer, 2001; Jackson, 2002). Nevertheless, it is generally believed that it is a limited number of DNA elements that is involved in nuclear organization, which suffices to reproduce essentials of the chromatin configuration. Understanding these elements and their cross talk would then allow to construct artificial domains containing the desired transgene. After integration this would assure a defined level of expression independent of the integration site and without interference with the host genome. Attempts to understand and apply these architectural and functional principles for an improvement of integrating or episomally maintained vectors will be the first major topic of this review. By principle, stable episomal vectors are free from the risk of insertional mutagenesis and in this case expression of a transgene should depend exclusively on its regulatory elements. Considerable efforts have been undertaken in the past to modify episomally replicating viruses, such as Simian Virus 40 (SV40), Epstein – Barr virus (EBV) or Herpes simplex virus, for their use as vector systems for human gene therapy. Limitations of cell specificity could be overcome by the insertion of chromosomal DNA-sequences in EBV-derived vectors (Calos, 1998). Yet, all these vectors require at least one virally encoded protein, such as the large T-antigen, Epstein –Barr nuclear antigen (EBNA) or Lana, for their replication and/or maintenance. Since these proteins cause immunological reactions in the host organism and can even lead to transformation of recipient cells, these constructs are only partially useful for gene therapy. Attempts to insert putative mammalian origins of replication into plasmids, an approach analogous to the autonomous replicating sequence (ARS) assay in yeast, failed in

most if not all cases suggesting that long term epigenetic features influence the complex process of eukaryotic DNAreplication (DePamphilis, 2000). Only recently the construction of small circular vectors based on chromosomal sequences could be achieved suggesting that it is possible to tailor these for use in mammalian cells. Immediately after the construction of the first yeast artificial chromosome (YAC; Murray and Szostak, 1983) efforts were undertaken to develop similar constructs (MACs) for mammalian cells. From there on it took 14 years until the first prototype of such a MAC was described (Harrington et al., 1997). Like for their yeast counterpart the functioning of a MAC relies on the presence of centromeric sequences, sequences that can initiate DNA replication and, in case of linear MACs, telomeric sequences. A MAC shares with the small circular episomal vectors the advantage that it does not interfere with other genomic sequences and that it should be mitotically stable in the absence of selection. In addition, it has a basically unlimited cloning capacity that allows not only the insertion of the gene of interest but of all sequences required for correct and cell specific expression. A large number of mammalian artificial chromosomes constructed by the assembly of functional chromosomal elements and minichromosomes, obtained by the fragmentation of existing natural chromosomes, does now exist. In this review both, prototypes of small circular non-viral vectors and of MACs, will be covered and their use for the genetic modification of eukaryotic cells and organisms and for gene therapy discussed.

2. Expression control: a concert with multiple players The regulation of eukaryotic gene expression involves the interaction of multiple trans-acting factors with specific cis-regulatory elements. There is recent evidence that for eukaryotic genes the chromatin structure is an important additional regulatory layer which is essential for authentic gene expression. In its default state it provides a tight packaging of the DNA and is repressive for transcription. Chromatin thereby serves a dual role: to compact the chromosomal DNA and to enable an efficient regulation of gene expression. 2.1. Simple and composite enhancers Enhancers are key regulatory elements which serve to relieve this chromatin-induced repression. Comparison with yeast suggests that, with the advent of higher eukaryotes, more and stronger elements became necessary by the formation of a powerful repressive nuclear compartment during differentiation. Enhancers were originally defined by transient transfection experiments in which an authentic chromatin structure was at most partially reconstituted. One early defining criterion was stimulation of transcription in a distance- and

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orientation-independent manner but the precise mode of action has largely remained an enigma. Using single cell detection techniques, there is recent data to support a mechanism according to which an enhancer affects transcription state (on or off) rather than transcription rate. Over time, most integrated constructs become silenced in cultured cells, a process that is markedly accelerated by deleting the enhancer. During such a process silencing is variegated meaning that in a population of cells a gene is silent in some and maximally active in other cells. These findings indicate that most enhancers act by increasing the probability that a gene will be active (Walters et al., 1996). Extensive evidence indicates that eukaryotic genomes are organized as sets of structural and thereby functional units (domains) that form the basis of independent gene activity. Actual models postulate a central switch (locuscontrol region, LCR), which determines whether or not a given domain is in the active, decondensed, histoneacetylated and DNA/histone demethylated state or converse. One central feature of these models is the existence of domain boundaries which delimit the action of enhancers and LCRs and thereby serve an insulating function. 2.1.1. LCRs Studies of the human b-globin locus first led to the discovery of an LCR which was able to overcome the integration-site dependent expression of transgenes, the so called position effect (PE). These observations led to its original designation as being a dominant control region (DCR). The property of this region suggests that an LCR can open silent chromatin and establish a transcriptionally competent domain (review: Li et al., 1999). Typically, LCRs are composed of multiple sequence elements including transcriptional enhancers, domain opening elements and tissue specific elements which are either clustered as for the b-globin locus or scattered throughout the domain as exemplified by the chicken lysozyme locus (Bonifer et al., 1994). The consistent appearance of DNAse hypersensitive sites (HS) demonstrates a local interruption of ordered chromatin structures. Like for classical enhancers, the activity of individual HSs is independent of orientation whereas the LCR as a whole acts in a manner which depends on both – orientation and distance. Since the human b-globin LCR is effective only in erythroid cells, the position-independent expression mediated by the element appears to depend on cell specific factors. Thus, while tissue-specific control of basic transcription resides in the promoter, tissue-specific transcriptional reinforcement appears as the central property of this prototype LCR. In the light of this widely accepted model it came as a surprise that, in the context of its normal localization, deletion of the entire mouse LCR (50 HS1 – 6) or most of the human LCR (50 HS2 – 5) does not impair the formation of HSs. Moreover, the general accessibility of the domain appeared to be conserved (Cimbora et al., 2000). This implies the existence of still more remote elements with a

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redundant function in opening the chromosomal environment. 2.2. Boundary elements Each eukaryotic gene is part of a chromatin environment (domain) the state of which determines its expression. Work over the last decade suggests the means by which a genomic unit with its own expression pattern defends itself against its neighbours. These studies revealed the existence of specialized boundary elements next to some genes, which are able to form a barrier to shield the coding region from its surroundings. Although these boundaries vary widely regarding the associated protein factors, most of them share two functional properties. First, if a gene has been moved from its native context, it has the ability to protect its expression against the influence of the individual integration site (position effect). Second they act as a positional enhancer blocker in the sense that an enhancer can act within but not across the boundaries. Early clues regarding the nature of these boundaries emerged from the architecture of independently regulated genomic regions, i.e. the packaging principles of the eukaryotic nucleus above the nucleosome level: eukaryotic DNA is organized into higher order chromatin structures which in turn are periodically attached by specific sequences to a subnuclear scaffold or matrix to form a series of 30 – 100 kb loops. Scaffold- or matrix-attachment regions (S/MARs) are devoid of a unique sequence consensus but are marked by structural features which are the consequence of their secondary structure formation potential and of a regular distribution pattern of AT-rich patches (Goetze et al., 2003). The first boundary elements ever described for vertebrates were the so called A-elements of the chicken lysozyme domain (Stief et al., 1989), prototypical scaffoldattachment regions with the whole range of S/MAR associated biological activities (Zahn-Zabal et al., 2001). Later on it became clear that not all elements with the capability to mediate position-independent expression or to block promoter-enhancer interactions are in fact S/MARs. Even today a subdivision of boundary elements into insulators and S/MARs remains a matter of contention. 2.2.1. Insulators The term ‘insulators’ has been coined for a group of genomic elements in Drosophila, among these examples with (gypsy) and without (scs, scs0 ) S/MAR activity. Well studied vertebrate insulators are found on both sides of the human apolipoprotein B domain in liver and intestine (Antes et al., 2001) and near the 50 ends of the chicken bglobin locus (HS4) and the human b-globin locus (HS5), respectively. Curiously, while cHS4 clearly has no S/MAR potential, HS5 does (Li et al., 2002a,b). Whereas the 30 boundary of the apo B domain is both a S/MAR and an insulator, the physical 50 boundary is a S/MAR whereas the

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functional boundary is a non-S/MAR insulator (Zhan et al., 2001; Antes et al., 2001). The most detailed studies have been performed on the 1.2 kb DNA insulator, HS4, 50 to the chicken b-globin locus: it prevents an external enhancer from acting on a promoter (‘enhancer blocking’) and functions as a barrier to chromosomal position effects (CPE) when it flanks a stably integrated reporter. Most of its enhancer-blocking activity resides in a 250 bp CpG island core region and here to a single CTCF binding site. On the other hand, full protection against CPE is also conferred by mutant 50 HS4 from which the CTCF-site has been deleted and it is associated with the maintenance of high levels of histone acetylation nearby (Recillas-Targa et al., 2002). 2.2.2. S/MARs Recent evidence supports the traditional view that S/MARs are in fact the elements, which provide the eukaryotic genome with a higher order organization by matrix attachment (Goetze et al., 2003). Even more important, the range of biological activities associated with S/MARs are in full agreement with the postulated role of a boundary element (Bode et al., 2003). Besides a context-dependent insulator function, these elements has also been implicated in the function of enhancers and replication origins. After transfection, the activity of a S/MAR on transcription initiation rates (Schuebeler et al., 1996) can first be traced when a defined chromatin structure has been attained during one or several rounds of replication, but not before. This applies to transgenes after integration and to replicated episomes (Ho¨rtnagel et al., 1995), but not to the phase of transient expression suggesting that the possibility to built up and release superhelical strain is a prerequisite of S/MAR function. In this context it is important that both the superhelicity and structure of the chromatin fiber are profoundly affected by the hyperacetylation of 12 or more histone tails per histone octamer (Tse et al., 1998) and that S/MARs support such a process (Fernandez et al., 1998, 2001). A dual-function protein which has first been described as hnRNP-U and which was later re-discovered as the major scaffold-attachment factor (SAF-A) appears to be involved. Martes et al. (2002) report a strong interaction between p300 and SAF-A which is interrupted by viral proteins such as E1A or SV40 large-T in the sense that binding is mutually exclusive. The proposed model ascribes the domain-opening action to p300. This forms a bridge between SAF-A at S/MAR elements and the transcription factors already bound to the promoter. Thus it enables a quick and complete histone acetylation once transcription is initiated. Several lines of evidence indicate that histone deacetylation and DNA methylation together serve to silence transcription by indirect mechanisms. DNA demethylation is mostly an active enzymatic process. For instance, the immunoglobulin kappa gene becomes specifically demethy-

lated during B-cell maturation in a process which utilizes cis-acting modules such as the intronic kappa enhancer element and the associated S/MAR (Kirillov et al., 1996). While any S/MAR sequence appears to be sufficient for this reaction, tissue specificity is clearly directed by the enhancer since a plasmocytoma cell line lacking nuclear factor kappa B (NF-kB) fails to demethylate the kappa locus. In their studies on the contribution of S/MARs to the enhancer-mediated control of the immunoglobulin-gene Fernandez et al. (2001) have demonstrated that the combination of enhancers and S/MARs constitutes the functional LCR. This suggests that S/MARs may be one class of the mentioned non-enhancer elements that have been implied in LCR function. Among all terrestrial life forms the activation of replication origins shares some common fundamental characteristics. An initiator protein, such as dnaA in Escherichia coli, T antigen in SV40, the E1 and E2 proteins for bovine papilloma virus (BPV) and EBNA1 in Epstein – Barr virus, directs the formation of a large initiation complex which requires that ORI sequences possess ATrich stretches. These base unpairing regions (BURs) undergo strand separation under superhelical tension. In mammalian cells, this is the role of a S/MARs which directs the ORI to the nuclear matrix where replication can be initiated. Such a role has become evident during the construction of artificial episomes: the potential of BPV vectors could be increased dramatically when a hybrid plasmid (BPV-BV1) was constructed, which contained a 69% subfragment of BPV and 2.7 kb of eukaryotic ‘stabilizing sequence’ which turned out to be a S/MAR later on (Bode et al., 2001). A S/MAR can only support ORI functions if the ORI has experienced a history of transcription underlining the tight coupling of these processes. For the amplified DHFR domain in CHO cells the chromatin structure undergoes dramatic changes as cells cross the G1/S boundary, but only for those copies of the amplicon that are affixed to the nuclear matrix (Pemov et al. 1998).

3. Artificial domains By now the components for the design of an independently regulated chromatin domain that can resume authentic expression at almost any genomic integration site have emerged. The gene of interest has to be posed under a promoter which provides the required tissue specificity. An enhancer element is necessary to target the construct to an actively transcribed nuclear compartment and to counteract the repressive forces of chromatin packaging (Francastel et al., 1999). If reinforcement of tissue-specific expression is required, a composite locuscontrol region has to be provided at the optimum distance and with the correct orientation relative to the promoter.

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Finally, the action of enhancer or LCR elements has to be delimited by insulators, which may be S/MARs in case of a strong chromatin domain (Dillon and Sabbattini, 2000). Whereas the action of these elements has been investigated in numerous studies at the tissue culture level (Bode et al., 2003), exploitation of these principles for gene therapy protocols has encountered unexpected difficulties. This is due to the fact that the genomic anchoring of transgenes occurs at low efficiency and that stable expression is based on an unpredictable copy number in a way that triggers cellular defense and silencing mechanisms. 3.1. Establishing a domain Over the last decade, a large number of DNA delivery systems – both viral and non-viral – have been developed for in vitro and in vivo applications. Among these the use of retroviral vectors has attracted particular attention since integration occurs at the single-copy level and at genomic loci with appropriate transcriptional properties. Therefore retroviral vectors have become the preferred tools both for the study and for the utilization of the mentioned elements. The well known inactivation in some cell types of transcription from the retroviral LTRs (Robbins et al., 1998) can be overcome, in part, by inhibiting histone deacetylases or DNA methyltransferases (Lorincz et al., 2000). Interestingly, the active state of these transgenes can also be maintained using the mentioned chromosomal elements for the improvement of retroviral vectors. Special attention is given to hematopoietic stem cellbased gene therapy protocols which enable the treatment of a variety of congenital and acquired diseases (Robbins et al., 1998). Gene therapy vectors that integrate one copy per cell should be regulated by elements that function in vivo at the single-copy level. Therefore LCR derivatives have been developed that are suitable for regulating b-globin genes as a prelude to the gene therapy of b-thalassemia and sickle cell anemia. These have been shown to overcome the action of silencer elements with a function in stem cells. After nearly 15 years of development LCR-beta globin vectors are now being tested in preclinical animal models. During the retroviral life cycle, the 30 LTR sequence is copied whereby it recurs at the 50 LTR position. Using this self-duplication principle, Rivella et al. (2000) have generated mouse erythroleukemia cell lines in which the reporter gene is flanked on both sides by the HS4 insulator element. It is shown that these borders increase the probability that integrated proviruses will be expressed. At the same time the level of de-novo methylation of the 50 LTR is dramatically decreased. Similarly, Emery et al. (2000) report the protection of a retroviral insert from position effects by virtue of the HS4 element. Related activities have also been reported for the S/MAR 50 from the human interferon-b gene (Auten et al., 1999; Dang et al., 2000). If inserted into a retroviral vector, the element improves transgene expression in human primary

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CD4(þ ) and CD8(þ ) T cells, and in primary monocytes, even in the resting state. The construct was found to be much more potent at inhibiting HIV-1 replication in infected CD4 þ peripheral blood lymphocytes than the S/ MAR free parent construct. Subsequent model studies demonstrated that this effect is explained by the DNA methylation status. Moreover, if cells were transfected with in vitro pre-methylated DNA, this modification completely abolished expression of the control vector but not of the S/ MAR derivative. These results indicate that S/MARs not only prevent methylation but that they are also in the position to interfere with the formation of methylationdependent inactive chromatin states. Human clinical trials have highlighted issues that limit the application of viral vector technology, therefore benefits of non-viral delivery systems attracts increasing attention. Here, the most widely used delivery methods still involve administration of double strand DNA as cationic lipid/DNA complexes or as naked plasmid (pDNA). In 1989, a concerted effort to adapt cationic lipid-mediated delivery for in vivo application yielded the surprising finding that direct injection of pDNA provided more transgene expression than a similar dose of lipid/DNA complex. We will therefore concentrate on certain improvements of this principle. Tissues for which efficient transfer methods are being developed are tumors, various epithelia or endothelia and organs as liver, heart and brain. The procedure is usually hampered by the lack of a safe, efficient system for the local application of the expression construct. Besides a direct injection, for subcutaneous delivery a ‘gene gun’ has been used to shoot microscopic gold-coated particles containing gene material into the skin. For an organ-specific application the genetic material has been encapsulated into albumin microspheres for its transport through the organism where it can be released by an intravascular destruction of the microbubbles by ultrasound directly in the affected organ or tissue (Skyba et al., 1998). Preferentially used for the delivery to skeletal muscle, another significant improvement of the direct injection method is electroporation. The process directs pulsed electric fields into the tissue whereby the permeability of cell membranes is transiently increased by two to three orders of magnitude (Mir et al., 1999). While conventional setups have used two parallel plate electrodes placed on both sides of the tissue, more advanced electrodes are now available. These are composed of six acupuncture needles arranged in a 1 cm circle. Sets of these needles can be energized sequentially. Still an alternative approach is the intra-arterial delivery of naked plasmid DNA (pDNA). This procedure leads to high expression levels throughout limb skeletal muscles and proved far superior to intramuscular injections, especially if supported by an external tourniquet. It is assumed that the intravascular pressure enables pDNA extravasation followed by receptor-mediated uptake. Gene delivery to skeletal muscle has raised special

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interest, not only for the correction of myopathies but also in order to use the muscle as an endocrine organ for the systemic secretion of therapeutic proteins. In some cases, expression as long as 2 years has been observed which has been ascribed to the postmitotic nature of muscle in which pDNA apparently persists in an extrachromosomal state without replication. For other tissues only transient expression has been reported and the state of transferred DNA (integrated or extrachromosomal) has remained largely elusive. It is a well established rule, however, that expression from circularly closed constructs is superior to linear DNA unless there are other means to maintain superhelicity (Dunaway and Ostrander, 1993), for instance on very extended templates (Krebs and Dunaway, 1996). Therefore it will be interesting to follow the status of the transgenes over time and to find out if for such a circular vector S/MAR elements are able to support expression either by targeting the vector to a transcriptionally active nuclear compartment or by reproducing the topological features of a matrix-attached chromatin domain. The next chapter will be devoted to this subject and will describe the elements and principles that have been used to construct an episomal vector which, in dividing cells, is able to replicate in synchrony with the host chromosomes. While, obviously, the fate of non-virally transferred, integrated DNA remains uncontrolled at the time being, it should be mentioned that tag and exchange procedures have been developed for the repeated use of pre-characterized genomic sites in murine ES cells (Bode et al., 2000). These procedures are based on a tagging step by homologous recombination, which is used to introduce short recognition sequences for the subsequent action of site specific recombinases. In a procedure called ‘RMCE’ (recombinase-mediated cassette exchange) the protocol permits the precise and irreversible introduction into this site of any gene cassette of interest. Regarding the recent success with somatic nuclear transfer into stem cells and the announcement of long-lived cultures from human embryonic stem cells (Gearhart, 1998) an entirely new avenue is now open which might finally lead to ‘therapeutical cloning’. It is one current vision that the modified cells will be used as a source of numerous cell types and tissues.

4. Episomal vectors Non-viral episomally replicating vectors, either small circular constructs, artificial chromosomes or minichromosomes share the advantage that they occur as an independent genomic unit in the recipient nucleus, that they do not interfere with the host genome and, consequently, that there is nothing like a position-dependent expression in the sense this term is conventionally used. Each of these vector types has its advantages and limitations as discussed below. Although they have been developed as expression systems,

up to now none of these constructs has been used in clinical gene therapy trials. 4.1. Small circular vectors In yeast a replication origin or ARS is defined by its primary sequence which can promote episomal replication after insertion into a plasmid. In higher eukaryotes the activity of an origin of replication is strongly influenced by epigenetic principles which are not yet fully understood. There are numerous reports describing the insertion of a mammalian origin of replication into a plasmid (for example: Krysan et al., 1989) but initiation of replication was almost always defective and the ultimate fate of all these constructs was either loss from the cell or integration. Therefore, this approach will not be further discussed here. In higher eukaryotes DNA replication occurs in tight association with the nuclear matrix and binding of an origin of replication precedes the onset of S phase (Cook, 1999). Most characterized mammalian origins of replication are associated with matrix-attached regions (S/MARs; DePamphilis, 1999). Therefore, various S/MAR-containing vectors were constructed and tested for episomal replication and stability as discussed for two examples. In the first example, three different mammalian origins of replication were cloned into a S/MAR containing YAC vector. Two clones were selected which were supposed to contain the vector as an episome. These episomes had a stability per generation of about 80% when kept under selection. Using high salt extraction of the nucleus for matrix preparation and fluorescence in situ hybridization (FISH) analysis it could be shown for one clone that it associates with the nuclear matrix (Cossons et al., 1997; Nielsen et al., 2000). In another construct the function of the SV40 large T-antigen could be replaced by a S/MAR-sequence isolated from the human b-interferon gene domain which, apparently, recruits components from the endogenous replication apparatus of the host cell. This vector, pEPI-1, replicates episomally at a copy number of about 10 in CHO cells and is stably retained in these cells in the absence of selection for over 100 generations (Piechaczek et al., 1999; Baiker et al., 2000). Further work showed that expression of transgenes encoded by the vector are not subject to silencing, even in the absence of selection, and that this construct also functions in other cell lines, including human and primary cells. This behavior must reflect a functional organization of genomic elements that include a reporter gene, a minimal SV40 replication origin and a human S/MAR sequence. The reasons why this combination of elements works so well are under present investigation and it has already become clear that interactions between the active promoter, the downstream S/MAR and the SV40 origin are essential since disrupting any of these elements results in the loss of episomal replication and integration. The interaction between promoter and S/MAR is very likely to be essential for maintaining expression and, at the same time, for

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replication. A point to be addressed is whether in this context, the SV40 origin functions as the origin and DNA unwinding element or if other sequences within the vector acquire or support this function. As this vector is stably retained over very many generations in the absence of selection it seems that replication and segregation must be very efficient, most likely since it can utilize the cellular replication machinery to permit its co-replication together with the genome of the host cell. In fact, it could be shown that pEPI-1 becomes exclusively replicated during S-phase. As shown by nuclear fractionation procedures and FISH analyses, pEPI-1 associates with mitotic chromosomes in a stable manner due to a specific interaction with the major nuclear matrix protein, SAF-A which has already been mentioned under Section 2.2.2. This interaction enables a ‘piggy-back’ mechanism by which it is passed efficiently to daughter cells (Baiker et al., 2000; Jenke et al., 2002). 4.2. Artificial chromosomes The design of vectors resembling natural chromosomes is an intriguing approach and it was once thought that such constructs would solve most problems related to safety and reproducibility during gene therapy. Like the small nonviral circular vectors they would not require viral proteins for their functioning. Instead they would be linear like natural chromosomes and would be stably maintained at a low and defined copy number in the cell in the absence of selection. Since their cloning capacity is infinite they would tolerate not only the insertion of a gene of interest but all control elements required for the regulated, cell specific expression of the transgene. More recently, it was argued that such constructs should be also ideal for the genetic modification of stem cells to be used for cell therapy. In fact, the description of the first prototype artificial chromosome 1997 has been regarded one of the great breakthroughs in animal biotechnology and human gene therapy (Harrington et al., 1997). Meanwhile a number of artificial chromosomes have become available and their construction has considerably contributed to our understanding of the functional elements of the eukaryotic chromosome, i.e. centromeres, telomeres and replication origins. In the past years numerous reviews on artificial chromosomes covering progress in this field up to about 2001 were published (Huxley, 1994; Ascenzioni et al., 1997; Vos, 1998, 1999; Schindelhauer, 1999; Cooke, 2001; Lewis, 2001; Lipps and Bode, 2001; Grimes et al., 2002a,b; Kuroiwa et al., 2002; Saffery and Choo, 2002). In this review, therefore, we concentrate on very recent advances in this field and especially on those constructs that have been actually used as cloning vehicles. Two principal procedures exist for the generation of mammalian artificial chromosomes. One approach, called ‘top down’, relies on the fragmentation of natural chromosomes either by low dose irradiation (Benham et al., 1989; Auriche et al., 2001) or telomere-directed

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truncation of chromosomes (Farr et al., 1992). Most of the presently available minichromosomes exist as a result of telomere fragmentation. This technique relies on the observation that integration of telomeric DNA can lead to the truncation of mammalian chromosomes, which in turn seed the formation of new telomeres on the truncated ends (Farr et al., 1992; Brown et al., 1994). Using this technique it has become possible to trim mammalian chromosomes down to sizes between 0.5 and 6 Mb (Farr et al., 1995; Heller et al., 1996; Yang et al., 2000; Shen et al., 2000; Saffery et al., 2001; Wong et al., 2002). These minichromosomes are structurally stable and consist, to a large extent, of centromeric DNA and some as yet non-characterized DNA sequences (Shen et al., 2000; Auriche et al., 2001). They can be either linear or in some rare cases circular and certainly more structural analyses are required before their use as a vector becomes feasible in gene therapy. Interestingly, fully functional centromeres can arise naturally in non-centromeric regions devoid of alpha satellite DNA demonstrating that epigenetic factors influence the formation of centromeres (Saffery et al., 2001; Wong et al., 2002). Other types of minichromosomes were generated from human cell lines that contain alpha satellite DNA (Raimondi et al., 1996; Voet et al., 2001) and large satellite-DNA-based artificial chromosomes (Csonka et al., 2000). A novel technology for the construction of minichromosomes relies upon integration of exogenous DNA into the centromeric region of mouse or human chromosomes. Amplification of this DNA can lead to chromosome breakage resulting in chromosomes with sizes between 60 and 400 Mb. They can be purified in quantities sufficient for microinjection or lipofection (Grimes et al., 2002a,b). Some of the minichromosomes have been shown to be efficiently transmitted through the mouse germ line (Tomizuka et al., 1997; Shen et al., 2000; Voet et al., 2001). During the alternative ‘bottom-up’ approach cloned centromeric DNA, telomeric DNA and, in some cases, putative replication origins are assembled in vitro or in vivo and introduced into mammalian cells. Using this approach the group of Willard obtained the first de novo artificial chromosome in HT1080 cells (Harrington et al., 1997). Using the same cell line other groups introduced circular or linear DNA constructs containing alpha satellite DNA and telomeric sequences cloned in YACs, BACs or PACs (Ikeno et al., 1998; Henning et al., 1999; Ebersole et al., 2000; Meija and Larin, 2000). Despite the presence of telomeric sequences most of the obtained de novo artificial chromosomes were circular. Many of those constructs showed a low structural stability. They continuously increased in size by some as yet not completely understood mechanism. Circular MACs were also created following the introduction of PACs containing about 70 kb alphoid DNA. These MACs had sizes between 1 and 10 Mb and since no evidence for the acquisition of host DNA was obtained, this increase in size was probably due to the formation of concatameres. The mitotic stability of those circular MACs was very high and

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immunocytochemical analysis showed that CENP-C and -E were present (Ebersole et al., 2000). This observation implies that alphoid DNA sequences are not only sufficient for mitotic stability but also for the replication of the MACs and demonstrate that it is the primary sequence for the de novo MAC formation in HT1080 cells. However, different alpha satellite DNA are different in their potential in forming a de novo MAC (Grimes et al., 2002a,b). Recently, major efforts have been undertaken to use those constructs as expression systems. Due to the large size of artificial chromosomes and the low amounts available this is not a trivial task. Strategies to incorporate genes into MACs include co-transfection, targeted site-specific recombination using the Cre-loxP- (Kuroiwa et al., 2000) or the FLP-FRT system from yeast (O’Gorman et al., 1991). There are reports from two groups that human artificial chromosomes containing the 40 kb hypoxanthine guanine phosphoribosyltransferase locus (HPRT1) can complement the metabolic defect in HPRT-deficient HT1080 cells. Grimes et al. (2001) cotransfected PACs containing either 70 kb of chromosome 21 alphoid DNA or containing a 140 kb HPRT1 genomic insert. Lines could be isolated containing a MAC hybridizing with both a-satellite and HPRT1 probes. These circular MACs expressed HPRT for at least 60 days without selection and the complementation of the parental HPRT deficiency could be confirmed by growth on HAT selection. Another group (Meija et al., 2001) transferred a 404 kb MAC consisting of 220 kb of alphoid DNA from the centromeric region of human chromosome 17, the HPRT genomic locus and human telomeres into HPRT-deficient HT1080 cells. Also in this case circular MACs arose which were mitotically very stable and expressed the transgene over a period of at least 60 days in the absence of selection. Another example is the incorporation of the entire genomic CFTR gene and its upstream sequences into a structurally well defined minichromosome (Auriche et al., 2001). Clones were identified showing the presence of both the CFTR transcript and protein. In these clones a chloride secretory response to cAMP could be detected (Auriche et al., 2002). These encouraging results clearly demonstrate that MACs can be modified for their use as expression systems of large genes. In contrast to cDNA-based vectors not only the gene of interest but all control elements required for regulated transgene expression can be incorporated.

5. Conclusion and perspectives One major aim of vector-based cellular modifications is the high-level long term and stable expression of a gene of interest. Traditional systems were derived from appropriate promoter-enhancer combinations and vectors were genomically anchored, preferentially at high copy numbers. With recent evidence for the existence of cellular defense mechanisms, particularly against tandem integrates

(repeat-induced silencing or co-suppression) the emphasis has changed significantly and has recently concentrated on systems which either integrate a single-copy transgene at an appropriate chromosomal locus or on concepts to provide it with the elements that guarantee its position-independent expression in any genomic context. Using the classical gene-transfer routes these approaches are severely limited by the unpredictable nature of illegitimate recombination systems in the host cell, which lead to frequent rearrangements at the site of integration, necessitating the isolation and characterization of single clones. With the advent of novel cassette exchange technologies the integration of single copy transgenes into a predetermined genomic location has come within reach. Meanwhile the precise and enzymatically controlled retroviral integration mechanism has been applied to investigate the performance of LCRs, insulators and scaffold-attachment elements under well-defined conditions. These studies clearly demonstrate the potential of chromosome-based vectors provided these elements are allowed to act under the authentic topological conditions of the host genome. For the time being, non-viral episomal vectors, either small circular vectors or artificial chromosomes, are an attractive alternative to conventional vectors currently used in gene therapy but also to integrating chromosome-based vectors. By virtue their covalently-closed or extended structure (Dunaway and Ostrander, 1993), these entities are ideal minimal systems to reveal certain functions for this group of elements. Therefore, all of these constructs already had a major impact on our understanding of chromosomal function. A number of prototype small circular vectors and artificial chromosome vectors have been developed but so far none of these constructs has been actually used in gene therapy trials. Each of these constructs has its advantages but also its specific limitations. Artificial chromosomes have the undoubted advantage of being mitotically stable in the absence of selection and have an indefinite cloning capacity allowing the insertion of all control elements for correct expression of the transgene. Due to their large size they are difficult to handle and can be recovered only in small quantities. If they will ever have a role in the therapy of human diseases, efficient delivery to primary cells and stem cells must be achieved and possibilities must be found to increase the proportion of cells maintaining these MACs as intact and stable entities. Small circular non-viral vectors have the advantage that they can be handled with ease and obtained in large quantities, but their cloning capacity may be restricted. Their functioning in vivo in various cell types is still under investigation. However, both types of vectors are constructed in several laboratories and it can be assumed that the problems and questions mentioned will be overcome in the near future. There is no doubt that the design of chromosome-based vectors will eventually be the solution of choice for the efficient, safe and reproducible modifi-

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cation of eukaryotic cells and eventually also for the therapy of many human diseases.

Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft and the Alfried von Bohlen und HalbachFoundation.

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