GB2538321A - Artificial chromosome for retroviral production - Google Patents

Abstract

This invention relates to a mammalian artificial chromosome ( also known as a MAC, satellite artificial chromosome, SATAC, an artificial chromosome expression system or an ACES) which is stably replicated and segregated alongside endogenous chromosomes in a mammalian host cell for creating more efficient retroviral packing cell lines. The artificial chromosome comprises nucleic acid sequences encoding a replication defective retroviral vector particle, preferably derived from HIV-1, HIV-2, SIV, FIV, EIAV or Visna. The retroviral vector particle comprises the RNA genome of the retroviral vector particle and genes encoding the gag (group specific antigen or HXB2 790-2292), pol (DNA polymerase) and env (viral envelope, GP160, GP120, GP41 or envelope glycoprotein) proteins. Further embodiments of the invention provide mammalian cells comprising such artificial chromosomes and methods for their use in producing retroviral particles.

Description

ARTIFICIAL CHROMOSOME FOR RETROVIRAL PRODUCTION

FIELD OF THE INVENTION

The invention relates to artificial chromosomes comprising genes required for retroviral production and uses thereof. Also provided are methods of making retroviral packaging cell lines comprising the artificial chromosomes described herein.

BACKGROUND TO THE INVENTION

In gene therapy, genetic material is delivered to endogenous cells in a subject in need of treatment. The genetic material may introduce novel genes to the subject, or introduce additional copies of pre-existing genes. Viral vector systems have been proposed as an effective gene delivery method for use in gene therapy (Verma and Somia (1997) Nature 389: 239-242).

In particular, these viral vectors are based on members of the retrovirus family due to their ability to integrate their genetic payload into the host's genome. Retroviral vectors are designed to keep the essential proteins required for packaging and delivery of the retroviral genome, but any non-essential accessory proteins including those responsible for their disease profile are removed. Examples of retroviral vectors include lentiviral vectors, such as those based upon Human Immunodeficiency Virus Type 1 (HIV-1), which are widely used because they are able to integrate into non-proliferating cells.

Currently, the majority of viral vectors are produced by transient co-transfection of viral genes into a host cell line. The viral genes are introduced using bacterial plasmids which exist in the host cell for only a limited period of time because the viral genes remain on the plasmids and are not integrated into the genome. As such, transiently transfected genetic material is not passed on to subsequent generations during cell division.

There are several drawbacks associated with transient transfection, such as batch-to-batch variability, the high cost of transfection reagents and the difficulty to maintain quality control (see Segura et al. (2013) Expert-Opin. Biol. Ther. 13(7): 987-1011). The process of transfection itself is also labour-intensive and challenging to scale up. There is also the difficult task of removing plasmid impurities which are carried over during vector preparation (see Pichlmair etas. (2007) 81(2): 539-47).

In order to address problems associated with transient transfection, there has been a desire to develop retroviral packaging and producer cell lines in order to simplify retroviral vector production.

Packaging cell lines have been generated by transfecting a cell line capable of packaging retroviral vectors with plasmids, where individual plasmids carry the retroviral packaging genes and unique eukaryotic selection markers. The packaging genes are integrated into the packaging cell line's genome and are described as being stably transfected. Over the past 20 years various attempts have been made to generate stable packaging and producer cell lines for retroviral vectors.

There have been many reported problems in the packaging and producer cell lines produced via integration of vector components into the host cell genome. In the first instance, sequential introduction of vector components can be laborious and inflexible. There have also been problems with genetic and/or transcriptional instability of vector components when they are integrated into the host cell genome because the site of integration is unpredictable (Ni et al. (2005) I. Gene Med. 7: 818-834.). A significant drop in viral vector productivity has also been reported during suspension adaptation and scale-up of the producer cell lines (Farson et al. (2001) Hum. Gene Ther. 12: 981- 997; Guy et al. (2013) Hum. Gene Ther. Methods. 24(2): 125-39).

It is therefore an object of the present invention to provide a method of making stable retroviral packaging and producer cell lines which overcomes one or more of the disadvantages associated with existing methods.

SUMMARY OF THE INVENTION

The present inventors have developed a new way of making packaging and producer cell lines which involves the use of an artificial chromosome comprising the retroviral genes essential for retroviral vector production. This allows expression of the retroviral genes required for production of replication defective retroviral vector particles while ameliorating problems associated with producer cell lines in which vector components are integrated into the host cell genome.

It has been found that problems associated with unpredictable integration of vector components into the host cell genome when developing producer cell lines can be overcome through the use of an artificial chromosome. Previously, artificial chromosomes have only been used for protein expression, not for the expression of retroviral vectors. By integrating the retroviral genes onto an artificial chromosome, retroviral gene expression can ensured and there is a lower risk of genetic and/or transcriptional instability associated with integration into the host cell genome. There are other advantages to using artificial chromosomes in order to produce retroviral packaging and producer cell lines. For example, because the retroviral genes are located in an independent, extra-genomic artificial chromosome they are stably maintained in an active transcription unit and are not ejected from the host cell via recombination or elimination during cell division. This avoids the need to continuously select for packaging and producer cells; such selection is required when introducing retroviral packaging genes via individual plasmids.

Furthermore, because the artificial chromosomes are capable of incorporating large segments of DNA, multiple copies of the heterologous gene and linked promoter element(s) can be retained in the artificial chromosomes, thereby providing a high-level of expression of the retroviral protein(s). This helps to combat problems associated with reduced viral vector productivity over time.

The use of an artificial chromosome therefore provides advantages in the generation of retroviral packaging and producer cell lines.

Therefore, according to a first aspect of the invention, there is provided an artificial chromosome comprising a genetic locus which enables the artificial chromosome to be stably replicated and segregated alongside endogenous chromosomes in a mammalian host cell, characterized in that said artificial chromosome comprises nucleic acid sequences encoding a replication defective retroviral vector particle, comprising: the RNA genome of the retroviral vector particle, gag and pol proteins, and env protein or a functional substitute thereof.

According to a further aspect of the invention, there is provided a mammalian host cell comprising the artificial chromosome as defined herein.

According to a further aspect of the invention, there is provided the use of the artificial chromosome or mammalian host cell as defined herein to produce a high titre of retroviral vector.

According to a further aspect of the invention, there is provided a method of producing a replication defective retroviral vector particle, comprising culturing a mammalian host cell comprising the artificial chromosome as defined herein under conditions in which the retroviral vector particle is produced.

According to a further aspect of the invention, there is provided a replication defective retroviral vector particle produced by the method defined herein.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entirety.

The term "comprising" encompasses "including" or "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y. The term "consisting essentially of limits the scope of the feature to the specified materials or steps and those that do not materially affect the basic characteristic(s) of the claimed feature.

The term "consisting or excludes the presence of any additional component(s).

The term "about" in relation to a numerical value x means, for example, x ± 10%, 5%, 2% or 1%.

The term "vector" refers to a vehicle which is able to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. Examples of vectors include plasmids and viral vectors, such as retroviral and lentiviral vectors, which are of particular interest in the present application. Lentiviral vectors, such as those based upon Human Immunodeficiency Virus Type 1 (HIV-1) are widely used as they are able to integrate into non-proliferating cells. Viral vectors can be made replication defective by splitting the viral genome into separate parts, e.g., by placing on separate plasmids. For example, the so-called first generation of lentiviral vectors, developed by the Salk Institute for Biological Studies, was built as a three-plasmid expression system consisting of a packaging expression cassette, the envelope expression cassette and the vector expression cassette. The "packaging plasmid" contains the entire gag-pol sequences, the regulatory (tatand rev) and the accessory (vif, vpr, vpu, net) sequences. The "envelope plasmid" holds the Vesicular stomatitis virus glycoprotein (VSVg) in substitution for the native HIV-1 envelope protein, under the control of a cytomegalovirus (CMV) promoter. The third plasmid (the "transfer plasmid") carries the Long Terminal Repeats (LTRs), encapsulation sequence (p), the Rev Response Element (RRE) sequence and the CMV promoter to express the transgene inside the host cell.

The second lentiviral vector generation was characterized by the deletion of the virulence 20 sequences vpr, WI; vpu and net. The packaging vector was reduced to gag, poi, tatand revgenes, therefore increasing the safety of the system.

To improve the lentiviral system, the third-generation vectors have been designed by removing the tatgene from the packaging construct and inactivating the LTR from the vector cassette, therefore reducing problems related to insertional mutagenesis effects.

The various lentivirus generations are described in the following references. First generation: Naldini et al. (1996) Science 272(5259): 263-7; Second generation: Zufferey et al. (1997) Nat. 8/otechnol. 15(9): 871-5; Third generation: Dull eta! (1998) J. V/ro/. 72(11): 8463-7, all of which are incorporated herein by reference in their entirety. A review on the development of lentiviral vectors can be found in Sakuma et al. (2012) Mochem. J. 443(3): 603-18 and Pican4o-Castro et al. (2008) Exp. Op/n. Therap. Patents18(5):525-539.

The terms "transfection", "transformation" and "transduction" as used herein, may be used to describe the insertion of the vector into the target cell. Insertion of a vector is usually called transformation for bacterial cells and transfection for eukaryotic cells, although insertion of a viral vector may also be called transduction. The skilled person will be aware of the different non-viral transfection methods commonly used, which include, but are not limited to, the use of physical methods (e.g. electroporation, cell squeezing, sonoporation, optical transfection, protoplast fusion, impalefection, magnetofection, gene gun or particle bombardment), chemical reagents (e.g. calcium phosphate, highly branched organic compounds or cationic polymers) or cationic lipids (e.g. lipofection). Many transfection methods require the contact of solutions of plasmid DNA to the cells, which are then grown and selected for a marker gene expression.

The term "artificial chromosome" refers to a fully functional chromosomal structure that can stably replicate and segregate alongside endogenous chromosomes so that it is transmittable to host cell progeny. In particular, the artificial chromosome comprises a genetic locus (e.g. comprising a centromere and a primary replication site) which enables it to be stably replicated and segregated alongside endogenous chromosomes in a host cell. This allows the artificial chromosome to be passed onto the host cell progeny, without the need for it to be integrated into the host cell genome. It will be understood that "genetic locus" as used herein encompasses both the centromere and primary replication site whether they are at the same or separate sites within the chromosome.

The artificial chromosome may also contain telomeres at each end, which allows it to be stably maintained side by side with the host cell's endogenous chromosomes. Therefore, in one embodiment, the artificial chromosome comprises at least one centromere, a primary replication site and two telomeres. It will be understood that "dicentric chromosome" refers to a chromosome with two centromeres and "multicentric chromosome" refers to a chromosome with multiple (i.e. more than two) centromeres. "Telomeres" refer to regions of repetitive DNA at the ends of a chromosome which protect it from degradation. In one embodiment, the artificial chromosome comprises a telomere, which may be a synthetic telomere, for example, a telomere containing a series of TTAGGG repeats.

Artificial chromosomes also have the capacity to accommodate and express heterologous genes inserted therein. Therefore, artificial chromosomes provide an extra genomic locus for targeted integration of heterologous DNA into a host cell.

For the avoidance of doubt, it will be understood that references to "artificial chromosomes" do not refer to plasmids (e.g. such as the plasmids currently used in transient transfection methods) because these are not passed onto subsequent generations of the host cell. Therefore, an artificial chromosome is not a plasmid (such as a bacterial or artificial plasmid) or episome.

Artificial chromosomes of the present invention can be derived from various naturally-occurring chromosomes, including both mammalian and non-mammalian originating chromosomes.

Mammalian origins include but are not limited to non-human primate, human, and mouse. Non-mammalian origins include eukaryotes such as yeast, and prokaryotes such as bacteria.

Artificial chromosomes of the present invention can be mammalian artificial chromosomes (MAC) which include an active mammalian centromere(s). This allows the MAC to be stably replicated and segregated alongside endogenous chromosomes of mammalian cells, such as mouse or human cells.

Among the MACs provided herein are satellite artificial chromosomes (SATACs) (which may also be known as artificial chromosome expression systems (ACES)), minichromosomes (also referred to as neo-minichromosomes), and in vitro synthesized artificial chromosomes.

The term "endogenous chromosomes" refers to genomic chromosomes found in the host cell prior to generation or introduction of an artificial chromosome.

The terms "euchromatin" and "heterochromatin" as used herein, have their recognized meanings. Euchromatin refers to chromatin which is less tightly bound and therefore stains diffusely, while heterochromatin refers to tightly packed DNA which stains intensely. Without being bound by theory, it is thought that euchromatin typically contains genes which are involved in active gene transcription, while heterochromatin contains mainly genetically inactive satellite sequences. Highly repetitive DNA sequences (also known as "satellite DNA") are usually located in regions of heterochromatin surrounding the centromere (le. pericentric heterochromatin).

The term "ribosomal DNA" or "rDNA" refers to DNA which encodes ribosomal RNA (rRNA). Ribosomal RNA is the specialized RNA that forms part of the structure of a ribosome and participates in the synthesis of proteins. The rDNA is tandemly arranged in units which are generally about 40-kilo base pairs (kb) in length and contain a transcribed region and a non-transcribed region known as an intergenic spacer (which can vary in length). In mice and humans, these tandem arrays of rDNA units are located adjacent to the pericentric satellite DNA sequences in regions known as the nucleolar organizing regions (NOR) which loop into the nucleolus. In the human genome, there are five endogenous chromosomes with nucleolus organizer regions: the acrocentric chromosomes 13, 14, 15, 21 and 22. In the mouse genome, there have been least 11 endogenous chromosomes shown to have rDNA present: chromosomes 5, 6, 9, 11, 12, 15, 16, 17, 18, 19 and X. In mammals, particularly mice and humans, these rDNA units are highly conserved and contain specialized elements, such as the origin of replication (or origin of bidirectional replication in mice) and amplification promoting sequences (APS) and amplification control elements (ACE) (see Gogel et al. (1996) Chromosoma 104:511-518; Coffman et al. (1993) Exp. Cell Res. 209:123-132; Little et al. (1993) Mot Cell. Biol. 13:6600-6613; Yoon et al. (1995) Mol. Cell. Biol. 15:2482-2489; Gonzalez and Sylvester (1995) Genomics 27:320-328; Miesfeld and Arnheim (1982) Nuc. Acids Res. 10:3933-3949; Maden et a! (1987) Biochem. 1 246:519-527).

The term "promoter" refers to a sequence that drives expression of the transgene. In order to drive a high level of expression, it may be beneficial to use a high efficiency promoter, such as a non-retroviral, high efficiency promoter. Examples of suitable promoters may include a promoter such as the human cytomegalovirus (CMV) immediate early promoter, spleen focus-forming virus (SFFV) promoter, Rous sarcoma virus (RSV) promoter, or human elongation factor 1-alpha (pEF) promoter.

A Tet operon (Tetracycline-Controlled Transcriptional Activation) may be used in a method of inducible gene expression, wherein transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline). In nature, the Ptet promoter expresses TetR, the repressor, and TetA, the protein that pumps tetracycline antibiotic out of the cell. In the present invention, the Tet operon may be present or absent, for example, in one embodiment the Tet operon may be present in the promoter.

The term "selectable marker" refers to a gene that will help select cells actively expressing the transgene. Examples of suitable selection markers include, enzymes encoding resistance to an antibiotic (i.e. an antibiotic resistance gene), e.g., kanamycin, neomycin, puromycin, hygromycin blasticidin, or zeocin. Another example of suitable selection markers are fluorescent proteins, for example green fluorescent protein (GFP), red fluorescent protein (RFP) or blue fluorescent protein (BFP).

The term "polyA signal" refers to a polyadenylation signal sequence, for example placed 3' of a transgene, which enables host factors to add a polyadenosine (polyA) tail to the end of the nascent mRNA during transcription. The polyA tail is a stretch of up to 300 adenosine ribonucleotides which protects mRNA from enzymatic degradation and also aids in translation.

Accordingly, the artificial chromosomes of the present invention may include a polyA signal sequence such as the human or rabbit beta globin polyA signals, the simian virus 40 (SV40) early or late polyA signals, the human insulin polyA signal, or the bovine growth hormone polyA signal.

The term "intron sequence" refers to a nucleotide sequence which is removed from the final gene product by RNA splicing. The use of an intron downstream of the enhancer/promoter region and upstream of the cDNA insert has been shown to increase the level of gene expression. The increase in expression depends on the particular cDNA insert. Accordingly, the artificial chromosome of the present invention may include introns such as human beta globin intron II or a chimeric human beta globin-immunoglobulin intron.

The term "packaging cell line" refers to a cell line with stably inserted gag and pol protein and envelope glycoprotein genes. Alternatively, the term "producer cell line" refers to a packaging cell line with a stably inserted transfer vector containing a transgene of interest.

The term "stably transfected" refers to cell lines which are able to pass introduced retroviral genes to their progeny (he. daughter cells), either because the transfected DNA has been incorporated into the endogenous chromosomes or via stable inheritance of exogenous chromosomes (e.g. artificial chromosomes). As used herein, stable maintenance of an artificial chromosome occurs when at least about 60%., about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% of daughter cells retain the artificial chromosome (Ie. per cell doubling time). Stable maintenance of an artificial chromosome may occur for over 10 cell doubling times, for example, for over 20, 30, 40, 50, 60, 70, 80, 90 or 100 cell doubling times. Stability may be measured by using a selective agent. Stable artificial chromosomes also retain their structure during cell culturing and suffer neither intrachromosomal nor interchromosomal rearrangements.

ARTIFICIAL CHROMOSOMES

According to a first aspect of the invention, there is provided an artificial chromosome comprising a genetic locus which enables the artificial chromosome to be stably replicated and segregated alongside endogenous chromosomes in a mammalian host cell, characterized in that said artificial chromosome comprises nucleic acid sequences encoding a replication defective retroviral vector particle, comprising: the RNA genome of the retroviral vector particle, gag and pol proteins, and env protein or a functional substitute thereof.

The artificial chromosome comprises the nucleic acid sequence encoding the RNA genome of the retroviral vector particle, the gag and pol proteins and the env protein or a functional substitute thereof. Therefore, in this aspect of the invention, the artificial chromosome comprises all of the nucleic acid sequences required to encode a replication defective retroviral vector particle and has the advantage of ensuring expression of the retroviral genes required for production of replication defective retroviral vector particles.

Current methods for generating retroviral vectors involve transient transfection of the retroviral genes into a host cell. However, many disadvantages have been associated with this method because it is costly, laborious and difficult to scale-up. One solution would be to engineer a packaging cell line that stably incorporates the retroviral packaging genes to avoid the problems associated with transient transfection and to reduce variable retroviral vector output.

The present inventors have found that an artificial chromosome can be used to generate a retroviral packaging cell line which ameliorates previous difficulties associated with retroviral vector production methods. In particular, the use of an artificial chromosome reduces the unpredictability associated with integrating retroviral genes into the host cell genome because variable position effects on gene expression are avoided. Previously, artificial chromosomes have only been used in protein production or to sequence the genome of organisms in genome projects There are other advantages associated with using artificial chromosomes in order to produce retroviral packaging cell lines. For example, because the heterologous DNA is located in an independent, extra-genomic artificial chromosome (as opposed to randomly inserted in an unknown area of the host cell genome or located as an extrachromosomal element providing only transient expression) it is stably maintained in an active transcription unit and is not ejected from the host cell via recombination or elimination during cell division.

Furthermore, because the artificial chromosomes are capable of incorporating large segments of DNA, multiple copies of the heterologous gene and linked promoter element(s) can be retained in the artificial chromosomes, thereby providing for high-level expression of the retroviral protein(s). Alternatively, multiple copies of the gene can be linked to a single promoter element and several different genes may be linked in a fused polygene complex to a single promoter for expression of all the retroviral genes at once. Alternatively, multiple copies of a single gene (i.e. a concatemer) can be operatively linked to a single promoter, or each copy may be linked to different promoters or multiple copies of the same promoter.

Another advantage of being able to introduce multiple copies of the nucleic acid sequences encoding a replication defective retroviral particle into an artificial chromosome is that the copy number ratio of each of the sequences can be controlled with precision. This allows the optimum ratio of sequences to be present on the artificial chromosome for efficient retroviral vector production. For example, with respect to the nucleic acid sequences encoding: (a) the RNA genome of the retroviral vector particle; (b) gag and pol proteins; and (c) env protein or a functional substitute thereof, the copy number of each of the sequences present on the artificial chromosome Examples of artificial chromosomes In one embodiment, the artificial chromosome is a mammalian artificial chromosome (MAC).

In a further embodiment, the mammalian artificial chromosome is a mouse or human artificial 20 chromosome.

WO 97/40183 and WO 2002/097059 provide heterochromatic artificial chromosomes designated therein as "satellite artificial chromosomes" (SATACs) or "artificial chromosome expression systems" (ACES) which may be used for the production of gene products. Developed by CHROMOS Molecular Systems Inc., these ACES were constructed through the induction of large scale amplifications of "satellite" DNA sequences in pericentromeric heterochromatin (e.g. see W097/40183, W02002/076508, W02002/097059, De Jong et al. (1999) Nat. Genet 35:129-133, Csonka et al. (2000) I. Cell So: 113:3207-3216 and Lindenbaum et al. (2004) Nuc. Acid Res. 32:21, which are herein incorporated by reference in their entirety). The ACES contains multiple integration sites using the lambda phage integration system (see Landy (1989) Annu. Rev. Biochem. 58: 913- 949). The ACES is predicted to contain more than 50 (approximately 50 to 70) recombination acceptor sites and can accept large "genetic payloads" from targeting vectors, therefore it can be used to introduce large genes into a cell. A high level of gene expression has also been observed when using ACES which makes it suitable for producing retroviral vector at high titre.

As an artificial chromosome, the ACES does not need to integrate into the mammalian host cell genome in order to replicate. This technology has been shown to be stably maintained in CHO cells (i.e. no translocations) for over 70 generations (see Kennard et al. (2009) Biotechnol. Bioeng. 104(3): 526-539).

may be in a ratio of at least 1:1:1, for example 2:1:1, 2:2:1, 3:1:1, 3:2:1, 3:2:2, 3:3:1, 3:3:2, 4:1:1, 4:2:1, 4:3:1, 4:3:2, 4:3:3, 4:4:1, 4:4:3, 5:1:1, 5:2:1, 5:2:2, 5:3:1, 5:3:2, 5:3:3, 5:4:1, 5:4:2, 5:4:3, 5:4:4, 5:5:1, 5:5:2, 5:5:3 or 5:5:4.

Size and content of artificial chromosomes The size of the artificial chromosome used can vary depending on its origin. For example, the artificial chromosome as described herein may be formed of repeating DNA units. In one embodiment, the artificial chromosome is made up primarily of repeating DNA units comprising tandem blocks of satellite DNA flanked by non-satellite DNA. The repeating DNA unit may vary in length, but typically would be on the order of about 7 to about 20 megabase pairs (Mb), for example between about 10 Mb to about 15 Mb.

The resulting artificial chromosome therefore, in one embodiment, contains at least 7 Mb, 10 Mb, 15 Mb, 20 Mb, 50 Mb, 70 Mb, 100 Mb, 150 Mb, 200 Mb, 250 Mb, 300 Mb, 400 Mb, 500 Mb, 600 Mb, 700 Mb, 800 Mb, 900 Mb or 1000 Mb. In a further embodiment, the artificial chromosome contains about 7 to about 450 Mb. In a further embodiment, the artificial chromosome contains about 15 Mb to about 60 Mb. In one example, the artificial chromosome is about 15 Mb, 20 Mb or 30 Mb.

In one embodiment, the artificial chromosome contains more heterochromatin than euchromatin, Le. less than 5%, 100/0, 20%, 3O%, 40% or 5O% euchromatin. In some cases, the artificial chromosome is predominantly heterochromatin, therefore in a further embodiment, the artificial chromosome contains substantially all heterochromatin, i.e. less than about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or 30% euchromatin. The advantage of providing an artificial chromosome which is substantially all heterochromatin is that it can be engineered to express the desired gene product without interference from potential genes coded in euchromatin.

Alternatively, in one embodiment, the artificial chromosome contains more euchromatin than heterochromatin, i.e. less than 50/0, 10%, 20%, 30%, 40% or 50% heterochromatin. In some cases, the artificial chromosome is predominantly euchromatin, therefore in a further embodiment, the artificial chromosome contains substantially all euchromatin, i e less than about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or 30% heterochromatin.

In one embodiment, the artificial chromosome contains one or a plurality of recombination site(s). This allows for target sequences to be integrated into the artificial chromosome in a site-specific manner in the presence of a recombinase enzyme. The recombinase enzyme catalyses the recombination reaction between two recombination sites.

Many types of site-specific recombination systems are known in the art, and any suitable recombination system may be used in the present invention. For example, in one embodiment the recombination site(s) are selected or derived from the int/aftsystem of lambda phage, the Cre/lox system of bacteriophage P1, the FLP/FRT system of yeast, the Gin/gix recombinase system of phage Mu, the Cin recombinase system, the Pin recombinase system of E. colland the R/RS system of the pSR1 plasmid, or any combination thereof. In a yet further embodiment, the recombination site is an aft site (e.g. from lambda phage), wherein the attsite is heterologous to the chromosome and the attsite permits site-directed integration in the presence of a lambda integrase. It will be understood that the reference to "lambda integrase" includes references to mutant integrases which are still compatible with the intfattsystem, for example the modified lambda integrases described in WO 2002/097059.

In this embodiment, the attsite may be selected from the group consisting of atzP and alley or variants thereof. In a further embodiment, the attP site is present on the artificial chromosome and the attB site is included with the target sequence to be incorporated (e.g. the transgene). In an alternative embodiment, the attB site is present on the artificial chromosome and the at/P site is included with the target sequence to be incorporated (e.g. the transgene). Examples of this system are described in more detail in WO 2002/097059 which is herein incorporated by reference in its entirety.

Methods of preparing artificial chromosomes It has previously been found that integration of heterologous DNA into the centromeric region or in close proximity thereto, initiates large-scale amplification of chromosomal segments which leads to de novo chromosome formation, thereby generating dicentric chromosomes. Cells with dicentric chromosomes may then be cultured under conditions whereby the chromosome breaks, generating a satellite DNA-based artificial chromosome (SATAC) containing satellite DNA interrupted by bands of transfected heterologous DNA.

As described in EP0929689, an exemplary method of producing an artificial chromosome in a mammalian cell comprises: introducing one or more heterologous DNA fragments into a mammalian cell, wherein the DNA fragment or fragments optionally comprises a selectable marker; growing the mammalian cell under selective conditions to produce mammalian cells that have incorporated the heterologous DNA fragment or fragments into their genomic DNA; identifying from among the resulting mammalian cells those that include chromosomes with more than one centromere and/or fragments of such chromosomes; selecting a mammalian cell or cells from among the identified mammalian cells and growing the mammalian cell or cells under conditions whereby mammalian cells that contain chromosomes with repeating DNA units comprising tandem blocks of satellite DNA flanked by non-satellite DNA, a primary replication site, and including heterologous DNA, are produced; subcloning mammalian cells that contain chromosomes with said repeating DNA units and growing the mammalian cells under selective conditions until a mammalian cell or cells containing a satellite DNA-based artificial chromosome, which contains said repeating DNA units, is produced; and selecting a mammalian cell that comprises a satellite DNA-based artificial chromosome which contains more heterochromatin than euchromatin and is made up primarily of repeating DNA units comprising tandem blocks of satellite DNA flanked by non-satellite DNA, a primary replication site, and including heterologous DNA.

In one embodiment, the method is performed in vitro. Selectable markers (which are well-known to the person skilled in the art) may be used throughout the method of preparing artificial chromosomes.

In one embodiment, the method additionally comprises ligating a telomere to each end of the artificial chromosome. This ligation may be performed in vitro, for example, in solution, LMP agarose or on microbeads.

In one embodiment, the heterologous DNA fragment or fragments are introduced into or adjacent to a centromere of an endogenous chromosome in the cell. In a further embodiment, the heterologous DNA fragment or fragments comprise a sequence of nucleotides that targets the fragment or fragments to a pericentric region or a heterochromatic region of a chromosome. In a further embodiment, the targeting sequence of nucleotides comprises satellite DNA.

In a preferred embodiment, the heterologous DNA fragment or fragments are introduced in the ribosomal DNA (rDNA) units that give rise to ribosomal RNA (rRNA). The rDNA have been shown to facilitate replication and/or amplification of chromosomal segments in the de novo formation of chromosomes in cells (see Keres6 et al. (1996) Chromosome Res. 4(3):226-39).

An exemplary sequence of rDNA in the mouse genome encompassing an origin of replication is in GENBANK accession no. X82564 at about positions 2430-5435. In human rDNA, a primary replication initiation site may be found a few kilobase pairs upstream of the transcribed region and secondary initiation sites may be found throughout the non-transcribed intergenic spacer region (see, e.g., Yoon et a/. (1995) Mot Cell. Biol. 15:24822489). A complete human rDNA repeat unit is presented in GENBANK as accession no. U13369.

A further advantage of targeting the fragment or fragments to the rDNA units is that they are AT-rich which means that the resultant artificial chromosome can be isolated from genomic DNA at a high rate and purity by flow cytometric sorting.

In one embodiment, the mammalian cell with a chromosome that has more than one centromere that is selected is a mammalian cell that has a dicentric chromosome that comprises a de novo centromere. The cell is then grown under conditions whereby a chromosome with a heterochromatic arm is produced. In a further embodiment, the method comprises culturing the cells that contains the dicentric chromosome with a heterochromatic arm under conditions that destabilize the dicentric chromosome, whereby a satellite DNA-based artificial chromosome is produced. The conditions that destabilize the chromosome may comprise a chemical agent that destabilizes the chromosomes, such as 5-bromodeoxyuridine (BrdU).

After treatment, the dicentric chromosome has formed two artificial chromosomes, a so-called minichromosome, and a formerly dicentric chromosome that has typically undergone amplification in the heterochromatin where the heterologous DNA has integrated to produce a satellite artificial chromosome (SATAC) or a "sausage" chromosome. These cells can be fused with other cells to separate the minichromosome and the formerly dicentric chromosome into different cells so that each type of artificial chromosome can be treated separately.

The artificial chromosomes described herein can be isolated by any suitable method known to those of skill in the art. In one embodiment, the artificial chromosomes may be isolated by fluorescence-activated cell sorting (FACS). This method may be used by virtue of the fact that artificial chromosomes, such as SATACs, have a higher heterochromatic DNA content compared to other chromosomes in a cell.

Alternative methods may be used which rely on the size and density differences between SATACs and endogenous chromosomes. Such methods involve techniques such as centrifugation (e.g. zonal rotor centrifugation and velocity sedimentation) or affinity-(e.g. immunoaffinity-) based methods for separation of artificial from endogenous chromosomes. For example, SATACs, which are predominantly heterochromatin, may be separated from endogenous chromosomes by using immunoaffinity procedures involving antibodies that specifically recognize heterochromatin, and/or the proteins associated therewith. This is particularly effective when the endogenous chromosomes contain relatively little heterochromatin, such as in hamster cells. These methods have the advantage that they are particularly well suited for large-scale isolation of artificial chromosomes.

A more specific method of generating an artificial chromosome may be, for example, the method described in WO 97/40183 which uses the mouse LMTK cell line and may be summarised as follows: (A) Transfecting cells with heterologous ("foreign") DNA in the centromeric region of a chromosome, such as chromosome 7, to generate a de novo centromere linked to the integrated foreign DNA, and form a dicentric chromosome; (B) Inducing breakage between the centromeres of the dicentric chromosome to generate a chromosome fragment with the de novo centromere, and a chromosome with traces of foreign DNA at the end; (C) Forming a stable neo-minichromosome by inverted duplication of the fragment bearing the de novo centromere; (D) Integrating exogenous DNA into the foreign DNA region of the formerly dicentric chromosome to initiate amplification (e.g. H-type amplification) and form a heterochromatic arm.

The new heterochromatic chromosome arm may be stabilized by capturing a euchromatic terminal segment (this forms a "sausage" chromosome); (E) Inducing further amplification (e.g. H-type amplification) by BrdU treatment and/or drug selection, which results in the formation of an unstable gigachromosome; and (F) Repeating BrdU treatments and/or drug selection to induce further amplification (e.g. H-type amplification), including centromere duplication, which leads to the formation of another heterochromatic chromosome arm. This may be split off from the chromosome by inducing chromosome breakage. A stable megachromosome (i.e. a SATAC) is formed by acquiring a terminal segment.

In one embodiment, the artificial chromosome used in the present invention is either the neo-minichromosome or the stable megachromosome, in particular the neo-minichromosome.

Once the artificial chromosome has been formed it may be used in the invention described herein. Optionally, the artificial chromosome may be moved from the cell line in which it is formed to a different cell line prior to use in the present invention. For example, the artificial chromosome may be formed in the LMTK cell line, as described above, and then transferred to a different cell line, for example a HEK cell line, before introducing the retroviral genes.

RETROVIRUSES

Retroviruses are a family of viruses which contain a pseudo-diploid single-stranded RNA genome. They encode a reverse transcriptase which produces DNA from the RNA genome which can then be inserted into the host cell DNA. The invention described herein may be used to produce replication defective retroviral vector particles. The retroviral vector particle of the present invention may be selected from or derived from any suitable retrovirus.

In one embodiment, the retroviral vector particle is derived from, or selected from, a lentivirus, alpha-retrovirus, gamma-retrovirus or foamy-retrovirus, such as a lentivirus or gammaretrovirus, in particular a lentivirus. In a further embodiment, the retroviral vector particle is a lentivirus selected from the group consisting of HIV-1, HIV-2, SIV, FIV, EIAV and Visna. Lentiviruses are able to infect non-dividing (te, quiescent) cells which makes them attractive vectors for gene therapy. In a yet further embodiment, the retroviral vector particle is HIV-1 or is derived from HIV-1. The genomic structure of some retroviruses may be found in the art. For example, details on HIV-1 may be found from the NCBI Genbank (Genome Accession No. AF033819). HIV-1 is one of the best understood retroviruses and is therefore often used as a viral vector.

Retroviral Genes The nucleic acid sequences common to all retroviruses may be explained in more detail, as follows: Long Terminal Repeats (LTRs): The basic structure of a retrovirus genome comprises a 5'LTR and a 3'-LTR, between or within which are located the genes required for retroviral production.

The LTRs are required for retroviral integration and transcription. They can also act as promoter sequences to control the expression of the retroviral genes. The LTRs are composed of three subregions designated U3, R, U5: U3 is derived from the sequence unique to the 3' end of the RNA; R is derived from a sequence repeated at both ends of the RNA; and U5 is derived from the sequence unique to the 5' end of the RNA. Therefore, in one embodiment, the artificial chromosome additionally comprises a 5'-and 3'-LTR.

In order to address safety concerns relating to the generation of replication-competent virus, a self-inactivating (SIN) vector has been developed by deleting a section in the U3 region of the 3' LTR, which includes the TATA box and binding sites for transcription factors Spl and NF-KB (see Miyoshi eta! (1998) J. Virol. 72(10):8150-7). The deletion is transferred to the 5' LTR after reverse transcription and integration in infected cells, which results in the transcriptional inactivation of the LTR. This is known as a self-inactivating lentiviral-based vector system.

y: Encapsidation of the retroviral RNAs occurs by virtue of a y (psi) sequence located at the 5' end of the retroviral genome. It is also well known in the art that sequences downstream of the psi sequence and extending into the gag coding region are involved in efficient vector production (see Cui et al. (1999) J. Virol. 73(7): 6171-6176). In one embodiment, the artificial chromosome additionally comprises a y (psi) sequence.

Primer Binding Site (PBS): The retroviral genome contains a PBS which is present after the U5 region of the 5'-LTR. This site binds to the tRNA primer required for initiation of reverse transcription. In one embodiment, the artificial chromosome additionally comprises a PBS sequence.

PPT: Retroviral genomes contain short stretches of purines, called polypurine tracts (PPTs), near the 3' end of the retroviral genome. These PPTs function as RNA primers for plus-strand DNA synthesis during reverse transcription. Complex retroviruses (such as HIV-1) contain a second, more centrally located PPT (Le. a central polypurine tract (cPPT)) that provides a second site for initiation of DNA synthesis. Retroviral vectors encoding a cPPT have been shown to have enhanced transduction and transgene expression (see Barry et al. (2001) Hum. Gene Ther. 12(9):1103-8). In one embodiment, the artificial chromosome additionally comprises a 3'-PPT sequence and/or a cPPT sequence.

The genomic structure of the non-coding regions described above are well known to a person skilled in the art. For example, details on the genomic structure of the non-coding regions in HIV-1 may be found from the NCBI Genbank (Genome Accession No. AF033819).

Gag/pot. The expression of gag and pdgenes relies on a translational frameshift between gag and gagpol. Both are polyproteins which are cleaved during maturation. The major structural matrix, capsid, and nucleocapsid proteins of the retroviral vector are encoded by gag. The po/gene codes for the retroviral enzymes: i) reverse transcriptase, essential for reverse transcription of the retroviral RNA genome to double stranded DNA, ii) integrase, which enables the integration of the retroviral DNA genome into a host cell chromosome, and iii) protease, that cleaves the synthesized polyprotein in order to produce the mature and functional proteins of the retrovirus.

Env The env("envelope") gene codes for the surface and transmembrane components of the retroviral envelope (e.g. glycoproteins gp120 and gp41 of HIV-1) and is involved in retroviral-cell membrane fusion. In order to broaden the vector's tissue tropism, the retroviral vectors described herein may be pseudotyped with an envelope protein from another virus. Pseudotyping refers to the process whereby the host cell range of retroviral vectors, including lentiviral vectors, can be expanded or altered by changing the glycoproteins (GPs) on the vector particles (e.g. by using GPs obtained from or derived from other enveloped viruses or using synthetic/artificial GPs). The most commonly used glycoprotein for pseudotyping retroviral vectors is the Vesicular stomatitis virus GP (VSVg), due to its broad tropism and high vector particle stability. However, it will be understood by the skilled person that other glycoproteins may be used for pseudotyping (see Cronin et al. (2005) Curr. Gene Ther. 5(4):387-398, herein incorporated by reference in its entirety). The choice of virus used for pseudotyping may also depend on the type of cell and/or organ to be targeted because some pseudotypes have been shown to have tissue-type preferences.

In one embodiment, the env protein or a functional substitute thereof is obtained from or derived from a virus selected from a Vesiculovirus (e.g. Vesicular stomatitis virus), Lyssavirus (e.g. Rabies virus, Mokola virus), Arenavirus (e.g. Lymphocytic choriomeningitis viruse (LCMV)), Alphavirus (e.g. Ross River virus (RRV), Sindbis virus, Semliki Forest virus (SFV), Venezuelan equine encephalitis virus), Filovirus (e.g. Ebola virus Reston, Ebola virus Zaire, Lassa virus), Alpharetrovirus (e.g. Avian leukosis virus (ALV)), Betaretrovirus (e.g. Jaagsiekte sheep retrovirus (JSRV)), Gammaretrovirus (e.g. Moloney Murine leukaemia virus (MLV), Gibbon ape leukaemia virus (GALV), Feline endogenous retrovirus (RD114)), Deltaretrovirus (e.g. Human T-lymphotrophic virus 1 (HTLV1)), Spumavirus (e.g. Human foamy virus), Lentivirus (e.g. Maedi-visna virus (MW)), Coronavirus (e.g. SARS-CoV), Coronavirus (e.g. Sendai virus, Respiratory syncytia virus (RSV)), Hepacivirus (e.g. Hepatitis C virus (HCV)), Influenzavirus A (e.g. Influenza virus) and Nucleopolyhedrovirus (e.g. Autographa californica multiple nucleopolyhedro virus (AcMNPV)). In a further embodiment, the env protein or a functional substitute thereof is obtained from or derived from Vesicular stomatitis virus. In this embodiment, the Vesicular stomatitis virus glycoprotein (VSVg) protein may be used which enables the retroviral particles to infect a broader host cell range and eliminates the chances of recombination to produce wild-type envelope proteins.

The structural genes described herein are common to all retroviruses. Further auxiliary genes may be found in different types of retrovirus. For example, lentiviruses, such as HIV-1, contain six further auxiliary genes known as rev, v/ vpu, vpr, nefand tat. Other retroviruses may have auxiliary genes which are analogous to the genes described herein, however they may not have always been given the same name as in the literature. References such as Tomonaga and Mikami (1996)1 Gen 77(Pt 8):1611-1621 describe various retrovirus auxiliary genes.

Rev The auxiliary gene rev(" regulator of virion") encodes an accessory protein which binds to the Rev Response element (RRE) and facilitates the export of retroviral transcripts. The gene's protein product allows fragments of retroviral mRNA that contain the Rev Responsive element (RRE) to be exported from the nucleus to the cytoplasm. The 250 nucleotide RRE sequence is predicted to form a complex folded structure. This particular role of rev reflects a tight coupling of the splicing and nuclear export steps. Rev binds to RRE and facilitates the export of singly spliced (env, vpr and vpu) or non-spliced (gag, po/and genomic RNA) viral transcripts, thus leading to downstream events like gene translation and packaging (see Suhasini and Reddy (2009) Curr. HIV Res. 7(1): 91100). In one embodiment, the artificial chromosome additionally comprises the auxiliary gene rev or an analogous gene thereto (i.e. from other retroviruses or a functionally analogous system).

Auxiliary genes are thought to play a role in retroviral replication and pathogenesis, therefore many current vector production systems do not include some of these genes. The exception is rev which is usually present unless a system analogous to the rev/RRE system is used. Therefore, in one embodiment, the nucleic acid sequences encoding one or more of the auxiliary genes vpr, vpu, tatand nef; or analogous auxiliary genes, are disrupted such that said auxiliary genes are removed from the RNA genome of the retroviral vector particle or are incapable of encoding functional auxiliary proteins. In a further embodiment, at least two or more, three or more, four or more, or all of the auxiliary genes vpr, vit; vpu, tatand nef, or analogous auxiliary genes, are disrupted such that said auxiliary genes are removed from the RNA genome of the retroviral vector particle or are incapable of encoding functional auxiliary proteins. Removal of the functional auxiliary gene may not require removal of the whole gene; removal of a part of the gene or disruption of the gene will be sufficient.

It will be understood that the nucleic acid sequences encoding the replication defective retroviral vector particle may be the same as, or derived from, the wild-type genes of the retrovirus upon which the retroviral vector particle is based, Le. the sequences may be genetically or otherwise altered versions of sequences contained in the wild-type virus. Therefore, the retroviral genes incorporated into the artificial chromosomes or host cell genomes, may also refer to codon-optimised versions of the wild-type genes.

ADDITIONAL COMPONENTS

The artificial chromosomes of the invention may comprise further additional components.

These additional features may be used, for example, to help stabilize transcripts for translation, increase the level of gene expression, and turn on/off gene transcription.

The retroviral vector particles produced by the invention may be used in methods of gene therapy. Therefore, in one embodiment, the artificial chromosome additionally comprises one or more transgenes. This heterologous transgene may be a therapeutically active gene which encodes a gene product which may be used to treat or ameliorate a target disease. The transgene may encode, for example, an antisense RNA, a ribozyme, a protein (for example a tumour suppressor protein), a toxin, an antigen (which may be used to induce antibodies or helper T-cells or cytotoxic T-cells) or an antibody (such as a single chain antibody).

In some cases more than one gene product is required to treat a disease, therefore in a further embodiment, the artificial chromosome additionally comprises two or more transgenes.

References herein to "transgene" refer to heterologous or foreign DNA which is not present or not sufficiently expressed in the mammalian host cell in which it is introduced. Therefore, the transgene may be a gene of potential therapeutic interest. The transgene may have been obtained from another cell type, or another species, or prepared synthetically. Alternatively, the transgene may have been obtained from the host cell, but operably linked to regulatory regions which are different to those present in the native gene.

The aim of gene therapy is to modify the genetic material of living cells for therapeutic purposes, and it involves the insertion of a functional gene into a cell to achieve a therapeutic effect. The retroviral vector produced using the artificial chromosomes and host cells described herein can be used to transfect target cells and induce the expression of the gene of potential therapeutic interest. The retroviral vector can therefore be used for treatment of a subject suffering from a condition including but not limited to, inherited disorders, cancer, and certain viral infections. In one embodiment, the artificial chromosome additionally comprises a transcription regulation element. For example, any of the elements described herein may be operably linked to a promoter so that expression can be controlled. Promoters referred to herein may include known promoters, in whole or in part, which may be constitutively acting or inducible, e.g. in the presence of a regulatory protein. In one embodiment, the artificial chromosome additionally comprises a high efficiency promoter, such as a CMV promoter. This promoter has the advantage of promoting a high level of expression of the elements encoded on the artificial chromosome. In an alternative embodiment, the artificial chromosome additionally comprises a pEF (human elongation factor la) promoter.

In one embodiment, the artificial chromosome additionally comprises a selectable marker. This allows the cells which have the artificial chromosome with the nucleic acid sequences encoding a replication defective retroviral vector particle to be selected. In a further embodiment, the selectable marker is an antibiotic resistance gene, such as a kanamycin or puromycin resistance gene.

In one embodiment, the artificial chromosome additionally comprises a polyA signal. The use of a polyA signal has the advantage of protecting mRNA from enzymatic degradation and aiding in translation. In a further embodiment, the polyA signal is obtained from or derived from SV40, Bovine Growth Hormone or Human Beta Globin.

In one embodiment, the artificial chromosome additionally comprises an intron sequence. The use of an intron downstream of the enhancer/promoter region and upstream of the cDNA insert (i.e. the transgene) is known to increase the level of expression of the insert. In a further embodiment, the intron sequence is a Human Beta Globin Intron II sequence.

In one embodiment, the artificial chromosome additionally comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The presence of WPRE has been shown to enhance expression and as such is likely to be beneficial in attaining high levels of expression.

In one embodiment, the artificial chromosome additionally comprises an internal ribosome entry site (IRES). An IRES is a structured RNA element that is usually found in the 5'-untranslated region downstream of the 5'-cap (which is required for the assembly of the initiation complex). The IRES is recognized by translation initiation factors, and allows for cap-independent translation.

HOST CELLS

According to a further aspect of the invention, there is provided a mammalian host cell comprising the retroviral particle-producing artificial chromosome as described herein. It will be understood that the term "retroviral particle-producing artificial chromosome" refers to an artificial chromosome comprising the nucleic acid sequences encoding the RNA genome of the retroviral vector particle, gag and pol proteins, and env protein or a functional substitute thereof, as described herein.

In one embodiment, the mammalian host cell is a HEK 293 cell, CHO cell, Jurkat cell, KS62 cell, PerC6 cell, HeLa cell or a derivative or functional equivalent thereof. In a further embodiment, the mammalian host cell is a HEK 293 cell, or derived from a HEK 293 cell. Such cells lines could be adherent cell lines (i.e. they grow in a single layer attached to a surface) or suspension adapted/non-adherent cell lines (i.e. they grow in suspension in a culture medium). In a yet further embodiment, the HEK 293 cell is a HEK 293T cell. The term "HEK 293 cell" refers to the Human Embryonic Kidney 293 cell line which is commonly used in biotechnology. In particular, HEK 293T cells are commonly used for the production of various retroviral vectors. Other examples of suitable commercially available cell lines include T-RExTm (Life Technologies) cell lines.

USES

According to a further aspect of the invention, there is provided the use of the retroviral particle-producing artificial chromosome as defined herein, or the mammalian host cell comprising the retroviral particle-producing artificial chromosome as defined herein, to produce a high titre of retroviral vector.

References herein to the term "high titre" refer to an effective amount of retroviral vector or particle which is capable of transducing a target cell, such as a patient cell. In one embodiment, a high titre is in excess of 106 TU/ml without concentration (TU = transducing units).

According to a further aspect of the invention, there is provided the retroviral particle-producing artificial chromosome defined herein for use in producing a retroviral packaging cell line.

The artificial chromosomes described herein may be used to create a retroviral packaging cell line which would greatly simplify vector production. It will be understood that if a transgene is included on the artificial chromosome, then this would be used to create a producer cell line.

METHODS

According to a further aspect of the invention, there is provided a method of producing a replication defective retroviral vector particle, comprising culturing a mammalian host cell comprising the retroviral particle-producing artificial chromosome as described herein under conditions in which the retroviral vector particle is produced.

It will be understood by the skilled person that the conditions used in the method described herein will be dependent upon the mammalian host cell used. Typical conditions, for example the culture medium or temperature to be used, are well known in the art.

In one embodiment, the method additionally comprises isolating the retroviral particle produced.

A method of preparing the retroviral particle-producing artificial chromosomes as defined herein comprises: (a) introducing an artificial chromosome comprising a genetic locus which enables the artificial chromosome to be stably replicated and segregated alongside endogenous chromosomes, into a mammalian host cell; and (b) integrating into the artificial chromosome nucleic acid sequences encoding: the RNA genome of the retroviral vector particle, gag and pol proteins, and env protein or a functional substitute thereof.

The skilled person will be aware that introducing the artificial chromosome into the mammalian host cell in step (a) may be performed using suitable methods known in the art, for example, lipid-mediated transfection, microinjection, cell (such as microcell) fusion, electroporation, microprojectile bombardment or direct DNA transfer. In one embodiment, the artificial chromosome is introduced into the mammalian host cell by lipid-mediated transfection.

As described above, the mammalian host cell in which the artificial chromosome is introduced may be a different cell type to the one used to generate the artificial chromosome.

The nucleic acid sequences may be integrated into the artificial chromosome sequentially. This allows for selection after each integration to ensure that all of the required nucleic acid sequences are successfully integrated into the artificial chromosome. Alternatively, at least two or more of the nucleic acid sequences are integrated into the artificial chromosome simultaneously (e.g. by triple transfection).

The skilled person will be aware that the integration step may be performed by transfection using a suitable vector. If recombination sites are present on the artificial chromosome then a targeting vector can be used for targeted recombination. For example, the artificial chromosome may contain site-specific recombination acceptor sites (such as acceptor att sites, in particular attP sites), therefore a vector with equivalent recombination donor sites (such as donor alt sites, in particular NO sites) can be used for targeted integration, potentially in the presence of a cofactor (such as an integrase, in particular an integrase obtained from, or derived from, lambda integrase).

The integration step may be performed using suitable methods known in the art, for example lipid mediated transfection. For example, in the case of the Intl att recombination system of lambda phage, lipid mediated co-transfection of the targeting vector and the integrase into the mammalian host cell may be used.

There are many methods known in the art to confirm the presence of the artificial chromosome in the mammalian host cell, for example using Fluorescence In Situ Hybridisation (FISH).

To increase expression of the replication defective retroviral vector particle, a second transfection or "double load" may be carried out after the integration step. In order to "double load" the artificial chromosome, the integration step is repeated on the primary transfectants in order to increase the number of target genes present on the artificial chromosome. Therefore, the integration step may be repeated one or more, such as two or more, three or more, or four or more times in order to increase the number of target genes present on the artificial chromosome and increase expression.

If the target genes are integrated into the artificial chromosome with a selective marker, such as an antibiotic resistance gene, it will be understood that different selective markers can be used with each repeat of the integration step in order to allow for selection after each integration.

According to a further aspect of the invention, there is provided a replication defective retroviral vector particle produced by the methods defined herein.

Claims (27)

1. CLAIMS1. An artificial chromosome comprising a genetic locus which enables the artificial chromosome to be stably replicated and segregated alongside endogenous chromosomes in a mammalian host cell, characterized in that said artificial chromosome comprises nucleic acid sequences encoding a replication defective retroviral vector particle, comprising: the RNA genome of the retroviral vector particle, gag and pol proteins, and env protein or a functional substitute thereof.
2. 2. The artificial chromosome of claim 1, additionally comprising the auxiliary gene rev or an analogous gene thereto or a functionally analogous system.
3. 3. The artificial chromosome of claim 1 or claim 2, wherein the retroviral vector particle is derived from a retrovirus selected from lentivirus, alpha-retrovirus, gamma-retrovirus or foamy-retrovirus.
4. 4. The artificial chromosome of claim 3, wherein the retroviral vector particle is derived from a lentivirus selected from the group consisting of HIV-1, HIV-2, SW, FIV, EIAV and Visna.
5. 5. The artificial chromosome of claim 4, wherein the retroviral vector particle is derived from HIV-1.
6. 6. The artificial chromosome of any one of claims 1 to 5, wherein the env protein or a functional substitute thereof is derived from Vesicular stomatitis virus.
7. 7. The artificial chromosome of any one of claims 1 to 6, which additionally comprises a transcription regulation element.
8. 8. The artificial chromosome of any one of claims 1 to 7, which additionally comprises aselectable marker.
9. 9. The artificial chromosome of any one of claims 1 to 8, which additionally comprises one or more transgenes.
10. 10. The artificial chromosome of any one of claims 1 to 9, which additionally comprises a polyA signal.
11. 11. The artificial chromosome of any one of claims 1 to 10, which additionally comprises an intron sequence.
12. 12. The artificial chromosome of any one of claims 1 to 11, which is made up primarily of repeating DNA units comprising tandem blocks of satellite DNA flanked by non-satellite DNA.
13. 13. The artificial chromosome of claim 12, wherein the repeating DNA units are about 7 to about megabases (Mb).
14. 14. The artificial chromosome of any one of claims 1 to 13, wherein the artificial chromosome is a mammalian artificial chromosome (MAC).
15. 15. The artificial chromosome of any one of claims 1 to 14, which contains at least 7 Megabase pairs (Mb), 10 Mb, 15 Mb, 20 Mb, 30Mb, 50 Mb, 70 Mb, 100 Mb, 150 Mb, 200 Mb, 250 Mb, 300 Mb, 400 Mb, 500 Mb, 600 Mb, 700 Mb, 800 Mb, 900 Mb or 1000 Mb.
16. 16. The artificial chromosome of any one of claims 1 to 15, which contains more heterochromatin than euchromatin.
17. 17. The artificial chromosome of any one of claims 1 to 16, which contains substantially all heterochromatin.
18. 18. The artificial chromosome of any one of claims 1 to 17, which contains one or a plurality of recombination site(s).
19. 19. The artificial chromosome of claim 18, wherein the recombination site(s) are derived from the intfattsystem of lambda phage, the Cre/lox system of bacteriophage P1, the FLP/FRT system of yeast, the Gin/gix recombinase system of phage Mu, the Cin recombinase system, the Pin recombinase system of E co//and the R/RS system of the pSR1 plasmid, or any combination thereof.
20. 20. The artificial chromosome of claim 18 or claim 19, wherein the recombination site is an att site which permits site-directed integration in the presence of lambda integrase.
21. 21. A mammalian host cell comprising the artificial chromosome of any one of claims 1 to 20.
22. 22. The mammalian host cell of claim 21, wherein the host cell is derived from a HEK 293 cell.
23. 23. Use of the artificial chromosome of any one of claims 1 to 20, or the mammalian host cell of claim 21 or claim 22, to produce a high titre of retroviral vector.
24. 24. The artificial chromosome of any one of claims 1 to 20 for use in producing a retroviral packaging cell line.
25. 25. A method of producing a replication defective retroviral vector particle, comprising culturing a mammalian host cell comprising the artificial chromosome of any one of claims 1 to 20 under conditions in which the retroviral vector particle is produced.
26. 26. The method of claim 25, additionally comprising isolating the retroviral vector particle.
27. 27. A replication defective retroviral vector particle produced by the method of claim 25.