WO2012156721A1

WO2012156721A1 - Methods for providing human cells comprising a human artificial chromosome

Info

Publication number: WO2012156721A1
Application number: PCT/GB2012/051075
Authority: WO
Inventors: Zoia Larin MONACO; Daniela MORALLI; Richard Wade-Martins
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 2011-05-16
Filing date: 2012-05-15
Publication date: 2012-11-22
Anticipated expiration: 2013-11-16
Also published as: GB201108116D0

Abstract

A method for providing a eukaryotic cell comprising a human artificial chromosome (HAC), the method comprising the step of: transducing the eukaryotic cell with at least one α-satellite DNA and at least one introduced DNA sequence comprised on at least two viral vectors, wherein the at least one α-satellite DNA and at least one introduced DNA sequence are able to form one or more HACs inside the cell.

Description

METHODS FOR PROVIDING HUMAN CELLS COMPRISING A HUMAN ARTIFICIAL CHROMOSOME

This invention relates to a method for providing a eukaryotic cell comprising a human artificial chromosome (HAC). In particular, it relates to a method for transducing a eukaryotic cell with DNA contained in one or more HSV- 1 amplicon vectors to form a HAC inside the eukaryotic cell.

For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties.

Human artificial chromosomes (HACs) are autonomous molecules that behave as normal chromosomes in human cells. Together with the endogenous chromosomes, HACs segregate during cell division and are maintained in the host cell. De novo HACs are generated by introducing defined sequences such as a- satellite (alphoid) DNA and specific introduced DNA sequences into eukaryotic cells. a-satellite DNA, containing higher-order repeat sequences, and a centromere protein B binding sequence (CENP-B box) are requirements for a functional centromere in a HAC.

Known methods for the delivery of large pieces of DNA are inefficient, often resulting in DNA shearing and degradation, which is a major obstacle in developing a HAC expression system in different cell types. Most gene expression studies in hESc utilize lentiviral, adenoviral and adeno- associated (AAV) viral vectors for gene delivery. However, lentiviral vectors integrate randomly at multiple sites within the host genome leading to insertional mutagenesis, and although adenoviral vectors remain episomal, silencing post transduction may occur. Another disadvantage is that the capacity of AAV and lentiviral vectors is limited to approximately 5 and 10 kb of DNA respectively.

Previous studies introduced input HAC DNA (404 kb) containing the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene into human HPRT deficient HT 1080 fibrosarcoma cells, and the HAC generated successfully complemented the deficiency. The input HAC DNA was delivered by standard transfection, but it was found that this method was inefficient for routine large DNA transfer and did not work in other cell types. The present invention provides a new approach to delivering large DNA sequences into cells to form a HAC. This delivery approach is based on the use of a viral vector system, preferably a herpes simplex virus type 1 (HSV- 1 ) amplicon vector system. The DNA may be delivered to the cell using two or more viral vectors which may be selected from vectors based on any virus, for example, an HSV virus, a retrovirus, an adeno-associated virus (AAV), an adenovirus, a vaccinia virus or a plant virus.

In one embodiment the two viral vectors may both be HSV- 1 amplicon vectors. HSV- 1 amplicon vectors differ from HSV- 1 vectors. HSV- 1 vectors are HSV- 1 viruses, which have been deleted of essential genes, but are still formed mostly of the viral genome. By contrast, in the HSV- 1 amplicon system, the vector may be, for example, a BAC or PAC input HAC DNA that only contains two short viral sequences, the origin of replication OriS, and the packaging signal Pac. HSV- 1 amplicon vectors are high-capacity vectors that can hold up to 150 kb of additional DNA sequence and successfully deliver a large introduced DNA sequence (for example bacterial artificial chromosomes (BACs) or P I artificial chromosomes (PACs)) intact into different cell types in the absence of contaminating viral genes. The advantage of the HSV- 1 amplicon system is that HSV- 1 amplicons have a high capacity for large DNA delivery (up to 150 kb) and can efficiently introduce HAC input DNA into cells, for example immortalized cell lines, or into stem cells. The efficiency of DNA delivery into cells using the HSV- 1 amplicon system is significantly greater than with other methods of chemical transfection (by a factor of 10⁴). Surprisingly the HSV- 1 amplicon vector system can be used to transduce stem cells. It is possible to co-transduce two HSV- 1 amplicon vectors into the same cell, both in differentiated cells and stem cells. While it is known that a cell where HSV- 1 has established latency can be superinfected by a second, different strain of HSV, it has generally been thought that the simultaneous infection by two identical HSV- 1 virions is not possible. This is because, since HSV- 1 directly delivers its DNA to the nucleus, even if two transgenes could enter the cell via different amplicons, they would not be able to interact, and so would probably integrate, or form HACs independently of one another. Surprisingly, the two HSV- 1 amplicon vectors delivered to cells were able to recombine and form a single HAC. This new approach opens up many possibilities for forming HACs inside cells because large amounts of DNA can be introduced into the cells efficiently and the DNA can form into HACs that are stably contained inside the cells.

According to a first aspect the invention provides a method for providing a eukaryotic cell comprising a human artificial chromosome (HAC), the method comprising the step of:

transducing the eukaryotic cell with at least one a-satellite DNA and at least one introduced DNA sequence comprised on at least two viral vectors, preferably at least two HSV- 1 amplicon vectors, wherein the a-satellite DNA and the at least one introduced DNA sequence are able to form one or more HACs inside the cell.

An α-satellite DNA is needed for each HAC that is formed. As well as the a- satellite DNA the HAC may comprise one or more introduced DNA sequences. All of the DNA that is required to form a HAC may be comprised on at least two viral vectors, preferably at least two HSV- 1 amplicon vectors. This means that the total DNA that is required to form a HAC is distributed between the two or more viral vectors, preferably two or more HSV- 1 amplicon vectors. The two or more viral vectors preferably two or more HSV- 1 amplicon vectors, may also comprise other DNA in addition to the DNA required to form a HAC. The DNA that is required to form a HAC may be comprised on or distributed between the two or more HSV- 1 amplicon vectors in any convenient way, for example, the a- satellite DNA may be on one of the vectors, while the one or more introduced DNA sequences may be on one or more additional HSV- 1 amplicon vectors. Alternatively, the α-satellite DNA may be on one of the HSV- 1 amplicon vectors with some of the introduced DNA and the remainder of the introduced DNA may be on one or more additional HSV- 1 amplicon vectors. In another embodiment the α-satellite DNA and the introduced DNA may each be divided between the one or more HSV- 1 amplicon vectors.

An introduced DNA sequence may be any piece of DNA (excluding the a-satellite DNA) that is included on an HSV- 1 amplicon vector, which is introduced into a cell in order to become part of a HAC. The introduced DNA may include a coding and/or a non-coding piece of DNA, for example all or part of one or more genes, regulatory regions and/or exons. The introduced DNA may be, for example, chromosomal DNA, an artificial DNA, a cDNA, or mitochondrial DNA. The introduced DNA may be identical to or different from the DNA of the host cell that it is introduced into. In one embodiment the introduced DNA comprises a gene that can express a protein or a peptide of interest inside the cell once a HAC is formed. Alternatively the introduced DNA may encode oligos or regulatory RNA molecules or a molecule that prevents transcription of a target gene, or interferes with the translation of a target protein or a non coding RNA/DNA controlling cellular processes. Preferably, the introduced DNA is configured to be at least transcribed, and preferably translated when the HAC is formed. The minimum requirement for forming a HAC in a cell is an α-satellite DNA sequence, for example the sequence of the core alpha satellite DNA from human chromosome 17 shown in Figure 12. In addition a HAC may include one or more introduced DNA sequences that may be expressed, or at least transcribed, in the host cell. Preferably the HAC has α-satellite DNA which provides the HAC with its own centromere and thus the HAC is maintained as an autonomous molecule in the cell without selection. In one embodiment telomeric sequences may also be included in the HAC. Preferably, the HAC behaves as a normal chromosome in a eukaryotic cell and segregates during cell division along with the endogenous chromosomes of the host cell. Optionally a HAC may also comprise a reporter gene, such as GFP, and selectable gene that help detection of HACs in the cells.

The minimum amount of input a satellite DNA sufficient to generate a HAC in the target cells is 40kb. Typically the total DNA needed to form a HAC (the HAC input DNA) ranges between 50 and 200 kb. If the HAC input DNA is delivered by HSV- 1 amplicons, then the upper limit of the amount of HAC input DNA is about 150kb or multiple of about 150 kb if more than one amplicon is used to deliver it, because each HSV- 1 amplicon vector can introduce about 150kb of DNA.

An advantage of forming a HAC in a cell is that it can contain and express large pieces of DNA. The large capacity of a HAC allows it to express whole genes. An important factor in the expression of some genes is that endogenous promoters or controlling regions may be located several kilobases upstream or downstream of the gene of interest. The position of the promoters or controlling regions with respect to the gene is important for overall gene regulation. Hence the larger the region that can be incorporated into the HAC, the higher the chance of obtaining physiological levels of expression. A HAC is maintained for a long time in the cells with or without selection. In the presence of selection a HAC may be maintained in the cell for more than four years. In the absence of selection a HAC is stable for longer than 9 months. The HAC replicates and segregates into the daughter cells when the cells divide. It has always been difficult to get large pieces of DNA into cells. Current methods of introducing DNA into cells include, use of calcium phosphate, electroporation and viral and non-viral vectors. Each of these methods shares the problem that only small pieces of DNA can be put into cells without the DNA being damaged. Large DNA fragments have very low transfection efficiencies or break up when entering cells using current methods. This has made it very difficult in the past to get sufficiently large pieces of DNA into cells to form a HAC.

If the DNA used to form a HAC is delivered by chemical or physical transfection, in theory there is no limit to the size of DNA. However, in practice, the larger the vector, the lower the delivery efficiency (for example an 8 kb vector can be delivered to HT 1080 cells by lipofection up to 10⁵ times more efficiently than a 400 kb vector). Furthermore, assembling a single vector containing at least 40 kb of alpha satellite and more than 200kb of other DNA is technically difficult and time consuming.

In order to use a HAC for gene therapy the efficiency of delivery of existing methods needs to be improved.

The DNA may be delivered to the cell using two or more viral vectors which may be selected from vectors based on any virus, for example an HSV virus, a retrovirus, an adeno-associated virus (aav), an adenovirus, a vaccinia virus or a plant virus. In one embodiment the two or more viral vectors may be based on different viruses. In another embodiment the two or more vectors may be based on the same virus. The advantage of delivering the DNA using viral vectors is that they deliver DNA efficiently to the cell. Two or more viral vectors may be introduced into the cell simultaneously and therefore more DNA can be introduced into the cell to form a HAC.

Using the HSV- 1 amplicon delivery system, a single HSV- 1 amplicon vector can deliver up to 150kb of DNA. Using more than one HSV- 1 amplicon vector introducing different pieces of DNA simultaneously, multiples of 150kb can be delivered with high efficiency to any cell type. Using standard delivery methods, DNA as large as 400kb can be delivered but with very low efficiency so that HAC formation has previously only been achieved in HT1080 fibrosarcoma cells.

In the present invention two or more HSV- 1 amplicon vectors, preferably HSV- 1 amplicon vectors, that comprise all of the DNA necessary to form a HAC are used to transduce the cells with the DNA needed to form a HAC and the HAC forms inside the transduced cell.

The requirements for the HSV- 1 amplicon system are the HSV- 1 origin of replication OriS and the packaging signal pac, in addition to a-satellite DNA required for HAC formation, for example the core α-satellite DNA from human chromosome 17 shown in figure 12. The DNA required to form a HAC, including the introduced DNA and at least 40 kb of α-satellite DNA, may be retrofitted in E. coli by loxP-Cre recombination with a smaller plasmid carrying the HSV- 1 origin of replication, OriS, and the HSV- 1 packaging signal, Pac. The vector DNA may be extracted from the E. coli bacteria by alkaline lysis, and lipofected using commercial reagents into the packaging cell line Vero 2-2, along with the fHSVApacA270+ vector and the pEBHICP27 constructs. fHSVApacA270+ vector and pEBHICP27 provide, in trans, the necessary proteins and enzymes to replicate and package the input HAC DNA which contains the OriS and Pac sequences. The input HAC DNA is thus packaged into HSV- 1 capsids (these infectious particles are now termed amplicons), which are released following Vero 2-2 cell lysis by ultrasonic disruption. Following centrifugation to concentrate the amplicons, they are applied to the target cells in a small volume of medium for 24 hours. After this period, excess amplicons may be removed by washing the cells with medium, and selection is applied to recover stably transduced clones. In one embodiment the cells are transduced with one a-satellite DNA sequence and one HAC is formed inside the cell. One α-satellite DNA is needed for each HAC, but any number of introduced DNA sequences may be on the HAC.

The HAC may comprise one or more than one introduced DNA sequence, for example one or more than one gene. The total DNA which forms the HAC (the input HAC DNA), including the α-satellite DNA and the one or more introduced DNA sequences, may be divided between the two or more HSV- 1 amplicon vectors. In one embodiment the α-satellite DNA may be on one of the HSV- 1 amplicon vectors and the one or more introduced DNA sequences may be on another one or more HSV- 1 amplicon vectors. In another embodiment the a- satellite DNA sequence may be divided between the two or more HSV- 1 amplicon vectors and the one or more introduced DNA sequences may be divided between the two or more HSV- 1 amplicon vectors. All or part of the α-satellite DNA sequence may be on the same HSV- 1 amplicon vector as one or more introduced DNA sequences.

In one embodiment an α-satellite DNA sequence may be on one HSV- 1 amplicon vector, while one or more introduced DNA sequences may be on another HSV- 1 amplicon vector. In another embodiment three, four, five, six or more than six HSV- 1 amplicon vectors may be transduced into the cells and may be introduced simultaneously into one cell. One of the HSV- 1 amplicon vectors may contain an a-satellite DNA sequence and the other co-transduced HSV- 1 amplicon vectors may comprise one or more introduced DNA sequences. These sequences may combine to form a HAC inside the cell. Preferably one a-satellite DNA sequence is required for each HAC that will be generated.

In one embodiment the at least one α-satellite DNA sequence and the at least one introduced DNA sequence may be divided between three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more HSV- 1 amplicon vectors.

Each HSV- 1 amplicon vector can deliver up to about 150 kb of introduced DNA. Therefore, if a large number of HSV- 1 amplicon vectors are used in the present invention more introduced DNA can be delivered into the cell. The introduced DNA from the large number of HSV- 1 amplicon vectors may join together in the cell to form one or more HAC. This is advantageous because a HAC with a larger introduced sequence or a larger number of introduced sequences may be formed in the cell.

A HAC may be used to express a gene that is lacking in particular cells. They are therefore useful in the treatment of genetic diseases, for example in gene therapy, where a particular gene is not expressed or a particular protein is expressed in cells in a form that does not have the normal activity. Furthermore, HACs can be used as model chromosomes to analyze carcinogenesis and tumour progression; to characterize the effect of compounds and treatments on chromosome structure, protein composition, replication, segregation and behaviour; to study biochemical or enzymatic pathways; to introduce extra copies of a gene to analyse the effects of increased copy number and overexpression of genes of certain biochemical or cellular assays; to introduce tagged copies of different proteins to analyse the assembly or structure of a protein complex; as vectors for vaccinia production and as vectors for induced pluripotent stem cell (iPS) generation. Other advantages of the use of HACs is that they behave as normal chromosomes in the cells, and assemble both euchromatin and heterochromatin, thus maintaining correct structure and behaviour. Due to their similarity to endogenous chromosomes they respond in a physiological way to cellular/extracellular stimuli and controlling systems. Furthermore, they occupy in the nucleus a specific position, thus ensuring that they are exposed to the correct proteins and/or modifying enzymes.

The cell that is transduced with one or more HSV- 1 amplicon vectors may be any type of eukaryotic cell for example a cell derived from a human and mammalian established or immortalized cell lines; embryonic stem cells; adult stem cells; induced pluripotent stem cells (iPS). In one embodiment the cell may be induced to have the characteristics of a stem cell, for example it may be an induced pluripotent stem cell. The cell may be made into an induced pluripotent stem cell before transduction with the HSV- 1 amplicon vectors according to the present invention. Alternatively, the cell may be induced to become an induced pluripotent stem cell by including the necessary genes on one or more of the HSV- 1 amplicon vectors introduced into the cell in the present invention. All or combinations of the following genes: Oct4, Sox-2, KLF4, c-myc, Nanog and Lin28, SV40 large T antigen, telomerase (hTERT) may be required to induce a cell to become an induced pluripotent stem cell. This is advantageous because a differentiated cell from a particular patient may be transduced according to the present invention, so that it includes a HAC and also is induced to form a pluripotent stem cell at the same time. The cell may then be caused to differentiate into the required cell type and be reintroduced into the patient. This allows cells comprising a HAC to be produced that are immunologically compatible with the patient.

In another embodiment one or more, prefereably two or more, HSV- 1 amplicon vectors according to the present invention may be transduced into a stem cell. The stem cell may be a human pluripotent stem cell or a human induced pluripotent stem cell. This is advantageous because the stem cell can be induced to differentiate into the required cell type. In one embodiment the eukaryotic cell transduced with the one or more HSV- 1 amplicon vectors is a mammalian cell, preferably a human cell. The mammalian cell may be a stem cell or an induced pluripotent stem cell. The stem cell may be a pluripotent or totipotent stem cell.

Preferably the eukaryotic cell is an adult stem cell or a cell derived from an adult, preferably a stem cell derived from brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, ovarian epithelium, or testis. Suitably the eukaryotic cell is a stem cell derived from umbilical cord blood or menstrual blood. Preferably the eukaryotic cell is a hematopoietic stem cell or a mesenchymal stem cell.

In one embodiment the eukaryotic cell may be a cell from a human or mammalian established or immortalized cell line. The eukaryotic cell may be an embryonic stem cell, an adult stem cell or an induced pluripotent stem cell (iPS). The embryonic stem cell may be a human embryonic stem cell. The embryonic stem cell may be derived from an embryo or from an embryonic stem cell line. Preferably the embryonic stem cell is derived from a commercially available embryonic stem cell line. Preferably the stem cell is not derived from a human embryo. Preferably the stem cell is a human stem cell that is not a human embryonic stem cell.

Human embryonic stem cells (hESc) are an important tool in clinical and basic research. Due to their high replicative lifespan and ability to differentiate into the three different germ layers, they are used for human developmental biology, tissue regeneration, transplant therapies and drug discovery studies. Safe and efficient in vitro genetic manipulation of hESc is an essential step in realizing their full potential for clinical applications. It is advantageous from the point of view of gene therapy to transform a human stem cell or induced pluripotent stem cell with the HSV- 1 amplicon to produce a HAC in the cell because the cell can be differentiated to form the required cell type. The cell may be obtained from a subject and, once the HAC is in place in the cell, the cell may be reintroduced into the subject where inserted genes may be expressed from the HAC in the cells. Alternatively, the HAC may be introduced in the cells in vivo.

It is possible to co-transduce various cell-types, including stem cell lines, with at least two different HSV- 1 amplicon vectors. This is advantageous because it allows a larger amount of DNA to be introduced into a cell at one time. The cells can be transduced with a number of HSV- 1 amplicon vectors each carrying part of the DNA needed to form a HAC. Once all of the vectors are inside the cell the DNA in the different HSV- 1 amplicon vectors can combine to form a HAC.

The transduction of cells may be done in vitro and the cells introduced into a patient once a HAC has been formed. Cells with the correctly formed HAC can be selected in vitro before introducing the selected cells into a patient. According to another aspect the present invention provides a method for providing a human cell comprising a human artificial chromosome (HAC) comprising the steps of:

providing a human pluripotent stem cell or an induced human pluripotent stem cell;

transducing the human stem cell with at least two different HSV- 1 amplicon vectors wherein one HSV- 1 amplicon vector comprises an a-satellite DNA sequence and the other HSV- 1 amplicon vector comprises an introduced DNA sequence, and wherein the a-satellite DNA and at least one introduced DNA sequence are able to form a HAC inside the cell.

The human stem cell may be transduced with a further one or more HSV- 1 amplicon vectors which comprise introduced DNA sequence and form part of the HAC inside the cell. In a further aspect the present invention provides a composition comprising two or more HSV- 1 amplicon vectors as defined in any one of the aspects of the invention for use in medicine. Preferably the composition comprises one or more a-satellite DNA sequences and one or more introduced DNA sequences comprised of two or more, preferably three, four, five, six, seven, eight, nine or ten or more HSV- 1 amplicon vectors. The composition may also comprise appropriate solvents, diluents, excipients or carriers.

The composition may comprise two or more HSV- 1 amplicon vectors as defined in any aspect of the invention for use in the treatment of diseases preventable or treatable by introducing introduced DNA sequences into cells. For example, the compositions and methods of the present invention may be used for the treatment of diseases due to the absence of large genes or genomic regions (for example Duchenne muscular dystrophy; hemophilia; DNA repair diseases) and for the treatment of diseases due to microdeletions or uniparental disomies (for example Prader-Willi syndrome, Angelman syndrome, Beckwith- Wiedemann syndrome). The compositions and methods of the present invention may be used to increase the expression of certain genes in cells that have regulatory mutations or variants which decrease normal gene expression. Furthermore HACs may be useful to efficiently deliver large interfering RNAs, to prevent the expression of large dominant genes.

In a still further aspect the present invention provides a eukaryotic cell as defined in any one of the preceding aspects for use in medicine. For example the present invention provides a eukaryotic cell, preferably a mammalian cell, more preferably a human cell, such as a stem cell, for example a human pluripotent stem cell, an induced pluripotent stem cell, an embryonic stem cell, a human adult stem cell or a cell derived from an adult, preferably a stem cell derived from brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, ovarian epithelium, or testis, suitably a stem cell derived from, umbilical cord blood or menstrual blood or a hematopoietic stem cell, or a mesenchymal stem cell comprising a HAC for use in medicine.

In another aspect the present invention provides a eukaryotic cell provided by the method according to any one of the preceding aspects for use in medicine, preferably for use in the treatment of diseases preventable or treatable by introducing introduced DNA sequences into cells.

In a yet further aspect the present invention provides a method of treating diseases treatable or preventable by introducing introduced DNA sequences into eukaryotic cells, the method comprising:

transducing the eukaryotic cell with at least one a-satellite DNA and at least one introduced DNA sequence comprised on at least two HSV- 1 amplicon vectors, wherein the at least one a-satellite DNA and at least one introduced DNA sequence are able to form one or more HACs inside the cell.

The method may further comprise the step of administering the transduced eukaryotic cells to a subject, such as a human. More preferably a method of treating diseases treatable or preventable by introducing introduced DNA sequences into cells, the method comprising administering at least one a-satellite DNA and at least one introduced DNA sequence comprised on at least two HSV- 1 amplicon vectors to the person, wherein the α-satellite DNA and at least one introduced DNA sequence are able to form one or more HACs inside the cells of the subject.

In a further aspect the present invention provides a kit comprising at least one a- satellite DNA and at least one introduced DNA sequence comprised on at least two HSV- 1 amplicon vectors, wherein the α-satellite DNA and the at least one introduced DNA sequence are able to form one or more HACs inside a eukaryotic cell.

The skilled person will appreciate that all preferred features of the invention described with reference to only some aspects of the invention can be applied to all aspects of the invention.

There now follows by way of example only a detailed description of the present invention with reference to the accompanying drawings, in which;

Figure 1 shows a diagram of the assembly of HAC vectors into HSV- 1 particles; Figure 2 shows a diagram of the alternative outcomes of double infection;

Figure 3 shows co-transduction of two vectors, one expressing the RFP gene (red) and one the GFP gene (green). Cells co-infected by both amplicon types are yellow (arrows).

Figure 4 shows (a): a schematic representation of vectors pHGNeo4 and pa40 not drawn to scale, (b): GFP expression in HUES-2, HUES- 10 and HT1080 cells 24 hours after transduction with pHGNeo4 (left) and pa40 (right) amplicons.

Figure 5 shows a HAC analysis, (a): FISH analysis with a 17a DNA probe (green), and a vector probe (red). The chromosomes are counterstained in DAPI, blue. The HAC are identified by yellow arrows. The insets show DAPI staining only (HAC, red arrows), in black and white, (b): Fibre FISH on HAC clones, with 17a DNA (green) and vector DNA (red) probes. C: ImmunoFISH with anti-CENP C antibody (green), and 17a HAC probe (red)

Figure 6 shows a pluripotency analysis, (a): RT-PCR with primers specific for pluripotency genes at time-points zero, 30, 60 and 90 days in culture (0, 30, 60, 90) in the presence (on) or absence (off) of selection. (RT-) control PCR performed in the absence of the reverse transcriptase, using GAPDH primers, (-) no template control, (b): Immuno- staining with pluripotency markers antibodies (red). The cells are counterstained with DAPI, blue, (c): RT-PCR analysis of germinal layer transcripts on cDNA from in vitro differentiated MM40.2 and SK40.19 cells, with primers specific for pluripotency (Oct4, Sox2, Nanog), endoderm (aFP, HFN3a, alAT), mesoderm (GATA-2), epidermis (CK- 14, CK-5, HSK), and neuronal markers (Pax6). U: undifferentiated cells, D : differentiated cells. (RT-): control PCR performed in the absence of the reverse transcriptase, using GAPDH primers. (-) no template control, (d): Neuronal differentiated cells stained with anti-βΙΙΙ tubulin antibody (red).

Figure 7 shows a teratoma assay, (a): Haematoxylin/eosin stained MM40.2- teratoma derived sections, (b): immuno staining with antibodies for ectoderm (βΙΙΙ-tubulin, green), mesoderm (a-actinin, red) and endoderma-feto protein, red) markers.

Figure 8 shows (a): Schematic representation of the pa60 and pal 00 vectors, not drawn to scale, and GFP expression in HUES-2, and HT 1080 cells 24 hours after transduction with pa60 (left) and pal 00 (right) amplicons. (b): FISH on metaphase spreads from HUES-2 HAC clone MM60.1 and MM100.3. In green, a 17a DNA probe, which also recognized the endogenous chromosomes 17; in red, a vector probe, which is HAC specific. The chromosomes are counterstained in DAPI, blue. The HAC are identified by yellow arrows. The small insets show the HAC DAPI staining only (red arrows), as black and white images.

Figure 9 shows FISH analysis on neuronal differentiated cells from MM40.2 and SK40.19, with a HAC specific probe (green signal) to identify HAC (yellow arrows) in cells stained with anti-βΙΙΙ tubulin antibody (red). The nuclei are counterstained in DAPI (blue).

Figure 10 shows real time qPCR analysis of HAC frequency in MM40.2 differentiated cells, using HAC specific primers.

Figure 11 shows heterogeneity of GFP expression in MM40.2 EB derived cells. Left, bright-field, right, GFP.

Figure 12 shows the sequence of the core alpha satellite DNA from human chromosome 17 (Seq ID No: 1 ).

Figure 13 shows the backbone sequence used in the vectors pa40 and pal 00. The backbone sequence is all of the sequence of the vector excluding the a- satellite DNA sequence. The backbone sequence is the same for vectors pa40 and pal 00 but pa40 has 40 kb of a-satellite DNA while pal 00 has 100 kb of a- satellite DNA (Seq ID No: 2). Figure 14 shows the backbone sequence of the vector pa60. The backbone sequence is all of the sequence of the vector excluding the a-satellite DNA sequence. pa60 has 60 kb of α-satellite DNA (Seq ID No: 4). Figure 15 shows the sequence of the vector pHG-Neo4 (Seq ID No: 4).

Figure 16 illustrates the rfficiency of transduction in hESc, iPSc and HT1080, compared to the control cell line G16-9. Figure 17 shows the Dual transduction in iPS cells. Left panel: cells expressing GFP (green). Mid panel: cells expressing RFP (red). Right panel: merged picture. Cells expressing both GFP and RFP are circled. If the GFP and RFP are expressed at the same level, the cells appear yellow (bottom circle). If the GFP fluorescent protein is expressed more than RFP, the cells will appear mostly green (top circle).

HAC formation by HSV-1 amplicon dual transduction

The HSV- 1 amplicon system is highly efficient at delivering DNA and forming a HAC in a variety of cell types. However, the size of the insert DNA is limited to 150 kb by the packaging capacity of the virion. Thus depending on the size of the initial vector, multiple copies of the HAC construct will be assembled as a linear molecule up to the size of 150kb, and packaged into the viral capsid as shown in Figure 1. It is essential to include α-satellite DNA (alphoid) in the HAC vector to seed the formation of a new centromere on the nascent HAC. The α-satellite DNA may be about 40 kb in length and this reduces the remaining capacity for incorporating therapeutic genes and their regulatory regions. To overcome the problem in the present invention cells are simultaneously co-infected with two different HSV- 1 vectors, one containing at least the α-satellite DNA and the other containing at least introduced DNA sequence, this may be a gene. The two vectors when transduced into a host cell can generate a single HAC containing both HSV- 1 constructs, and thus carrying the introduced DNA sequence, gene of interest and the α-satellite DNA. A series of experiments were set up, as outlined in Figure 2, in each case using a vector carrying a-satellite DNA and the red fluorescent protein gene, RFP, as a marker, and a second vector carrying the green fluorescent protein gene, GFP, and a either a selectable marker gene or the HPRT minigene. Several independent experiments were conducted, using the HSV- 1 amplicon at different multiplicities of infection (MOI), to transduce human HT1080 cells. In all of the experiments, the two independent amplicons were able to co-infect the same cell, with various efficiencies, as estimated by counting the number of cells expressing GFP+RFP, compared to those expressing either GFP or RFP only (corresponding to cells transduced by one or the other vector) (examples are shown in Figure 3, and reported in Table 1 ). The efficiency of co-transduction was slightly improved by subjecting the cells to centrifugation at low speed (750g for 45 minutes) (Table 1 ). Table 1 : examples of transduction efficiency in two different HSV- 1 delivery experiments

Experiment 1 Vector 1 (GFP)/ Vector 2 (RFP)

MOI 1 MOI 5

Vector 1 GFP 1 1 % 15 %

pHGHPRT

Vector 2 RFP 4 % 13.3 %

227bsdRFP

Vector 1+vector 2 0.39 % 4.6 %

GFP+RFP

Experiment 2

MOI 1 MOI 5

Vector 1 GFP 18 % 23%

Vector 2 RFP 13 % 6 %

Vector 1+vector 2 2 % 10 %

Stable clones were recovered in each double transduction experiment, and the presence of a HAC was verified by fluorescence in situ hybridization (FISH) with specific probes on chromosome metaphase spreads.

In the most recent set of experiments, the majority of the recovered clones (19 out of 20) contained a HAC, in percentages ranging from 20% to 70%. In all cases, the HAC was composed of both vectors.

In summary, the results show that when two HSV- 1 amplicons are used to deliver different HAC constructs to cells, the exogenous DNAs recombine to generate a single HAC. This demonstrates that co-infection can at least double the capacity of the artificial chromosome vectors.

Transduction in Human Embryonic Stem Cells.

In this study, input HAC DNA vectors (ranging from 55 to 1 15kb) containing the marker gene for GFP were delivered into HUES-2 and HUES- 10 human embryonic stem cells (hESc) lines following transduction with HSV- 1 amplicons. Mitotically stable, gene expressing, functional HACs were generated. The HACs were present in up to 70% of the hESc, and gene expression was maintained in the absence of selection over a period of 60 days and following cell differentiation. No DNA integrated into the hESc genome, in contrast to HT1080 cells, where the HAC DNA frequently integrated into the host chromosomes. More importantly, the HSV- 1 HAC hESc retained their pluripotency and differentiation capabilities.

This is the first successful study to establish non-integrating, stable, gene expressing HACs using HSV- 1 amplicons in human embryonic stem cells, and in particular in cells of a human embryonic stem cell line, and is a significant step forward for the use of HACs in gene therapy. The present invention provides an alternative efficient approach, based on direct HAC formation in two hESc lines utilizing HSV- 1 amplicons for delivery. HACs can accommodate large introduced DNA sequences containing potentially therapeutic genes along with their regulatory sequences, and have successfully been used as gene transfer vectors to complement genetic deficiencies in human cultured cells. HACs are also composed of heterochromatic and euchromatic regions in a similar pattern to endogenous chromosomes. While the presence of heterochromatin is necessary for the correct segregation of HACs, the euchromatin potentially ensures the prolonged expression of introduced DNA sequences.

In the present invention input HAC DNA vectors were delivered to the HUES -2, HUES- 10 and HT 1080 cells and to induced pluripotent cells PF19.9, using HSV- 1 amplicon mediated transduction. This technique is up to 10⁴ times more efficient at delivering large DNA than chemical transduction, in several different cell types.

FISH analysis revealed that HACs were formed in both HUES-2 and HUES- 10 cells. This is the first report of de novo HAC formation in a karyotypically normal primary human cell line, and is highly significant for developing HACs as gene expression vectors for gene therapy applications. Most importantly, the input HAC DNA vectors never integrated into the hESc genome, compared to HT1080, where integrations were found in most of the clones. It is possible that the presence in hESc of systems actively guarding genome integrity may prevent or reduce the frequency of large vector integration events, thus giving an advantage to cells where the exogenous DNA forms a episomal HAC, in the presence of selective pressure. In this scenario, the smaller vector pHGNeo4 integrated in hES cells, because no other outcome would have allowed the cells to survive selection, as pHGNeo4 is incapable of forming a HAC. On the other hand, HT1080 cells are tumour derived, and lack an efficient control of genome integrity, thus explaining why both HAC formation and integration events were observed. The hESc HACs were fully stable, and formed an active centromere. This showed that although the HAC were not detected in 100% of the cells, it was not the result of instability. It is possible that the cloning procedure for hESc did not produce pure clones (hESc have a low survival rate as single cells, even when ROCK inhibitor Y-27632 is used), and hence the HAC frequency in the cells was lower than 100%, as different sublines may have been present within the clones.

The analysis of HAC gene expression showed that the RNA levels of GFP reporter gene on or off selection were different in the clones characterized, containing either HAC or integrated pHGNeo4 DNA. This confirmed the existence of clonal variability between different lines, possibly due to epigenetic effects. In one of the clones, MM40.2, GFP expression was highly stable, and did not change over prolonged time in culture, both on and off selection. Furthermore, the reporter gene expression was maintained following MM40.2 differentiation, although heterogeneity was observed in the GFP levels among the differentiated cells. The process of de novo HAC formation generally results in the multimerization of the input DNA, as shown by the alternate pattern of vector and alpha satellite signals observed in the FISH on chromatin fibres, and the possibility that more than one copy of the introduced DNA sequence is present on the HAC. This ensures that copies of the introduced DNA sequence will be localized away from the centromeric heterochromatic area, and thus escape potential silencing. The spreading of heterochromatin, which is a stochastic event, may explain the variability observed in the GFP reporter gene expression following differentiation in clone MM40.2, and in prolonged culture in clone SK40.19.

Importantly, neither the HSV- 1 transduction nor the HAC formation led to a loss of pluripotency in the HUES-2 or HUES- 10 cells, as suggested by the staining with hESc specific markers, expression of three germinal layer markers in EB- derived cells, by the differentiation into neuronal types, and by MM40.2 teratoma formation. The HAC was present in the neuronal differentiated cells in both MM40.2 and SK40.19. The HAC frequency in MM40.2 neuronal cells was slightly lower than in the undifferentiated cells, yet the frequency in SK40.19 remained unchanged following differentiation. The HSV- 1 amplicon particles package DNAs up to 150kb. This is far larger than the size which can be accommodated in lentiviral, AAV or adenovirus based vectors. In this study, the pa40 containing the shortest tract of 17 alpha satellite DNA (40 kb) was the most efficient vector at HAC formation in hESc, and since its total size is approximately 55 kb, it can accommodate a large introduced DNA sequence region containing gene loci of up to 100 kb. HSV- 1 HAC replicate at the same rate as the endogenous chromosome, making the gene dosage level more easily controllable.

In summary, high capacity, gene-expressing HAC formation was observed in two hES cell lines, using a high efficiency delivery method based on the HSV- 1 amplicon technology. The HACs obtained were stable and sustained long-term gene expression. The lack of integrated DNA in hESc following HSV- 1 delivery is an important and exciting finding. These findings also suggest that HAC vectors may be used with induced pluripotent stem (iPS) cells.

RESULTS

Vector Construction

To generate HSV- 1 based input HAC DNA vectors that were highly proficient at HAC formation, the BAC hBAC495J24 (containing 220 kb of chromosome 17 core a DNA) used in a previous study to construct an efficient input HAC DNA vector (pJM2256), was modified. As hBAC495J24 was approximately 70 kb larger than the 150 kb packaging limit of HSV- 1. The aim was to reduce it while retaining its HAC-forming properties. Three derivatives of the BAC hBAC495J24 were generated, two of which arose spontaneously (containing 40 and 100 kb of 17a DNA) during culture, and the third derivative (containing 60 kb of 17a DNA) by utilizing the RED/ET recombination system. All three derivatives were modified by LoxP-Cre recombination with pHGNeo4 to include the essential HSV- 1 elements and reporter genes (GFP), thereby generating pa40 (55 kb, including 40 kb of 17a DNA), pa60 (75 kb, including 60 kb of 17a DNA), and pal OO (1 15 kb, includingl OO kb of 17a DNA) (Figure 4a and Figure 8a).

Introduction of Input HAC DNA Vectors into hES cells Among the three HSV- 1 HAC input DNA vectors that were generated, pa40 (40 kb of 17a DNA) was the most useful, as its size allowed up to 100 kb of introduced DNA sequence to be accommodated within the vector for packaging into HSV- 1 amplicons (limit capacity of 150 kb). For this reason, the HAC forming efficiency was investigated by delivering pa40 to HUES-2 (at passage 40) or HUES- 10 (at passage 29) by HSV- 1 amplicon transduction at multiplicity of infection (MOI) 2. In a parallel control experiment pHGNeo4 amplicons were also delivered to both cell lines. The other 17a input HAC DNA vectors, pa60 and pal 00, were delivered only to the HUES-2 line, in the same conditions described above.

The efficiency (%) of transduction, summarized in Table 2, was determined after 24 hours by FACS or counting GFP expressing cells. The results are also shown in Figure 4b and Figure 8b. In HUES-2, the average transduction efficiency was approximately 40% for both pa40 and the control vector pHGNeo4. The two larger vectors, pal 00 and pa60 were delivered to HUES-2 with an efficiency of 16% and 20% respectively (Table 2 and Figure 8b). In HUES- 10, the delivery efficiency was 27% for both pHGNeo4 and pa40 (Table 2, Figure 4b). In parallel control experiments, the input HAC DNA amplicon vectors were delivered by HSV- 1 mediated transduction to HT1080 cells, which efficiently form HAC. The delivery efficiency of the input HAC DNA vectors was similar to that observed in hESc (Table 2, Figure 4b and 8b). Table 2. Average efficiency of HSV- 1 amplicon transduction at MOI 2, and HAC formation in HUES-2, HUES- 10 and HT1080 cells. NA, not applicable.

Cell Line Input HAC Efficiency of No. of HAC % of

DNA Transduction Clones Positive HAC/Cells

Vector Analyzed Clones pa40 40% 10 5 10-70 pa60 20% 9 5 10-25

HUES-2 pal OO 16% 7 3 10-20

pHGNeo4 40% 5 NA NA pa40 27% 5 5 35-50 HUES- 10 pHGNeo4 27% 1 NA NA pa40 19% 10 6 5-30 pa60 34% 3 1 20

HT 1080 pal OO 25% 4 1 15

pHGNeo4 28% 3 NA NA

Viability of hESc Following Transduction with HSV-1 Amplicons

In addition to the experiments outlined above, to determine if the HSV- 1 amplicon transduction affected hESc viability, 2.5x 10⁵ HUES-2 cells were transduced with pHGNeo4 amplicons at MOI 1 , 2 and 5. The average efficiency of transduction was determined after 24 hours by FACS or counting GFP expressing cells, and found to be approximately 27% for MOI 1 , 42% for MOI 2, and 48%) for MOI 5. Since transduction efficiencies were similar at MOI 2 and 5, the HUES-2 cell lines probably reached transduction saturation in this range.

The cells were monitored for six days post transduction and the average growth rate was calculated by measuring the rate of population increase divided by the initial number of cells, and compared to that of an untreated control. The growth rate and morphology of HUES-2 were not affected post HSV- 1 amplicon transduction, with a 5% reduction in viability, detected only for MOI 1 .

Generation and Analysis of Stable Clones in hES Cells

Overall, 10 HUES-2 and 5 HUES- 10 clones were isolated following G41 8 selection, derived from the pa40 transduction (Table 2) . In addition clones 9 and 7 were isolated from pa60 and pal 00 respectively following transduction into HUES-2 cells. The stable clone formation efficiency of HSV- 1 transduction was relatively high for both hESc lines, at 10^"4, as calculated by the ratio between the number of stable clones and GFP positive cells 24 hours post transduction. Chromosome metaphase spreads were prepared from stable clones, and analyzed by two colour FISH with vector and 17a DNA probes (Table 2 and Figure 5a and Figure 8b). Following HSV- 1 transduction with pa40, HACs were detected in 5 of the 10 stable clones obtained in HUES-2 cells, and in all 5 clones isolated in HUES- 10. The HACs were present in up to 70% of the cells from each clone. In the HUES-2 cells transduced with either pa60 or pal 00, HACs were detected in approximately half of the clones, with a frequency of up to 25% of the cells of each clone (Figure 8b). The lower HAC frequency per cell observed with pa60 or pal 00 indicated that the pa40 vector was the most efficient at HAC formation in HUES-2 following HSV- 1 transduction. Most importantly, in none of the 31 clones (HUES-2 or HUES- 10) analysed, the HAC DNA had integrated into the host genome. However, in all of the analyzed hESc clones derived from the HSV- 1 delivery of the control vector pHGNeo4, the exogenous DNA had integrated into the host genome.

In HT1080, the HSV- 1 amplicon transduction successfully generated several hundred clones from each of the 17a HSV- 1 HAC input DNA vectors, and several clones were selected for analysis from pa40, pa60 and pal 00 (Table 2). The stable clone formation efficiency of HSV- 1 transduction was 5xl 0^~3. Positive clones were analyzed by two colour FISH with vector and 17a DNA probes (Table 2). The HSV- 17a input DNA vectors generated HACs in most of the clones following transduction, but were present at a lower frequency in cells (up to 30%)), and concomitant integrations in the HT1080 genome were found in all of the clones.

Karyotypic analysis of hESc HAC clones

Two hESc HAC clones were chosen for further studies. Clone MM40.2 derived from the HSV- 1 mediated delivery of pa40 into HUES-2, and contained a HAC in 70%) of the cells. (Figure 5a). Clone SK40.19 was generated by the transduction of pa40 in HUES- 10 cells and contained a HAC in 50%> of the analyzed metaphase (Figure 5a). The structure of the HACs present in both clones was analyzed further by FISH on extended chromatin fibres. The HAC contained repeat units of input DNA, arranged in an alternate fashion as seen in previous studies, but the vector backbone DNA was less abundant in clone SK40.19. (Figure 5b).

The percentage of GFP positive cells in the two HAC clones was established by FACS analysis, and found to be 33% for SK40.19 and 83% for MM40.2. These values are in concordance with the estimated HAC frequency. HAC Stability and CENP C Staining

The mitotic stability of the HAC in MM40.2 and SK40.19 was monitored by FISH for 90 days (corresponding to approximately 30 passages) in the absence of selection. In both clones, the HAC frequency did not change significantly over this period, with a daily loss rate of 0.03% for MM40.2 and 0.24% for SK40.19 (calculated by the formula N_n=N₀ x (1 -R)ⁿ, where N₀ is the number of metaphase chromosome spreads showing HAC in the cells cultured under selection, N_n is the number of HAC -containing metaphase chromosome spreads after n days of culture in the absence of selection, and R is the daily rate of loss).

To confirm that an active centromere was present on the HAC, cells from the MM40.2 and SK40.19 clones were stained with anti- centromere protein C (CENP C) antibody, coupled to FISH with HAC specific probes. A positive CENP C signal was identified on the HACs in both clones at a similar intensity of those observed on endogenous chromosomes (Figure 5c), thus confirming that an active centromere was present, similar to the HSV- 1 HAC in HT1080 cells.

Pluripotency Studies

To determine if the MM40.2 and SK40.19 cells expressed pluripotency epitopes compared to the parental lines, cells from each clone (at approximately passage 70 and 60 respectively) were stained with antibodies against the embryonic stem cell markers Oct4, Sox2, Nanog and TRA- 1 -60 (Figure 6b). The HAC clones and the parental lines were stained similarly by the antibodies while HT1080 and MEF (mouse embryonic fibroblast) control cells were completely negative. The result indicate that the HACs containing hESc clones expressed embryonic stem cell markers.

The hESc markers expression was sustained over time in both HAC clones, as confirmed by RT-PCR analysis on RNA extracted from the MM40.2 and SK40.19 cells, over a period of 90 days, cultivated in the presence and absence of selection (Figure 6a). To confirm that the HAC containing clones were pluripotent, differentiation of the three embryonic germ layers was induced through embryoid body formation of MM40.2 and SK40.19. Total RNA was extracted from the pool of differentiated cells and analysed for the presence of endoderm (a Feto-protein, HNF3a, al anti-trypsin), mesoderm (GATA-2) and ectoderm (CK-5, CK- 14, high sulphur keratin, Pax6) specific transcripts. RT-PCR experiments revealed that mRNAs for all the markers were present (Figure 6c), indicating that both HAC clones retained pluripotency. HAC Clones Directed Differentiation

Furthermore, neuronal differentiation was induced in clone MM40.2 and SK40.19 by treatment with medium containing noggin and fibronectin. After 25 days of directed differentiation, the cells were fixed in formaldehyde and stained with anti-βΙΙΙ tubulin antibody, a neuronal cell marker. Neuronal cells were detected that were highly positive for βΙΙΙ tubulin staining, in both HAC clones, ranging approximately between the 18% and the 40% of the treated cells (Figure 6d). On average, the SK40.19 was up to 2 times more efficient at forming neuronal cells, compared to MM40.2. The control cells (untreated MM40.2 and SK40.19; HT1080; MEF) never displayed positive cells. To determine if the HAC was still present in the cells following neuronal differentiation, FISH experiments were conducted on the interphasic cells stained with the anti-βΙΙΙ tubulin antibody. In clone SK40.19 the HAC frequency remained the same (50%) while a positive HAC signal was detected in 44% of MM40.2 the cells (Figure 8). Teratoma Formation Assay

Since the MM40.2 cells contained the highest HAC frequency, teratoma were generated using these cells in immunodeficient mice, as this constituted the most rigorous test of pluripotency for human ES cells. Sub-cutaneous injection into immunodeficient mice generated tumours between the 5^th and 7^th week post- treatment. The subsequent histological analysis of haematoxylin/eosin stained tumour sections revealed the presence of ectodermal (neural tube), mesodermal (muscle and blood vessels) and endodermal (gut epithelium, alveoli, and glandular epithelium) structures (Figure 7a). Moreover, immuno staining using suitable antibodies confirmed the presence of all three germinal layers in thes e sections (Figure 7b) thereby confirming the tumour growth as a teratoma.

As the sections prepared for the immunological staining were not suitable for FISH experiments, the presence of HAC in the teratoma cells was confirmed by Real Time qPCR on total genomic DNA extracted from the teratoma mass. HAC- specific primers (Neo and GFP) were used, and the HAC DNA content in the teratoma cells was normalized against that of the undifferentiated MM40.2 cells (Figure 10). No difference in the abundance of HAC sequences was detected between the undifferentiated MM40.2 and teratoma cells, suggesting that the HAC had not been lost during the in vivo cell replications. A similar analysis was conducted on the neuronal differentiated MM40.2 cells, previously analysed by FISH. The qPCR results closely reflected the FISH quantification of HAC distribution (Figure 10).

HAC Gene Expression Analysis

The expression level of the reporter gene for GFP from the MM40.2 and SK40.19 HAC was investigated by Real Time qPCR experiments on cDNA, in cells grown either on or off selection. The HAC gene expression was compared to that of a ubiquitously expressed gene (GAPDH). In SK40.19 the GFP expression was found to be decreased by about 50% after approximately 40 days on or off selection (Table 3). By contrast, in MM40.2, the GFP gene expression level remained constant for a prolonged period of time (60 days) on or off selection (Table 3). To further analyse the reporter gene expression clonal variability, the GFP relative amount in three pHGNeo4 stable clones was measured by Real Time qPCR. Compared to the MM40.2, one clone had approximately 5 times lower levels of GFP, and two had 5 times and 10 times more GFP respectively (Table S I ).

Table SI. Analysis by qPCR of the GFP reporter gene expression in pHGNeo4 derived HUES-2 clones. The qPCR -fold difference values are expressed in reference to the MM40.2 clone, using GAPDH as internal control.

Clone Relative GFP Expression Standard Deviation

MM40.2 1 0.03

pHGl 0.21 0.13 pHG4 4.8 0.73 pHG6 1 1.3 1.52

Taken together, these data suggest that stochastic events may have affected the level of expression of the reporter gene in different clones. To confirm that the MM40.2 clone had very stable levels of reporter gene expression, GFP presence was further monitored by FACS over the course of 90 days. The percentage of GFP expressing cells remained relatively constant and ranged from 81 -89%. The fold increase in the geometric mean of fluorescence intensity compared to the untreated control ranged from -8- 12, indicating that the average GFP expression per cell also remained relatively constant.

Table 3. Analysis by qPCR of the GFP reporter gene expression in clones MM40.2 and SK40.19, in the presence or absence of selection. The qPCR -fold difference values are expressed in reference to the day zero time-point, using GAPDH as internal control. SD, standard deviation. NA, not analyzed

*37 days

Furthermore, the GFP reporter gene expression levels of the MM40.2 clone were characterized by qPCR following differentiation, and compared to the levels present in the undifferentiated MM40.2 parental. The GFP gene was still expressed in both EB derived-, and neuronal differentiated cells, at approximately 60-70% of the level present in the undifferentiated parental (Table 4). The level of GFP expression was consistent with the HAC frequency observed in the neuronal differentiated MM40.2 cells. Based on direct microscopic observation of the EB derived and neuronal cells, the derivative differentiated cells exhibited a heterogeneous GFP expression: while some cells were still highly GFP positive, in others the GFP fluorescence appeared reduced or absent (Figure 1 1). This suggests that stochastic events in the early stages of hESc differentiation had an effect in the level of expression of the reporter gene during the later stages.

Table 4. Analysis by qPCR of the GFP reporter gene expression, in differentiated MM40.2 cells. The values are expressed in reference to the undifferentiated parental, using GAPDH as internal control. SD, standard deviation

HSV-1 amplicon dual transduction in human embryonic stem cells and induced pluripotent stem cells

To demonstrate that the simultaneous transduction with two different amplicons is feasible in human stem cells, two vectors, containing either GFP or RFP, were delivered to the human embryonic stem cells (hESc) HUES2 (Cowan et al. 2004 Engl. J. Med., 350, 1353- 1366) and the induced pluripotent stem cells (iPSc) DF 19.9 (WiCell Research Institute), at MOI 2. In control experiments, HT1080 cells, and the highly transducible human glioma cell line G16-9 (Moralli et al. 2006 EMBO Rep., 7, 91 1 -918) were transduced with the same two vectors. The results, summarized in Figure 16, show that in hESc and iPSc the frequency of dual transduction events is comparable to that observed in the highly transducible cells G16-9, and is approximately 2-3 times higher of that observed in HT1080 cells. Examples are shown in Figure 17.

MATERIALS AND METHODS

Input HAC DNA Vector Assembly - HSV-1 Amplicon Vector Assembly

The pHGNeo4 vector, whose sequence is shown in Figure 15, carries the HSV- 1 amplicon origin of replication (Ori) and packaging signal (pac), the reporter GFP gene, under control of the I/E promoter from HSV- 1 , and the G418 resistance gene (Neo), controlled by the SV40 promoter. Vectors BAC 17a40 and BAC17al 00, containing respectively 40kb and l OOkb of a satellite DNA from human chromosome 17 (17a), were derived by spontaneous deletions of hBAC495J24. PAC17a60 was obtained by RED/ET recombination based on a commercial kit: Red/ET BAC subcloning kit, Gene Bridges GmbH, which transferred 60kb of 17a DNA from hBAC495J24 to pCYPAC2 vector (commercially available from the BACPAC Resource Center, CHORI http://bacpac.chori.org/pcypac2.htm). Briefly, the pCYPAC2 vector was used as a template to generate a 9.5kb PCR fragment, using primers containing 50bp homologous tails to the alpha 17 satellite DNA consensus (Table S2). The linear 9.5kb pCYPAC2 PCR product was then transformed into E. coli cells containing hBAC495J24, and expressing the RED/ET system proteins.

Table S2. Primers used for PCR and Real Time qPCR amplification.

The BAC 17a40, PAC17a60, and BAC 17al 00 constructs were then retrofitted with pHGNeo4 by LoxP-Cre recombination (Moralli, D. et al., (2006) Rep., 7, 91 1 -918), to generate three new HSV- 1 based HAC vectors: pa40, pa60, and pal 00 respectively. The backbone sequence of vectors pa40 and pal 00 is shown in Figure 13. The backbone sequence is all of the sequence of the vector excluding the a-satellite DNA sequence. The backbone sequence is the same for vectors pa40 and pal 00 but pa40 has 40 kb of a-satellite DNA while pal 00 has 100 kb of α-satellite DNA. Figure 14 shows the backbone sequence of the vector pa60. The backbone sequence is all of the sequence of the vector excluding the α-satellite DNA sequence. pa60 has 60 kb of α-satellite DNA. An example of the core α-satellite DNA is shown in Figure 12. A 40, 60 or 80 kb a-satellite DNA sequence may, for example, be made up from repeats of the sequence shown in Figure 12. Vectors comprising α-satellite DNA may be obtained from the BACPAC Resources Center (BPRC) at the Chori Institute (http://bacpac.chori.org/). The α-satellite DNA sequences used in the vectors described in this example derive from vector RP11-495J24, which is available from (BPRC). Cell Culture

The human embryonic stem cell lines HUES-2 and HUES- 10 were obtained from Douglas Melton (Harvard University, Cambridge, MA, USA) and grown under license from the UK Stem Cell Steering Committee as described (Cowan, C.A., Klimanskaya, I., et al. (2004) N. Engl. J. Med., 350, 1353- 1366.; Karlsson, K.R., et al. (2008) Exp. Hematol. , 36, 1 167- 1 175.) on mitomycin C inactivated mouse embryonic fibroblasts (MEF) or SNL76/7 cells. Feeder independent HUES-2 and HUES- 10 cells were grown on Matrigel (BD Biosciences) coated wells using the mTeSR medium (STEMCELL Technologies). TrypLE Express (Invitrogen) was used to enzymatically passage the hESc. Cells were maintained and passaged at high densities on Matrigel. To increase single cell survival, ROCK (Rho- associated kinase) inhibitor Y-27632 (Merck Biosciences) was added during each passaging step, at a final concentration of 10 μΜ. The HT1080 (ATCC-CCL- 121 ) cells were grown using standard techniques in DMEM medium (Invitrogen), supplemented with 10% FBS and 1 % Penicillin/Streptomycin.

HSV-1 Amplicon Preparation

HSV- 1 amplicons were prepared as described (Moralli, D. et al., (2006) Rep., 7, 91 1 -918. ; Wade-Martins, et al. (2001 ) Nat. Biotechnol., 19, 1067- 1070.). Briefly, the packaging cell line Vero 2-2 was transduced with each input HAC vector DNA, fHSVApacA270 0+ and pEBHICP27 by Lipofectamine (Invitrogen) and Plus Reagent (Invitrogen). The cells were harvested, sonicated and then amplicons concentrated as described. The pellet was resuspended in 500 μΐ_^ of PBS. The titre of the HSV- 1 amplicon preparation was determined by transducing the glioma cell line G16-9.

HSV-1 Amplicon Transduction

Transduction of HUES-2 (passage 40), HUES- 10 (passage 29) and HT1080 cells with HSV- 1 HAC amplicons, was carried out as described (Moralli, D. et al., (2006) EMBO Rep., 7, 91 1 -918., Wade-Martins, R., et al. (2001 ), Nat. Biotechnol., 19, 1067- 1070). The HUES-2 and HUES- 10 on Matrigel coated plates, and the HT1080 cells were seeded at 2.5 >< 10⁵ cells per 24 well plate the day before transduction. On the day of transduction, HSV- 1 HAC amplicons were inoculated at a multiplicity of infection (MOI) of 1 , 2 or 5 in 250 μΕ of media (mTeSR or DMEM).

To improve the transduction efficiency, upon addition of the HSV- 1 amplicons, the cells were centrifuged under low gravitational forces. The plates were covered with sterile adhesive films, to avoid aerosol escape during centrifugation, and centrifuged at 750 g for 45 minutes. Transient expression was monitored at 24 hours post transduction either under the microscope or by flow cytometry. Three days after HSV- 1 HAC transduction, the HUES-2 cells were transferred onto G418 resistant inactivated MEF or SNL-76/7 cells. Two days later, 50μg/mL of G418 (Invitrogen) was added as selection. After seven days, individual hESc clones were observed and cells were removed from selection. The clones were allowed to grow for an extra seven days, then each clone was isolated and expanded on inactivated MEF. Upon reaching confluency in a 24 well dish, the cells were transferred to feeder-free growing conditions on Matrigel and mTeSR. The HT1080 cells were selected with 350μg/mL of G418.

Embryoid Body (EB) Formation and Germ Layer Differentiation

For germ layer differentiation of each hESc line, approximately 3 x 10⁷ cells were used to form 192 uniform sized embryoid bodies (EB) of size ~ 1.5 >< 10⁵ cells/EB. The hESc were seeded in non-adherent 96 well V-bottom plates (Nunc) in mTeSR and Ι ΟμΜ ROCK inhibitor Y-27632. After 3 days the EBs were released into suspension on non-adherent plates for 8- 10 days in DMEM, 20% FBS. The EBs were then plated on Matrigel coated plates and le771 1717ft to adhere and expand for a further 15-20 days.

Neuronal Differentiation

Neuronal differentiation was carried out as described (Iacovitti, L. et al. (2007) Brain Res., 1127, 19-25.) with minor modifications. Uniform sized EBs composed of approximately 4000 cells were formed using Aggrewell™400 plates (STEMCELL Technologies) as described by the manufacturer. After 2 days, the EBs were released and left in suspension in non-adherent plates for 5 days in DMEM F/12 (Invitrogen), 1 % N2 supplement (Invitrogen), containing human plasma fibronectin (5μg/mL) (Sigma) and recombinant human noggin (200μg/mL) (RDI/Fitzgerald Industries). The EBs were then transferred onto Matrigel coated plates under the same medium for a further 8 days. Recombinant human bFGF (20ng/mL) (BD Biosciences) was then added to the medium, and the cells were incubated for further 8- 10 days for expansion of neuronal rosettes. Neuronal rosettes were lifted using TrypLE Express (Invitrogen) and plated on Matrigel coated slides with the addition of ROCK Y-27632 inhibitor.

Teratoma Formation Assay

For the teratoma formation assay, l xl O⁶ Matrigel-grown MM40.2 cells were injected subcutaneously into immunodeficient mice (common gamma-chain-/-, RAG2-/-, C5-/-). The mice were sacrificed between 5-7 weeks after injection and the teratoma was dissected and processed for haematoxylin/eosin staining and immuno staining with the following primary antibodies: anti-tubulin beta III isoform (Tuj l ) (ectodermal derivatives), anti- alpha- actinin (mesodermal derivatives), anti-alpha fetoprotein (endodermal derivatives) (all from Millipore). Total genomic DNA was prepared from the teratoma mass by phenol/chloroform extraction. The HAC abundance was estimated by Real Time qPCR analysis of the DNA, using the GFP and GAPDH primers listed in Table S2, with the kit SYBR Green Supermix IQ (Quanta Biosciences), on an ICycler (Bio-Rad) machine. The DNA relative amount was measured using the 2^"AACt method. Fluorescence-Activated Cell Sorting (FACS)

Cells were fixed in 4% formaldehyde in PBS. Samples were then run through a FACS Calibur and a minimum of 10,000 events was captured for each sample. Acquired data was analyzed using FlowJo 7.6 software. The percentage of GFP expressing cells was calculated by setting the gate at 0.5% for the negative control.

Fluorescence in situ Hybridization (FISH) and Immuno-FISH

Chromosome preparation and FISH analyses were carried out as described (Moralli, D., et al, (2006) EMBO Rep., 7, 91 1 -918; Moralli, D., and Monaco, Z.L. (2009) PLoS One., 4, e4483 10.1371/journal.pone.0004483.; Moralli D. et al., (2010) Stem Cell Rev. First published on December 29, 2010, 10.1007/s l 2015-010-9224-4.). For each experiment, up to 40 metaphases were scored, and the number of HAC containing cells was recorded. For HUES-2 karyotype analysis, metaphase spreads were subjected to FISH with whole chromosomes paint probes for chromosomes 12 and 17 (Aquarius Whole Chromosome Paint Probes, Cytocell), according to the manufacturer instructions. The HUES- 10 karyotype was analyzed on Affymetrix Cytogenetics Whole- Genome 2.7M Arrays, following the manufacturer instructions.

The binding of Centromere Protein C (CENP C) to chromosome metaphase spreads was carried out by immuno-FISH as previously described (Moralli, D. et al., (2006) Rep., 7, 91 1 -918), using an anti-CENP C primary antibody. Cytological preparations were analyzed with an Olympus BX-51 epifluorescence microscope coupled to a JAI CVM4+ CCD camera, with CytoVysion software system (Genetix).

Immunofluorescence Staining of Fixed Cells

Actively growing cells were fixed in 2% formaldehyde in PBS. After permeabilization in PBS, 0.1 % Triton X- 100 the following antibodies were used: mouse-anti-TRA- 1 -60 (Abeam); rabbit-anti-Oct4 (Abeam); rabbit-anti-Nanog (Abeam); rabbit-anti-Sox2 (Abeam); mouse-anti-βΙΙΙ tubulin (R&D Systems), followed by TRITC conjugated anti-rabbit or anti-mouse antibodies (Molecular Probes, Invitrogen).

The cells were analyzed with a wide-field inverted Nikon TE2000U fluorescence microscope. Images were acquired using the IPLab software, and pseudo-coloured using Adobe Photoshop.

RNA Preparation and Analysis

Total RNA was extracted from approximately 6xl 0⁶ cells using the RNeasy kit (Qiagen), following the manufacturer's instructions. The RNA was treated with DNase I (Qiagen) and reverse transcribed into cDNA, using the RETRO Script system (Ambion), with random decamer primers. Gene expression levels were quantified by Real Time qPCR analysis of the cDNA, using the GFP and GAPDH primers listed in Table S2, as described for the introduced DNA sequence analysis. The RNA relative amount was measured using the 2^"AACt method.

Claims

1. A method for providing a eukaryotic cell comprising a human artificial chromosome (HAC), the method comprising the step of:

transducing the eukaryotic cell with at least one a-satellite DNA and at least one introduced DNA sequence comprised on at least two viral vectors, wherein the at least one α-satellite DNA and at least one introduced DNA sequence are able to form one or more HACs inside the cell.

2. The method according to claim 1 wherein the at least two viral vectors are both HSV- 1 amplicon vectors.

3. A method according to claim 1 or claim 2, wherein the cells are transduced with one α-satellite DNA sequence and one HAC is formed inside the cell.

4. A method according to any one of claims 1 to 3, wherein the at least one a-satellite DNA sequence and the at least one introduced DNA sequence are comprised on three or more vectors.

5. A method according to any one of claims 1 to 4 wherein the α-satellite DNA sequence and the at least one introduced DNA sequence are on different vectors.

6. A method according to any one of claims 1 to 5, wherein the at least two vectors also comprise DNA sequences for inducing the cell to become an induced pluripotent stem cell.

7. The method according to any one of claims 1 to 6, wherein the eukaryotic cell is a human cell, preferably a human stem cell or a human induced pluripotent stem cell.

8. A method according to any one of the preceding claims wherein the cell is a human stem cell.

9. A method for providing a human cell comprising a human artificial chromosome (HAC) comprising the steps of:

providing a human pluripotent stem cell or an induced human pluripotent stem cell; transducing the human stem cell with two HSV- 1 amplicon vectors wherein one HSV- 1 amplicon vector comprises an a-satellite DNA sequence and the other HSV- 1 amplicon vector comprises an introduced DNA sequence, and wherein the a-satellite DNA and at least one introduced DNA sequence are able to form a HAC inside the cell.

10. A method according to claim 9 wherein the human stem cell is transduced with a further one or more HSV- 1 amplicon vectors which comprise introduced DNA sequence and form part of the HAC inside the cell.

1 1. A composition comprising two or more viral vectors, preferably HSV- 1 amplicon vectors as defined in any one of the preceding claims for use in medicine.

12. A composition comprising at least two viral vectors, preferably at least two HSV- 1 amplicon vectors as defined in any one of claims 1 to 10 for use in the treatment of diseases preventable or treatable by introducing introduced DNA sequences into cells.

13. A eukaryotic cell as defined in any one of claims 1 to 10 for use in medicine.

14. A eukaryotic cell provided by the method according to any one of claims 1 to 10 for use in medicine.

15. A eukaryotic cell provided by the method according to any one of claims 1 to 10 for use in the treatment of diseases preventable or treatable by introducing introduced DNA sequences into cells.

16. A method of treating diseases treatable or preventable by introducing introduced DNA sequences into cells, the method comprising:

transducing the eukaryotic cell with at least one α-satellite DNA and at least one introduced DNA sequence comprised on at least two viral vectors, preferably at least two HSV- 1 amplicon vectors, wherein the α-satellite DNA and at least one introduced DNA sequence are able to form one or more HACs inside the cell.

17. A method according to claim 16 further comprising the step of administering the transduced eukaryotic cells to a person.

18. A method of treating diseases treatable or preventable by introducing introduced DNA sequences into cells, the method comprising administering at least one a- satellite DNA and at least one introduced DNA sequence comprised on at least two viral vectors, preferably at least two HSV- 1 amplicon vectors to the person, wherein the at least one a-satellite DNA and at least one introduced DNA sequence are able to form one or more HACs inside the cells of the person.

19. A kit comprising at least one α-satellite DNA and at least one introduced DNA sequence comprised on at least two viral vectors, preferably at least two HSV- 1 amplicon vectors, wherein the at least one α-satellite DNA and the at least one introduced DNA sequence are able to form one or more HACs inside eukaryotic cells.

20. A method as described herein with reference to the figures.

21. A composition as described herein with reference to the figures.

22. A eukaryotic cell as described herein with reference to the figures.

23. A kit as described herein with reference to the figures.