WO2012156721A1 - Procédés pour fournir des cellules humaines comprenant un chromosome humain artificiel - Google Patents

Procédés pour fournir des cellules humaines comprenant un chromosome humain artificiel Download PDF

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WO2012156721A1
WO2012156721A1 PCT/GB2012/051075 GB2012051075W WO2012156721A1 WO 2012156721 A1 WO2012156721 A1 WO 2012156721A1 GB 2012051075 W GB2012051075 W GB 2012051075W WO 2012156721 A1 WO2012156721 A1 WO 2012156721A1
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cells
hac
cell
hsv
dna
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Zoia Larin MONACO
Daniela MORALLI
Richard Wade-Martins
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Isis Innovation Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/20—Pseudochromosomes, minichrosomosomes
    • C12N2800/208—Pseudochromosomes, minichrosomosomes of mammalian origin, e.g. minichromosome

Definitions

  • This invention relates to a method for providing a eukaryotic cell comprising a human artificial chromosome (HAC).
  • HAC human artificial chromosome
  • 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.
  • HACs 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.
  • lentiviral, adenoviral and adeno-associated (AAV) viral vectors for gene delivery.
  • AAV adeno-associated viral vectors
  • 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.
  • HAC DNA 404 kb
  • HPRT hypoxanthine-guanine phosphoribosyl transferase
  • 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.
  • viruses for example, an HSV virus, a retrovirus, an adeno-associated virus (AAV), an adenovirus, a vaccinia virus or a plant virus.
  • 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.
  • the vector 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 10 4 ).
  • 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.
  • 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.
  • the invention provides a method for providing a eukaryotic cell comprising a human artificial chromosome (HAC), the method comprising the step of:
  • 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.
  • the ⁇ -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.
  • the ⁇ -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.
  • the introduced DNA comprises a gene that can express a protein or a peptide of interest inside the cell once a HAC is formed.
  • 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.
  • 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 ⁇ -satellite DNA sequence, for example the sequence of the core alpha satellite DNA from human chromosome 17 shown in Figure 12.
  • a HAC may include one or more introduced DNA sequences that may be expressed, or at least transcribed, in the host cell.
  • the HAC has ⁇ -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.
  • telomeric sequences may also be included in the HAC.
  • 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.
  • 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.
  • the total DNA needed to form a HAC 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.
  • 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.
  • 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 10 5 times more efficiently than a 400 kb vector).
  • 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.
  • 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.
  • the two or more viral vectors may be based on different viruses.
  • 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.
  • a single HSV- 1 amplicon vector can deliver up to 150kb of DNA.
  • multiples of 150kb can be delivered with high efficiency to any cell type.
  • 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.
  • 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 ⁇ -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 ⁇ -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.
  • the cells are transduced with one a-satellite DNA sequence and one HAC is formed inside the cell.
  • One ⁇ -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 ⁇ -satellite DNA and the one or more introduced DNA sequences, may be divided between the two or more HSV- 1 amplicon vectors.
  • the ⁇ -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.
  • 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 ⁇ -satellite DNA sequence may be on the same HSV- 1 amplicon vector as one or more introduced DNA sequences.
  • an ⁇ -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.
  • 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.
  • the at least one ⁇ -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.
  • 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.
  • chromosomes 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).
  • 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.
  • 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.
  • hTERT SV40 large T antigen, telomerase
  • 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.
  • 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.
  • 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.
  • the eukaryotic cell is a stem cell derived from umbilical cord blood or menstrual blood.
  • the eukaryotic cell is a hematopoietic stem cell or a mesenchymal stem cell.
  • 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.
  • the embryonic stem cell is derived from a commercially available embryonic stem cell line.
  • the stem cell is not derived from a human embryo.
  • the stem cell is a human stem cell that is not a human embryonic stem cell.
  • hESc Human embryonic stem cells
  • 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.
  • the HAC may be introduced in the cells in vivo.
  • HSV- 1 amplicon vectors 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.
  • the present invention provides a method for providing a human cell comprising a human artificial chromosome (HAC) comprising the steps of:
  • 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.
  • 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.
  • 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.
  • 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.
  • HACs may be useful to efficiently deliver large interfering RNAs, to prevent the expression of large dominant genes.
  • the present invention provides a eukaryotic cell as defined in any one of the preceding aspects for use in medicine.
  • 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.
  • a stem cell derived from, umbilical cord blood or menstrual blood or a hematopoietic stem cell, or a mesenchymal stem cell comprising
  • 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.
  • the present invention provides a method of treating diseases treatable or preventable by introducing introduced DNA sequences into eukaryotic cells, the method comprising:
  • 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 ⁇ -satellite DNA and at least one introduced DNA sequence are able to form one or more HACs inside the cells of the subject.
  • 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 ⁇ -satellite DNA and the at least one introduced DNA sequence are able to form one or more HACs inside a eukaryotic cell.
  • 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
  • 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
  • Figure 6 shows a pluripotency analysis
  • (RT-) control PCR performed in the absence of the reverse transcriptase, using GAPDH primers, (-) no template control,
  • the cells are counterstained with DAPI, blue,
  • U undifferentiated cells
  • D differentiated cells.
  • Figure 7 shows a teratoma assay, (a): Haematoxylin/eosin stained MM40.2- teratoma derived sections, (b): immuno staining with antibodies for ectoderm ( ⁇ -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.
  • 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- ⁇ 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 ⁇ -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).
  • the HSV- 1 amplicon system is highly efficient at delivering DNA and forming a HAC in a variety of cell types.
  • the size of the insert DNA is limited to 150 kb by the packaging capacity of the virion.
  • 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.
  • the ⁇ -satellite DNA may be about 40 kb in length and this reduces the remaining capacity for incorporating therapeutic genes and their regulatory regions.
  • cells are simultaneously co-infected with two different HSV- 1 vectors, one containing at least the ⁇ -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 ⁇ -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.
  • FISH fluorescence in situ hybridization
  • 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.
  • 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.
  • HAC DNA vectors ranging from 55 to 1 15kb
  • hESc human embryonic stem cells
  • 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.
  • 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.
  • 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 10 4 times more efficient at delivering large DNA than chemical transduction, in several different cell types.
  • 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.
  • 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.
  • MM40.2 GFP expression was highly stable, and did not change over prolonged time in culture, both on and off selection.
  • 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.
  • 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.
  • HAC vectors may be used with induced pluripotent stem (iPS) cells.
  • 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)
  • 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).
  • GFP essential HSV- 1 elements and reporter genes
  • the efficiency (%) of transduction was determined after 24 hours by FACS or counting GFP expressing cells. The results are also shown in Figure 4b and Figure 8b.
  • 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).
  • HUES- 10 the delivery efficiency was 27% for both pHGNeo4 and pa40 (Table 2, Figure 4b).
  • 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.
  • 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
  • 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 .
  • HUES-2 and 5 HUES- 10 clones were isolated following G41 8 selection, derived from the pa40 transduction (Table 2) .
  • 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).
  • 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.
  • 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.
  • 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.
  • endoderm a Feto-protein, HNF3a, al anti-trypsin
  • mesoderm GATA-2
  • ectoderm CK-5, CK- 14, high sulphur keratin, Pax6
  • 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- ⁇ tubulin antibody, a neuronal cell marker. Neuronal cells were detected that were highly positive for ⁇ 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.
  • 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).
  • 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.
  • 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).
  • GFP expression was found to be decreased by about 50% after approximately 40 days on or off selection (Table 3).
  • the GFP gene expression level remained constant for a prolonged period of time (60 days) on or off selection (Table 3).
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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 ⁇ -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 ⁇ -satellite DNA sequence.
  • pa60 has 60 kb of ⁇ -satellite DNA.
  • An example of the core ⁇ -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 ⁇ -satellite DNA may be obtained from the BACPAC Resources Center (BPRC) at the Chori Institute (http://bacpac.chori.org/).
  • BPRC BACPAC Resources Center
  • the ⁇ -satellite DNA sequences used in the vectors described in this example derive from vector RP11-495J24, which is available from (BPRC).
  • 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 ⁇ . 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 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 ⁇ ⁇ of PBS. The titre of the HSV- 1 amplicon preparation was determined by transducing the glioma cell line G16-9.
  • 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.
  • the HUES-2 cells were transferred onto G418 resistant inactivated MEF or SNL-76/7 cells. Two days later, 50 ⁇ g/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 350 ⁇ g/mL of G418.
  • EB embryoid bodies
  • DMEM fetal bovine serum
  • Uniform sized EBs composed of approximately 4000 cells were formed using AggrewellTM400 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 (5 ⁇ g/mL) (Sigma) and recombinant human noggin (200 ⁇ g/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.
  • l xl O 6 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)
  • FISH Fluorescence in situ Hybridization
  • 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.
  • 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.
  • Triton X- 100 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- ⁇ 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.

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Abstract

L'invention concerne un procédé pour fournir une cellule eucaryote comprenant un chromosome humain artificiel (HAC), le procédé comprenant l'étape de: transduction de la cellule eucaryote avec au moins un ADN α‑satellite et au moins une séquence d'ADN introduite comprise sur au moins deux vecteurs viraux, l'au moins un ADN α-satellite et l'au moins une séquence d'ADN introduite étant capables de former un ou plusieurs HAC à l'intérieur de la cellule.
PCT/GB2012/051075 2011-05-16 2012-05-15 Procédés pour fournir des cellules humaines comprenant un chromosome humain artificiel WO2012156721A1 (fr)

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JP2018196398A (ja) * 2018-09-25 2018-12-13 国立研究開発法人産業技術総合研究所 人工染色体ベクター及び形質転換哺乳類細胞

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JP2015119643A (ja) * 2013-12-20 2015-07-02 国立研究開発法人産業技術総合研究所 人工染色体ベクター及び形質転換哺乳類細胞
JP2018196398A (ja) * 2018-09-25 2018-12-13 国立研究開発法人産業技術総合研究所 人工染色体ベクター及び形質転換哺乳類細胞

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