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WO2007034169A2 - Selection de cellules et systeme rapporteur - Google Patents

Selection de cellules et systeme rapporteur Download PDF

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Publication number
WO2007034169A2
WO2007034169A2 PCT/GB2006/003484 GB2006003484W WO2007034169A2 WO 2007034169 A2 WO2007034169 A2 WO 2007034169A2 GB 2006003484 W GB2006003484 W GB 2006003484W WO 2007034169 A2 WO2007034169 A2 WO 2007034169A2
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WO
WIPO (PCT)
Prior art keywords
cell
cells
fusion protein
domain
fusion
Prior art date
Application number
PCT/GB2006/003484
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English (en)
Other versions
WO2007034169A3 (fr
Inventor
Timothy Allsopp
Lillian Alexandra Hook
Original Assignee
Stem Cell Sciences (Uk) Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Stem Cell Sciences (Uk) Limited filed Critical Stem Cell Sciences (Uk) Limited
Publication of WO2007034169A2 publication Critical patent/WO2007034169A2/fr
Publication of WO2007034169A3 publication Critical patent/WO2007034169A3/fr

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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a cell selection and reporter system, to methods and tools for labelling cells, preferably stem cells, so that they and their progeny can be selected and/or identified at various stages during growth, development and use.
  • the invention relates in particular to tools and methods that enable selection of stem cells and subsequent identification of those cells or their derivatives after transplantation and subsequent recovery of the cells or their derivatives.
  • Transplanted cells may be affected by the environment into which they are placed and it is therefore desirable to be able to recover the cells or their progeny after transplantation to determine the extent to which any changes in the cells have taken place. Transplanted cells may migrate, and so again it is desired to identify migration and fate of the transplanted cells.
  • a problem with existing constructs and reporter systems is that they do not allow sufficient flexibility in both selecting for cells in vitro and subsequently identifying cells in vivo or recovering cells or their progeny after, say, transplantation.
  • the invention is based upon the realisation of a need for and devising of a strategy for genetic modification of a cell enabling isolation or purification of the cell and subsequent analysis or recovery, or both, of the cell or its progeny.
  • the modification provides the option, with respect to a given cell, to isolate live cells, use these for transplantation purposes and then recover transplanted cells or analyze a section of tissue containing transplanted cells - for simplicity, reference to transplanted cells is intended to embrace, unless the context demands otherwise, both transplanted cells and progeny of the transplanted cells.
  • the invention hence provides a fusion protein comprising the domains :-
  • a cell expressing the fusion protein can be sorted by FACS or MACS methods.
  • the cell can also be grown in in vitro culture under selection to eliminate cells not expressing the fusion - especially if the third domain confers resistance to a cytotoxic agent or similar.
  • This cell can be transplanted and afterwards tissue into which the cell has been transplanted can be sectioned and stained — for example to determine the localization of the cell, its development and/or growth in situ.
  • Cells expressing fusion protein can also be sorted from transplanted tissue
  • the first domain suitably comprises a cell surface antigen or a fluorescent protein.
  • the cell surface antigen can be more or less any antigen that comprises an extracellular domain.
  • the fusion protein will include a signal sequence, conveniently comprised within the first domain, to enable the fusion protein when made to traffick to the cell surface.
  • it will also include a transmembrane region, for attaching the fusion protein to the cell membrane, with the extracellular region on the outside of the cell and the second and third domains being intracellular.
  • an antibody raised to the extracellular region of the first domain binds to that region and can be used for sorting of cells.
  • the antibody can be linked to a fluorescent protein for FACS sorting.
  • the antibody can be linked to a magnetic bead or other suitable component for MACS sorting.
  • cell surface antigens include CD2, CD4 and FLAG epitopes, and as will be appreciated a very large number of cell surface antigens are suitable, so long as an antibody will recognise the antigen to enable sorting.
  • the fusion protein is expressed in mouse host cells and comprises human CD2 as a cell surface antigen.
  • Antibodies are raised which are specific to human CD2 and which bind the fusion protein but do not bind to mouse CD2, and as a result there is ready sorting of cells which express the fusion from those which do not, and very little if any background.
  • the first domain encodes a protein not normally expressed in the intended host cell - such as a human protein when the fusion is for use on cells of another species, such as mouse, or vice versa.
  • the first domain can also be or comprise a fluorescent protein, such as a green fluorescent protein or red fluorescent protein, though others are also available and suitable.
  • the first domain enables sorting by FACS.
  • the second domain typically comprises an enzyme which converts a substrate into a product for use in staining a tissue section or for use in staining e.g. a piece of tissue or even a whole organism - staining can for example be used to visualise an entire embryo.
  • a tissue section or for use in staining e.g. a piece of tissue or even a whole organism - staining can for example be used to visualise an entire embryo.
  • Cells grown in vitro, on tissue culture plastic or glass slides can also be visualised.
  • enzymes examples include ⁇ -galactosidase, alkaline phosphatase,
  • horse radish peroxidase and luciferase horse radish peroxidase and luciferase.
  • a preferred enzyme is ⁇ -galactosidase and this is used widely for staining applications.
  • the third domain is suitably an enzyme which destroys a drug that is toxic to mammalian cells.
  • a typical example is an enzyme that confers resistance to antibiotic introduced into culture medium.
  • the third domain can confer resistance to hygromycin, puromycin, blasticidin or zeomycin.
  • the domain can also encode the neomycin phosphotransferase gene, giving resistance to G418/geneticin.
  • the third domain encodes neomycin phosphotransferase, giving resistance to G418, and in a very specific embodiment, the second and third domains of the fusion are made up of or comprise ⁇ -geo being a known fusion of ⁇ -galactosidase and neomycin.
  • the fusion protein comprises CD2, ⁇ -galactosidase and neomycin.
  • the CD2 region as is generally the case for the first domain, is N-terminal.
  • the ⁇ -galactosidase and neomycin domains can be interchanged.
  • a signal sequence ensures that the fusion trafficks to the cell membrane and a transmembrane domain which is part of the first domain or part of the second domain or between the first domain and the second domain anchors the fusion in position in the membrane.
  • the second and third domains including in this case ⁇ -galactosidase and neomycin, are positioned intracellularly but are membranes-bound. Testing of this fusion has confirmed that the functionality of all three domains is retained in the fusion.
  • a genetic construct is prepared that will express the fusion protein in a desired cell.
  • a cell is transfected with the construct so that the fusion is expressed. It is then possible to select for cells expressing the construct by adding to the culture medium a factor which would otherwise be toxic. Thus, typically, antibiotic is added to the culture medium and those cells expressing the fusion, and thus cells which are resistant to antibiotic, survive and can be selected.
  • the third domain encodes neomycin phosphotransferase G418 is added to culture medium and over a culture duration of typically about one week a purified population of cells expressing the construct can be obtained.
  • the transfected cell may or may not be the cell to be selected.
  • an ES cell may be transfected, wherein the construct links expression of the fusion to activity of an ES cell specific promoter. Selection then can be carried out to obtain a population purified in respect of ES cells.
  • An ES cell may be transfected, wherein the construct links expression of the fusion to activity of a promoter specific for a differentiated cell, eg. a neural cell. Selection then can be carried out, generally after a period of culture which supports differentiation, to obtain a population purified in respect of neural cells.
  • linking expression of the fusion to a promoter specific for a given cell lineage can be used to select for cells of that lineage.
  • the invention may hence be used in combination with the lineage selection methods described in EP-A- 1115840.and US-A-6929948.
  • This step can assist selection of the desired cells and can assist purity of the cell culture.
  • the cells can be sorted, using the first domain, into populations based on the activity of the promoter used to express the fusion protein. If the fusion protein is expressed under control of the Sox-1 promoter then Sox-1 positive and Sox-1 negative populations can be obtained. If ES cells are transfected with the fusion under control of the sox-1 promoter and then differentiated, the culture is a mixture of sox-1 positive and sox-1 negative cells. These cells can be sorted into their respective populations which can be compared for various parameters such as differential gene expression. This is an example of a further advantage of the invention, for if cells are selected using a method in which cells are killed, e.g. using a neo domain, then differential expression can not be compared because sox-1 negative cells would be killed.
  • the purified and/or sorted cells can then be used for transplantation.
  • Post transplantation the fate of cells can be analysed by a staining procedure. For example, tissue into which the cells were transplanted is stained and visualised based on the presence of the second domain (e.g. ⁇ -galactosidase). This technique can usefully be employed to determine the fate of the cells and whether and to what extent they have migrated.
  • the second domain e.g. ⁇ -galactosidase
  • a method of analysing an effect of transplantation on a cell hence comprises transplanting the cell into a subject; removing tissue from the subject, wherein the tissue contains the cell or a progeny of the cell; sorting the cell or the progeny of the cell from other cells in the tissue; and analysing the cell or progeny; wherein the cell expresses a fusion according to the invention.
  • a method of transplantation of a cell comprises transplanting the cell into a subject; removing tissue from the subject, wherein the tissue contains the cell or a progeny of the cell; sorting the cell or the progeny of the cell from other cells in the tissue; and transplanting the sorted cell or progeny into a subject; wherein the cell expresses a fusion according to the invention.
  • the cell can be transplanted back into the original recipient, or can first be transplanted into a first subject and the sorted cell or progeny then transplanted into a second subject, different from the first.
  • sorted and/or purified cells can be cultured in vitro, optionally in co-culture with other cells.
  • the invention hence provides a method of analysing an effect of culture on a cell, comprising culturing the cell, optionally in co-culture with another cell; removing an extract from the culture, wherein the extract contains the cell or a progeny of the cell; sorting the cell or the progeny of the cell from other cells in the extract; and analysing the cell or progeny; wherein the cell expresses a fusion according to the invention.
  • the first domain of the fusion can be used to pick out the cells expressing the fusion protein and, in a similar way to that described above, the cells can be tested to determine whether there has been any effect of culture or co-culture.
  • Another application of the invention is to condition cells through culture with or contact with other cells, and then recover the cells of the invention or their progeny and use them.
  • One such method comprises culturing cells containing a construct of the invention in combination or co-culture with other cells or tissue, so as to condition the cells. Then cells expressing the fusion protein are recovered, optionally purified. These cells can be tested to determine the extent if any of conditioning.
  • Thus-conditioned cells form further aspects of the invention, and can be used for transplantation, cell therapy, drag testing or otherwise.
  • Also provided by the invention is a method of conditioning a cell, comprising culturing the cell, optionally in co-culture with another cell; removing an extract from the culture, wherein the extract contains the cell or a progeny of the cell; and sorting the cell or the progeny of the cell from other cells in the extract; wherein the cell expresses a fusion of the invention.
  • a composition of the invention comprising a thus conditioned cell.
  • the cells of the invention are allowed to grow in vivo for a period so as to condition the cells.
  • One example is to grow neural cells in the brain. Then cells can be recovered, and, again, optionally purified. These cells can be tested to determine the extent if any of conditioning.
  • Thus-conditioned cells form further aspects of the invention, and can be used for transplantation, cell therapy, drug testing or otherwise.
  • the invention thus provides a number of advances and advantages in sorting / purifying / selecting / testing and subsequent use of cells for cell therapies.
  • the fusion protein can be used to study the fate and properties of cells post transplantation.
  • the fusion can be used to select for and obtain highly pure populations of transfected cells ready for transplantation.
  • the construct enables the user to have a number of different sorting, selection and staining options available following a single transfection event.
  • FACS and MACS work well for individual sorting events, they are awkward for repeated sorting and can not be used for sorting of particular cell types, such as neurons, without significant cell damage.
  • MACS may be better for sorting delicate cells as it is not as physically demanding on the cells as FACS.
  • the third domain offers the ability to apply continuous selection over a period of time during in vitro culture.
  • the construct provides options to the user that enables different sorting options in accordance with the cells to be sorted.
  • a cell is transfected with the construct and the fusion protein is preferentially expressed in target cells, optionally target stem cells or specific progeny of transfected cells. This is conveniently achieved by operatively coupling expression to a promoter which is preferentially active in the target cells but not active or less active in other cells. Cell-specific activity of the given promoter leads to cell specific expression of the fusion - which cells can then be selected, sorted, identified etc in accordance with the teachings herein.
  • the invention thus provides for cell-specific expression of the fusion protein, optionally stem cell - specific expression.
  • the invention provides for expression of the construct driven by tissue — specific or stem cell — specific promoters.
  • a construct of the invention combines a nucleic acid encoding the fusion protein operably linked to a tissue - specific promoter or a stem cell - specific promoter.
  • the invention also optionally provides a vector comprising the nucleic acid, which may be RNA or DNA.
  • a large range of suitable promoters are of application to the present invention.
  • specific expression in dopaminergic neurons can be obtained when the promoter is selected from AHD2, Aldhla, PITX3 and TH.
  • Specific expression in serotonin - producing neurons can be obtained when the promoter is PETl .
  • the Oct4 or nanog promoters can be used for ES cells
  • the Flkl promoter can be used for mesodermal cells
  • the AmI-I or CD45 or Seal promoters can be used for haemopoietic cells, including haemopoietic stem cells
  • the a-MHC promoter can be used for cardiac cells
  • the VE-cadherin promoter can be used for endothelial cells
  • the Pdx-1 promoter can be used for pancreatic cells.
  • the invention provides also a nucleic acid encoding a fusion protein of any embodiment of the invention.
  • the nucleic acid preferably comprise a cell-specific promoter, for example selected from the AHD2, Aldhla, PITX3, TH, PETl, Oct4, nanog, Flkl, AmI-I, CD45, Seal, a-MHC, VE-cadherin, and Pdx-1 promoters.
  • a cell-specific promoter for example selected from the AHD2, Aldhla, PITX3, TH, PETl, Oct4, nanog, Flkl, AmI-I, CD45, Seal, a-MHC, VE-cadherin, and Pdx-1 promoters.
  • Other cell and stem cell specific promoters are known and are of use in the invention.
  • a vector provided as a further aspect, comprises a nucleic acid of the invention, and the invention additionally provides cells comprising such vectors.
  • the invention provides uses of the nucleic acids, vectors and cells, including cells containing the vector and cells expressing a three domain - containing fusion protein of the invention.
  • the technology is of application generally to cells, including bird, reptile, fish and mammal cells, though in particular to mammalian cells, including primate, especially human, murine, bovine, ovine, caprine and porcine, more especially human and mouse.
  • fusion proteins are expressed in human cells to enable selection of the cells in antibiotic-containing culture medium.
  • Cells are optionally further purified by FACS, using an antibody that binds to the cell surface antigen of the first domain.
  • Cells are transplanted into a patient in a cell-based therapy. Tissue is recovered from the patient and stained to determine the localization of cells. Tissue is recovered from the patient and cells expressing the fusion purified by FACS.
  • a method of the invention for selecting or purifying a cell comprises transfecting the cell with a vector of the invention, leading in practice to expression of the fusion protein. The method then optionally includes selecting in vitro or in vivo for cells expressing the fusion protein.
  • cells can be sorted cells by FACS, and where it comprise a cell surface antigen, cells can be sorted by FACS or MACS using an antibody that binds to the antigen.
  • the methods can also include staining a cell, tissue or organism comprising the cells.
  • a fusion protein according to the invention in monitoring a cell in vivo after transplantation, and use of a fusion protein of the invention in monitoring a cell in vitro.
  • the invention also provides a method of preparing and monitoring a cell for use in cell therapy, comprising expressing in that cell a fusion protein of the invention.
  • the invention has been carried out in a representative range of cells using various promoters.
  • Constructs of the invention have been transfected into mouse NS cells and human NS cells.
  • Constructs have been transfected into mouse ES cells and human ES cells.
  • Constructs have been placed under control of stem cell specific promoters, in mouse and human cells, and have been designed based upon promoters specific for somatic cells which are not stem cells, again in mouse and human cells.
  • a construct has been prepared for transfection of human dopaminergic neuron progenitor cells. In all cases, expression of a functional fusion protein of the invention has been confirmed.
  • Fig. 1 shows a cloning strategy for preparation of a fusion protein of the invention
  • Fig. 2 shows expression of ⁇ -galactosidase (a) and human CD2 (b) in an ES cell clone isolated after transfection of ES cells with the TFM2 construct described in the examples and selection with G418; and
  • Fig. 3 shows a cloning strategy for preparation of a fusion protein of the invention for expression under control of an ES cell specific promoter.
  • a 890bp fragment containing the human CD2 signal sequence, extracellular domain and transmembrane domain regions was PCR amplified from the plasmid BigTnotchICiresCD2 using the PCR primers i) CD2SalIFw: 5'gagtcgacggacgtgtttttcctttg3' (SEQ ID NO: 1) and ii) CD2SalIRev: 5'gtgtcgacttggaagttgctggattc3' (SEQ ID NO: 2) at 94 0 C for 2 mins followed by 30 cycles of 94 0 C 15s, 55 0 C 30s, 68 0 C 60s with Platinum Pfu polymerase (Invitrogen). Taq polymerase (Qiagen) was then added and the sample incubated for 8 mins at 72 0 C. Each primer contains an exogenous Sail site to aid cloning.
  • the PCR product was gel purified and cloned into Topo2.1 vector (Invitrogen) according to manufacturer's instructions to generate Topo2.1CD2PCR.
  • Topo2.1CD2PCR containing the human CD2 sequences was inserted into Sail digested Topo2.1CAGlacz to create Topo2.1CAGCD2LacZ. This placed the lacZ gene downstream of and in frame with the human CD2 fragment.
  • the TFM2 construct was linearised with Hindl ⁇ l, the digested DNA ethanol precipitated and resuspended in lOO ⁇ l of PBS. 2. 8xlO 6 E14Tg2a ES cells were electroportated with the digested TFM construct at 0.8V, 3 ⁇ F.
  • a 3628bp fragment containing the mouse Oct4 promoter was PCR amplified from the plasmid pOct4GFPIP using the PCR primers i) oct4promfw: 5'acgacgttgtaaaacgacgg3' (SEQ ID NO: 3) and ii) oct4promrev: 5'tggaaagacggctcacctac3' (SEQ ID NO: 4) at 94 0 C for 2 mins followed by 30 cycles of 94 0 C 15s, 55 0 C 30s, 68 0 C 60s with Platinum Pfu DNA polymerase (Invitrogen). Taq polymerase (Qiagen) was then added and the sample incubated for 8 mins at 72 0 C.
  • the PCR product was gel purified and cloned into Topo2.1 vector (Invitrogen), according to manufacturer's instructions, to generate Topo2.10ct4promPCR
  • This construct is for mouse ES cell specific expression of a fusion protein containing CD2 extracellular region, resistance to G418 and ⁇ -galactosidase.
  • a trifunctional marker reporter as described in Example 1 was transfected into mouse NS cells and, separately, human NS cells under control of the CAG promoter.
  • FACs and X-gal staining confirmed functional expression in both species.
  • a trifunctional marker reporter as described in Example 1 was transfected into mouse and, separately, human ES cells under control of the Oct4 promoter. X-gal staining has not been carried out but selection in the presence of G418 confirmed functional expression of the fusion protein in both species under the respective ES cell specific promoters.
  • Example 5
  • a construct was prepared for expression of the reporter of Example 1 under control of human Nanog promoter (ES cell-specific).
  • a separate construct was prepared for expression of the reporter of Example 1 under control of human Aldhla, a dopaminergic neuron specific promoter.
  • the invention hence provides in a single construct and a single genetic modification tools and methods for selecting, sorting, analyzing and recovering of cells, including stem cells, pre- and post transplantation.

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Abstract

Selon l'invention, une protéine de fusion comprend trois domaines, soit un antigène de surface cellulaire extracellulaire ou une protéine fluorescente utilisé(e) à des fins de tri, une enzyme permettant la coloration de cellules dans des tissus ou des sections de tissu, et une enzyme, telle qu'une enzyme de résistance aux antibiotiques, qui protège les cellules contre les effets d'un facteur toxique dans un milieu de culture. La fusion est utilisée pour la purification et le tri de cellules exprimant la fusion sous le contrôle d'un promoteur spécifique de cellule ou de tissu, puis pour le suivi et les analyses des cellules après transplantation.
PCT/GB2006/003484 2005-09-20 2006-09-20 Selection de cellules et systeme rapporteur WO2007034169A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0519197A GB0519197D0 (en) 2005-09-20 2005-09-20 Cell selection and reporter system
GB0519197.8 2005-09-20

Publications (2)

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WO2007034169A2 true WO2007034169A2 (fr) 2007-03-29
WO2007034169A3 WO2007034169A3 (fr) 2007-05-31

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TW (1) TW200804598A (fr)
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US5789653A (en) * 1995-01-10 1998-08-04 University Of Edinburgh Secretory gene trap
CA2450780A1 (fr) * 2001-08-02 2003-02-20 Altana Pharma Ag Nouveau procede d'expression genique de recombinaison par suppression du codon de terminaison

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TW200804598A (en) 2008-01-16
WO2007034169A3 (fr) 2007-05-31

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