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WO2011042181A1 - Système de modèle de poursuite cellulaire, transgénique pour variants marqueurs - Google Patents

Système de modèle de poursuite cellulaire, transgénique pour variants marqueurs Download PDF

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Publication number
WO2011042181A1
WO2011042181A1 PCT/EP2010/006115 EP2010006115W WO2011042181A1 WO 2011042181 A1 WO2011042181 A1 WO 2011042181A1 EP 2010006115 W EP2010006115 W EP 2010006115W WO 2011042181 A1 WO2011042181 A1 WO 2011042181A1
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marker
donor
recipient
alpp
cell
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Reinhold G. Erben
Verena Proell
Thomas RÜLICKE
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Veterinaermedizinische Universitaet Wien
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Veterinaermedizinische Universitaet Wien
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests

Definitions

  • the present invention relates to diagnostic models for cell therapy and cell based gene therapy.
  • Cell and gene therapy are thought to revolutionise tissue repair in various organs, cancer therapy, and therapy of genetic diseases in the near future. Therefore, cell and gene therapy holds great promise to cure or ameliorate a large variety of diseases of various organ systems.
  • Cell therapy involves the syngeneic (genetically identical), allogeneic (different indi ⁇ viduals of the same species), or xenogeneic (between species) transplantation of cells for therapeutic purposes.
  • liver diseases for example cell therapy of liver cirrhosis or other severe liver diseases
  • Lung diseases for example cell-based gene therapy of cystic fibrosis .
  • Skin diseases for example cell therapy of skin defects with epidermal cells
  • Cardiovascular diseases for example cell therapy of
  • Kidney diseases for example cell therapy or cell-based gene therapy of glomerulonephritis or of renal failure
  • Orthopaedic diseases for example cell therapy of osteoarth ritis, disc degeneration, ligament injuries, or cell-based gene therapy of genetic myopathies
  • Diabetes mellitus for example cell therapy of diabetes by transplantation of autologous beta cells grown from precursor cells extracted from pancreas, liver, or bone marrow
  • neurodegenerative diseases such as Parkinson' s disease or amyotrophic lateral sclerosis, cell therapy of cerebral infarction (palsy) , cell therapy of multiple sclerosis
  • Cancer for example cell therapy with ex vivo conditioned autologous immune cells, cell-based gene therapy with autologous cells expressing for example immunostimulatory cytokines such as interleukin 2
  • immunostimulatory cytokines such as interleukin 2
  • GFP green fluo ⁇ rescent protein
  • HStk Herpes simplex thymidine kinase
  • ALPP human placental alkaline phosphatase
  • the host can be treated with immunosuppressive drugs such as cyclosporins or glucocorticoids. It is clear that with this approach it is difficult to tell whether immune-mediated phenomena were actually absent or not. In addition, immunosuppressive drugs have to be introduced into the experimental system, possibly influencing the outcome of the experiments.
  • immunosuppressive drugs such as cyclosporins or glucocorticoids.
  • WO 2006/113962 Al the use of marker tolerant animals for use in a cell-tracking system is disclosed.
  • Marker tolerance was a novel in vivo technology for studying labelled cells in the complete absence of immune-mediated rejection in immunocompetent hosts. This technology has a very broad applicability.
  • the idea of this invention was to overcome the problem of immune-mediated rejection by inducing specific tolerance to the marker protein in immunocompetent hosts.
  • tolerance can be induced by injection of marker-carrying, irradiated cells into normal animals of the same inbred strain directly after birth, i.e., before immunological self-non-self recognition is completed.
  • the present invention relates to a cell-tracking model system comprising
  • donor marker a marker or being capable of expressing said donor marker, said donor harbouring said donor marker or cells of said donor capable of expressing the donor marker being transferable to a recipient animal;
  • non-human recipient animal which is tolerant against a marker which is an immunologically neutral variant of the donor marker ("recipient marker”) , wherein "immunologically neutral” means that said donor marker and said recipient marker are not distinguishable by the immune system of the recipient animal, said recipient marker being encoded in the genome of the recipient animal in a form being capable of expression of said recipient marker and said recipient marker being physicochemically or biologically distinguish- able from the donor marker.
  • the present invention provides an improved model system for cell tracking which allows the study and tracking of cells which contain a suitable marker without any bias with respect to such markers, i.e. without that the marker would have a negative effect or otherwise influence the reaction of the model animals.
  • the model is easy to establish and to standardise and therefore allows a broad applicability in various fields for observing cells and their movement inside the body of animals.
  • the recipient animals are characterized by a normal immune system ("normal" in terms of being immunologically analogous to the non- tolerant or wild-type animal, and there is no need for immunosuppression or other measures which reduces the reliability of the model system.
  • the recipient animals can be transgenic animals which have the recipient marker as transgene and are therefore tolerant to this marker, because these animals recognise the transgene as "self” (if the transgene has been appropriately expressed during the critical period in the development of the immune system of the recipient animal); the recipient marker can also be an endogenous protein whereto the donor marker is a (n immunologically neutral) variant of this endogenous protein which is biochemically or physicochemically distinguishable so that the prerequisite of the present invention (that the recipient marker is an immunologically neutral variant of the donor marker) is fulfilled.
  • the donor harbours or is capable of expressing an immunologically neutral variant of the transgene; in the latter case, the recipient animal can be a wild type animal; the donor then harbours the immunologically neutral variant of the endogenous protein as donor marker or is capable of expressing this donor marker.
  • the recipient animals can also be made tolerant against the recipient marker by usual ways of induction of tolerance (e.g. described French et al., Diabetes 46(1) (1997), 34-39; Xu et al., Clin. Immunol.
  • transgenic animals with the recipient marker as transgene are used as recipient animals
  • stable innate tolerance to the recip- ient marker is induced in the recipient animal by transgenic expression of a mutated donor marker (as recipient marker) which is an immunologically neutral variant of the donor marker.
  • marker tolerant recipient animals are preferably generated by establishment of transgenic vertebrate (mammal, bird, fish; especially mouse, rat, rabbit, pig, frog, zebrafish, chicken) lines, expressing the recipient marker under the control of a ubiquitous, tissue-specific, or inducible promoter.
  • transgenic vertebrate mimal, bird, fish; especially mouse, rat, rabbit, pig, frog, zebrafish, chicken
  • the present model system is based on a rodent (preferably mouse, rat, and rabbit) or pig system, especially on a system of two inbred mouse/rat /rabbit lines, a transgenic donor mouse/rat /rabbit line and a transgenic marker tolerant recipient mouse/rat /rabbit line.
  • rodent preferably mouse, rat, and rabbit
  • pig system especially on a system of two inbred mouse/rat /rabbit lines, a transgenic donor mouse/rat /rabbit line and a transgenic marker tolerant recipient mouse/rat /rabbit line.
  • other vertebrate systems can be based on the present invention, especially in those model systems which allow the provision of transgenic animals, such as Xenopus, Zebrafish (or other fish, such as salmonids, carps and tilapias), chicken, and other mammals, such as goat, sheep, cow, etc.) .
  • novel marker tolerant, immunocompetent animal models can also be generated for multi-modality cell tracking.
  • the model according to the present invention significantly improves the ability to track labelled cells in immunocompetent hosts.
  • the present invention is a breakthrough technology for cell tracking in life sciences, especially in regenerative medicine, because such models are very important to assess the efficacy and safety of novel treatment approaches, especially for investigating the therapeutic targets mentioned above.
  • the term "animal” exclusively refers to non-human animals as far as the model animal is concerned. It is further noted that any treatment of the animals referred to herein is exclusively related to non-therapeutic treatment, since the aim of the model is to analyse the movement of the cells which - at least terminally - is generally observed in tissue analysis af- ter sacrifice of the recipient animal.
  • final termination of the model animal is mandatory, even if cell tracking is made in the living organism.
  • the system according to the present invention is, however, also suitable to be used in cell tracking of cell therapy, both in the model system and in humans (of course, in humans, the re ⁇ cipient marker is always an endogenous protein and the donor marker is an immunologically neutral variant thereof which is biochemically or physicochemically distinguishable from the en ⁇ dogenous marker protein chosen) . It is important that the donor and recipient marker as used for the present invention are also as in all other uses of the present invention - non- therapeutic marker in such systems.
  • the donor according to the present invention can be any kind of unit harbouring the donor marker, or any kind of unit capable to transfer the donor marker to (cells of) the recipient animal.
  • the expression “harbouring” or “capable to transfer” includes the constitutive expression of the donor marker in a cell of the donor to be transferred, the induced expression of the donor marker in the cells to be tracked after application of an induction stimulus, the ability (e.g. of a gene therapy vector) to express the donor marker in an expression system (e.g. a cell) in the recipient, or nano-vehicles or nano-containers delivering the protein marker to cells of the recipient.
  • the donor can be a gene therapy vector, nano-vehicles, nano- containers, transduced cells, expression vectors in liposomes, transgenic cells or tissue of a transgenic animal.
  • the donor is a cell or tissue which expresses the donor marker (or wherein the expression of the donor marker can be induced) , especially preferred as a donor is a transgenic animal expressing the donor marker (or wherein the expression of the donor marker can be induced) .
  • the donor especially cells or tissues of a donor animal, can be transferred to the recipient animal wherein the donor marker is or can be expressed (e.g. under the control of a given promoter which can be an inducible promoter (in this case, ex ⁇ pression of the donor marker in the recipient animal has to be actively induced) ) .
  • a given promoter which can be an inducible promoter (in this case, ex ⁇ pression of the donor marker in the recipient animal has to be actively induced) ) .
  • the recipient according to the present invention is always a vertebrate animal, preferably a mammal, especially a rodent . If the recipient is a transgenic animal, the recipient is characterized by life-long, innate tolerance to the recipient marker, and, because recipient and donor marker are immunologically neutral, the respective matched donor marker.
  • Donor marker and recipient marker have to be immunologically neutral, i.e. that the two markers are not discriminated (are not distinguishable) by the immune system of the recipient.
  • the immunological neutrality in the recipient can be tested by a tolerance test using skin transplants.
  • the rodent skin transplantation system as disclosed in Hedrich, "Genetic Monitoring of Inbred Strains of Rats” (Hedrich ed., G. Fischer, Stuttgart (1990), pages 102-114 (Chapter 4.1.3. Testing for Isohistogenei- ty (Skin Grafting) ) is used for determining immunological neutrality according to the present invention.
  • the skin transplantation test described in the example section herein is used for this purpose. Presence of no statistical significant difference in leukocyte numbers in transplants between wt —> wt and donor marker — recipient marker proves immunological neutrality.
  • the donor marker and the recipient marker have to be physicochemically/biologically distinguishable, i.e. the two markers can be distinguished by a physicochemical or biological method.
  • the corresponding markers usually differ with respect to their amino acid sequence. Possible alterations in the amino acid sequence in the recipient relative to the donor marker can be amino acid substitutions by point mutations, insertions, or deletions.
  • the amino acid difference causes a functional and/or structural difference, e.g.
  • the differences do not amount to immunological differences in the recipient; this means that the difference is not recognised by the immune system of the recipient animal (i.e. the distinguishing feature is immunologically neutral as the immune system of the recipient animal is concerned) .
  • the "variant" of the donor marker according to the present invention generally refers to an immunologically neutral, structurally differing marker compared to the donor marker which is - due to this structural difference - distinguishable in any of its physicochemical or biological properties from this donor marker, but not distinguishable by the immune system of the recipient animal.
  • the recipient marker is a mutant of the donor marker (or vice versa) . This usually enables a immunological neutrality between the two marker forms. In any way, immunological neutrality between marker pairs (donor marker in recipient animal) can always be determined by the tolerance test using skin transplants as described above.
  • the donor or recipient marker is a fluorogenic or chromogenic marker, especially Escherichia coli lacZ, green fluorescent protein (GFP) , luciferase, human placental alkaline phosphatase (ALPP) , or Herpes simplex virus 1 thymidine kinase (HSVl-tk) .
  • GFP green fluorescent protein
  • APP human placental alkaline phosphatase
  • HSVl-tk Herpes simplex virus 1 thymidine kinase
  • the system of the present invention is characterised in that the donor marker has a higher/varying heat stability, a higher enzymatic activity, different sensitivity to enzyme inhibitors, different fluorescence or luminescence characteristics, or a different acid stability than the recipient marker.
  • physicochemical/biological differences include: higher/lower stability with respect to acids/bases or chaotropes; higher/lower enzyme activity with respect to ionic strength; sensitivity with respect to enzyme inhibitors or cofactors; however, preferred marker pairs should be distinguishable by clear-cut differences, such as distinctive features, such as different emission/extinction spectra, different substrate or antibody affinities; one member of the marker pair is heat /acid/base labile, the other is stable; one member of the enzyme marker pair is inhibited by a specific inhibitor, the other is not; one member of the marker pair is recognised by a specific (monoclonal) antibody, the other is not (and/or is recognised by a different (monoclonal) antibody; etc.) .
  • a typical example for the latter is disclosed in Hoylaerts et al. (Eur. J. Biochem. 202 (1991), 605 to 616) with respect to alkaline phosphatase.
  • donor and recipient marker can differ (e.g. through a point mutation) in specificity with respect to a given substrate (e.g. by a different rate of enzymatic turnover) or with respect to two given substrates (e.g. the donor marker is able to process substrate A and the recipient marker is able to process substrate B; each without significant or relevant ability to process the other substrate) .
  • the present invention provides "pairs" of markers which can act as donor and recipient marker being immunologically neutral in the recipient animal, i.e. the recipient animal which has an intact immune system cannot distinguish immunologically between the two markers.
  • Preferred "pairs" as donor/recipient markers according to the present invention are selected from the group consisting of: human placental alkaline phosphatase (ALPP) and a heat labile ALPP mutant, especially ALPP E 29G ; green fluorescent protein and a GFP mutant with altered fluorescence or shifted spectra, especially GFP , GFP or GFp Y66H.
  • fi re fiy luciferase mutant S284T and firefly luciferase HSVl-tk and a HSVl-tk mutant with altered substrate specificity or two HSVl-tk mutants with different substrate specificity, es ⁇ pecially HSVl-R176Q-sr39tk and HSVl-A167Y-sr39tk.
  • Donor animals ubiquitously express the marker enzyme ALPP under a fragment of the murine ROSA26 promoter (Zambrowicz et al., PNAS 94 (1997), 3789- 3794), whereas recipients are transgenic for a mutated derivative of ALPP, named ALPP E429G (recipient marker), which is also expressed under the same fragment of the ROSA26 promoter.
  • ALPP E429G transgenic for a mutated derivative of ALPP
  • the rationale behind this strategy is a single amino acid substitution in the ALPP enzyme which results in the loss of its heat resistant properties, a difference in physicochemical properties which is easily detectable.
  • ALPP is virtually identical to its derivative ALPP E429G , recipients (recognising the recipient marker as self) are also tolerant to donor ALPP. Therefore, the recipient marker is an immunologically neutral mutant of the donor marker. Due to the heat sensitivity of the mutated ALPP E429G the presence of the mutated enzyme does not interfere with de ⁇ te
  • the multi-modality in vivo cell tracking systems allows the combination of e.g. fluorescent and bioluminescent imaging with excellent histological tracking capabilities in marker tolerant hosts.
  • fluorescent or bioluminescent marker such as GFP and luciferase permits optical non-invasive in vivo imaging of transplanted cells. Therefore, the present invention allows the combination of the advantages of the different marker proteins in a multi-modality cell tracking model.
  • Suitable marker pairs which can be tracked by different (monoclonal) antibodies are described in Hoylaerts et al . , 1991 cited above. For example, substitution in wt placental AP of Glu429 for Gly429 or even for His429 (found at this position in tissue-nonspecific alkaline phosphatase) and Ser429 (found in the intestinal alkaline phosphatase) induced a general decrease in affinities as detected by 16 of the 18 antibodies tested.
  • [Leu254] PLAP, [Leu297 ] PLAP, [His429] PLAP, [Ser429]PLAP and [Gly429]PLAP may be combined to suitable marker pairs which can be differentiately detected by the monoclonal antibodies Fll, D10, C2, H7, BIO G10, B2, H5, A3, F6, E5, C4, 17E3, 7E8, 327, 151, 130 and E6 (as referred to in Hoylaerts et al.).
  • marker pairs of mutant/wild type marker protein or of two different mutant marker proteins are the following:
  • the ⁇ -galactosidase from E. coli has been extensively studied, and represents one of most widely used enzymes in molecular biology. Based on sequence homologies, site-directed mutagenesis studies, and the elucidation of its three-dimensional structure, three highly conserved amino acid residues (Glu-461, Glu-537 and Thy-503) are considered to be essential for stability and catalytic activity. Therefore, amino acid substitutions at one or more of these positions ⁇ result in loss of catalytic activity of ⁇ -galactosidase .
  • substitutions of the amino acid positions His- 357 and His-540 result in reduced stability to heat (52°C), which provides an alternative possibility to pursue a strategy analogous to ALPP.
  • GFP green fluorescent protein
  • GFP a protein isolated from the jellyfish Aequorea victoria, emerged as one of the most often used fluorescent marker proteins for in vitro as well as in vivo cell labelling. Due to the widespread usage of GFP, different mutants have been engineered, resulting in altered fluorescence intensity and shifted spectra. In particular, the widely used EGFP variant is a very suitable mutated marker for the present invention. Due to two amino acid substitutions (F64L and S65T) this variant of wild- type GFP is shifted to lower excitation energies (excitation GFP/EGFP 395/488 nm; emission 509 nm for both) .
  • EGFP is optimized for brighter fluorescence (35-fold increase) and higher expression in mammalian cells. It has been shown that an additional amino acid substitution (Y66H) results in blue-shifted excitation at 382 nm and emission maxima at 458 nm (Blue fluorescent protein, BFP) . Therefore, animals, especially mice, transgenic for BFP should also be tolerant to EGFP. In this system, e.g. EGFP transgenic mice can serve as donors, while BFP transgenic mice can serve as marker tolerant recipients. In addition, BFP displays less fluorescence intensity compared to EGFP due to low extinction coefficients and quantum yields, and undergoes rapid photobleaching .
  • BFP expression endogenous cells of the recipient
  • EGFP expression transplanted donor cells
  • BFP emission is BFP emission at 458 nm able to excite EGFP
  • the excitation wavelength of EGFP is 488 nm.
  • blue shifted emission by introducing Y66F can be achieved, or a non-fluorescent variant by introducing Y66L.
  • fluorescent proteins from other species than Aequorea victoria can be used according to the present invention.
  • variants from GFPmx (GFP from Aequorea macrodactyla) has been shown to produce stronger fluorescence intensities than EGFP at 37°C, show partially red-shifted emission spectra, and can also be expressed in mammalian cells.
  • Further relevant amino acid substitutions will also result in changed emission spectra (CN 1321689) , rendering possible the differentiation between donor marker and its derivative by their specific emission spectra.
  • Bioluminescent imaging represents a very good tool for in vivo imaging, because animal, especially mammalian tissue completely lacks intrinsic bioluminescence . Furthermore, BLI shows a very good signal-to-noise ratio, because it does not require an external excitation light source but depends on ATP- driven conversion of luciferin to the light-emitting product ox- yluciferin. Up to now many luciferase genes from different spe ⁇ cies have been isolated and sequenced, but the strong tissue- absorbance of blue-green light still hampers in vivo monitoring of luciferase-expressing cells.
  • FLuc firefly Photinus pyralis
  • thermostabilizing mutations For instance the single mutation E354K improved thermostability as well as the combination of the following amino acid substitution Thr214Ala, Ala215Leu, I-le232Ala, Phe295Leu, and Glu354Lys increased thermostability of WT and S284T luciferase mutant.
  • Mutations I288A and S286N in Luciola cruciata (Japanese Gen- ji-botaru) luciferase result in red shifts from 560 nm to 613 and 605 nm, respectively.
  • substitution of asparagine at position 230 in Pyrocoelia miyako luciferase to serine or threonine provokes a shift from 562 to 616 nm (pH 7) and from 547 to 605 nm (pH 8) .
  • the mutation T217I in Lampyris cruciata significantly improved thermostability.
  • a preferred embodiment of the present invention refers to a combination of mutations resulting in maximum emission wavelength shift and intensity, thermostability, pH insensitivity and enzyme activity for the marker and non-interfering emission spectrum for the derivative. Additionally, it is advantageous to avoid any interfering emission with the fluorescent marker ( GFP ) , because this might be a confounding factor in multimodal- ity tracking models.
  • GFP fluorescent marker
  • Wild-type HSVl-tk recognizes a variety of pyrimidine and acycloguanosine nucleoside analogues and is widely used as a reporter gene for nuclear imaging (positron emission tomography,
  • PET with a variety of derivates (e.g. 29-deoxy-29-fluoro-5- iodo-l-b-D-arabino-furanosyluracil (FIAU) , 2 ' -fluoro-2 ' -deoxy-1- b-D-arabinofuranosyl-5-ethyluracil (FEAU) , 2 ' -deoxy-2 ' -fluoro-5- methyl-l-b-D-rabinofuranosyluracil ( FMAU) , 2 ' -fluoro-2 ' -deoxy-5- fluoro -ethyl-l-b-D-arabinofuranosyluracil or 9- [ 4 -fluoro-3- (hydroxymethyl ) butyl ] guanine (FHBG)) differentially labelled with either 131 I, 123 I, 124 I, 125 I or 18 F.
  • derivates e.g. 29-
  • HSV1- sr39tk differs from HSVl-tk by seven nucleotide substitutions leading to five different nonpolar . amino acids. HSVl-sr39tk shows enhanced activity relative to HSVl-tk.
  • the two mutants of HSVl-sr39tk carry one additional substitution each, either R176Q or A167Y.
  • the two derivatives HSVl-R176Qsr39tk and HSV1- A167Ysr39tk exhibit nucleoside specificity to acycloguaniosine- based and pyrimidine-based radiotracers, respectively. In this way, discrimination between both enzymes can be achieved by employing different tracers.
  • Immunologically, HSVl-R176Qsr39tk and HSVl-A167Ysr39tk are expected to be neutral.
  • HSVl-tk expression of an inactive mutant of HSVl-tk in recipient animals would also meet the requirements.
  • Random mutagenesis by several groups have identified putative essential amino acid residues for thymidine or thymidilate kinase activity of HSVl-tk: residues 165-177 and 155-165, 159-173 especially D162, R163, H164, L17057, Q125, H58, 128, Y172 and E225. More over, complete loss of activity has been shown for the substitution M128F.
  • a preferred embodiment of the present invention is an immunocompetent, marker tolerant animal (especially mouse) model which can be used at the same time for histological, fluorescent, and bioluminescent cell tracking. Therefore, multicistron- ic transgene constructs are used, where AL PP , EGFP , and lucifer- ase are driven by the same ubiquitous ROSA promoter. It is clear that such a multi-modality tracking model can also be obtained by interbreeding the separate donor and recipient models. Howev- er, when the separate lines are interbred, differential integration effects can occur, so that expression of the different markers may not be uniform in certain types of cells. This problem can be ruled out by de novo generation of an animal model based on a multicistronic transgene construct. However, both approaches can be followed according to the present invention.
  • Simple interbreeding of various combinations of marker and marker-derivate expressing mouse lines can also be performed to permit the possibility to visualize putative cell fusion events post cell or tissue transplantation.
  • luciferase transgenic mice can be interbred with tg (ALPP E429G ) mice to achieve luciferase/ALPP E429G -expressing mice, which can be transplanted with ALPP positive cells.
  • ALPP positive cells fusion between endogenous (luciferase expressing) and exogenous (ALPP expressing) cells can be monitored, as double positive cells will only be available due to fusion events.
  • Single ALPP positive cells on the other hand are then definitely of exogenous origin.
  • GFP recipient: ALPP E429G + A GFP + Luc, ALPP E429G + A GFP + LacZ or ALP pE 29G + A GFP + HStk; etc > (i. e . and all further combinations possible; ⁇ denotes immunologically neutral variant of the superscript marker protein) .
  • transgenic marker tolerant animal Central to the applications for using the system according to the present invention is the transgenic marker tolerant animal as recipient, independent of the mode of employment as well as of the marker-pair used.
  • the vertebrate animal to be used according to the present invention is not critical in principle, however, animals, especially mammals, which are already established in laboratory practice or which are known as animal models for other scientific or industrial purposes.
  • animals to be used as recipients are rabbits, rodents, especially hamster, mice, guinea pigs or rats; primates, especially chimpanzees; or pigs.
  • donor and recipient are inbred animals.
  • autologous cells can be modified ex vivo to receive the donor marker and then act as donor cells which may be transplanted to the recipient .
  • the marker is controlled by a specific promoter.
  • a preferred promoter is an inducible promoter (reviewed by Romano, Drug News Per- spect 17 (2004), 85-90; Clackson, Gene Therapy 7 (2000), 120- 125; Pollock et al . , Current Opin Biotechnol 13 (2002), 459- 467).
  • the promoter is a ubiquitous constitutive promoter, especially ROSA26 (R26) promoter, ⁇ - actin promoter, especially human, rat, or chicken ⁇ -actin promoter, or ⁇ -actin promoter with cytomegalovirus enhancer, cytomegalovirus promoter, ubiquitin promoter, or SV40 promoter.
  • the promoter is a cell- or tissue- specific promoter, especially a constitutive promoter.
  • a specific advantage of a constitutive promoter is that all cells derived from a genetically labelled cell express the marker regardless of differentiation status.
  • a specific advantage of an inducible promoter and a cell- or tissue-specific promoter in the current invention is that marker gene expression can be limited in space and/or time.
  • Rosa26Promoter full length or truncated (Zambrowicz et al . PNAS 94(1997), 3789- 3794)
  • CMV cytomegalovirus; US 5,168,062
  • promoters truncated promoters, mutated promoters for promoter/reporter studies
  • any promoter suitable for specific additional transgene expression in conjunction with marker expression e.g. via IRES any promoter suitable for specific additional transgene expression in conjunction with marker expression e.g. via IRES
  • the present invention also relates to the use of the system according to the present invention for tracking cells; it also relates to a method for tracking cells wherein the donor with the donor marker is trans ⁇ ferred to the recipient animal, whereafter the donor marker (in cells) is tracked in the recipient animal.
  • a "donor” may be e.g. an animal, tissue or cell that expresses at least one marker (donor marker) suitable for detection within the compatible recipient post administration that does not induce immune mediated rejection in the recipient.
  • Recipient can be (i) an animal expressing compatible marker-derivate ( s ) to the chosen marker (s) of donor, being therefore tolerant to the chosen donor's marker (s) or (ii) an animal expressing compatible marker-derivate ( s ) to the chosen marker (s) of donor, being therefore tolerant to the chosen donor's marker (s) and marker (s) suitable for detection and different from the marker (s) expressed by the donor, to allow monitoring of fusion events of endogenous and exogenous cells.
  • the model according to the present invention is the "next generation" of in vivo cell tracking models, including the preferred embodiments, combines several important advantages: (i) stable genetic labelling of cells of interest, (ii) unlimited source of genetically tagged cells, (iii) immunocompetent hosts, and (iv) most importantly, absence of immune-mediated rejection of labelled cells, allowing valid long-term studies.
  • An additional advantage of the approach according to the present invention is - in the embodiment that also the donor is an animal of the same kind as the recipient (e.g. both are mice) - that the heterozygous transgenic donor and recipient can be interbred with gene-targeted knockout or knock-in animals, e.g. of the same strain.
  • liver diseases for example cell therapy of liver cirrhosis or other severe liver diseases
  • lung diseases for example cell-based gene therapy of cystic fibrosis
  • skin diseases for example cell therapy of skin defects with epidermal cells
  • cardiovascular diseases for example cell therapy of myocardial infarction with mesenchymal cells, gene therapy of cardiomyopathies, for example with vascular growth factors
  • kidney diseases for example cell therapy or cell-based gene therapy of glomerulonephritis or of renal failure
  • orthopaedic diseases for example cell therapy of osteoarthritis, disc degeneration, ligament injuries, or cell-based gene therapy of genetic myopathies
  • diabetes melli- tus for example cell therapy of diabetes by transplantation of autologous beta cells grown from precursor cells extracted from pancreas, liver, or bone marrow
  • diseases of the central nervous system for example cell therapy of the central nervous system
  • Fig. 1 shows the homology of human alkaline phosphatases.
  • A Heat properties of human APs .
  • B The sequences of GCAP and PLAP differ in only seven amino acids.
  • C An exchange of the C- terminal part of GCAP results in a single amino acid substitution at position 429 and in resistance to heat of mutant C5;
  • Fig. 2 shows histochemical ALPP detection in tissue sections of different organs after heat inactivation at 72°C for the in ⁇ dicated time.
  • Tissues from ALPP transgenic donors show strong staining, whereas, similar to wild-type mice, no enzyme activity can be detected in tissues of ALPP E429G mice; and
  • Fig. 3 shows that tg (ALPP E429G ) recipients are tolerant to the marker ALPP.
  • CD45R represents a transmembrane protein expressed on leukocytes and is used here to monitor immune-mediated rejection of skin transplants.
  • the aim of the present examples is to generate marker tolerant mouse models as novel tools for regenerative medicine, cancer research, and cell tracking studies in general.
  • labelled cells can be tracked for long periods of time in the complete absence of immune-mediated rejection of labelled cells.
  • the first example is based on the ALPP/ALPP E429G mouse model. Further embodiments involve the gener ⁇ ation of donor and marker tolerant recipient mouse lines for the most widely used protein markers GFP, lacZ, luciferase, and HSV- tk, as well as a mouse model for multi-modality cell tracking. All these marker proteins are non-toxic at appropriate doses/expression levels and developmentally neutral in transgenic animals .
  • Each example involves generation of a marker tolerant animal model for a specific marker protein (a "pair” of donor and recipient marker) or for multi-modality cell tracking (more than one "pair”) .
  • Design of the constructs is followed by in vitro testing in cell culture. Thereafter, appropriate constructs are used for generation of transgenic animals. Tolerance of recipient lines is tested by skin transplantation.
  • site-directed mutagenesis is used for generation of the models. This strategy is taking advantage of the detailed structure- function information already available for all the marker proteins used in the present example section.
  • site-directed mutagenesis in the present examples is restricted to a single amino acid substitution, if possible, or alternative to as few mutations as necessary to meet the crite ⁇ ria for physico-chemical or biological differentiation of donor marker and recipient marker, but maintain immunological neutrality between donor and recipient marker.
  • the present invention is based on a dual marker system consisting of a donor and a corresponding marker tolerant recipient.
  • a mouse model this can preferably be accomplished by a mouse line on the same inbred background (e.g. C57BL/6) :
  • the donor line expresses the wild-type marker protein and serves as an unlimited source for genetically labelled cells and tissues, whereas the recipient line expresses a mutated form of the marker which can be easily distinguished from the wild-type form by its physicochemical or biological properties.
  • the mutation can preferably be introduced into the wild-type form by site- directed mutagenesis of one amino acid.
  • the immune system of the recipient will usually not recognize donor cells labelled with the wild-type marker as foreign. In other words, the recipient mouse line will be tolerant to the wild-type marker.
  • APP is known to be a superb marker for histological detection of labelled cells
  • APs human alkaline phosphatases
  • Fig. 1 All four human alkaline phosphatases (APs) are highly homologous (Fig. 1) . However, only placental alkaline phosphatase is heat resistant. It was demonstrated that one particular amino acid is mainly responsible for the different sensitivity of mam ⁇ malian APs to heat. It. was further shown that germ cell AP can be made heat resistant by substitution of the C-terminal part of the molecule by the corresponding ALPP sequence which differs only in one amino acid (Fig. 1). For the purposes of the present invention, advantage was taken of this unique feature of ALPP. By site-directed mutagenesis, a heat sensitive form of ALPP was generated by changing glutamic acid 429 to glycin in the C- terminal part of the enzyme.
  • the heat-sensitive derivative ALp pE429G is v r tually identical to normal ALPP, and differs only in one amino acid. Accordingly, a transgenic animal expressing ALP pE 29G does not recognize ALPP as a foreign protein (ALPP E 29G is "immunologically neutral" compared to ALPP) .
  • FIG. 3a Similar results were obtained when skin was transplanted from male tg(ALPP) to female C57BL/6 or tg (ALPP E429G ) (sex mismatched; smm) , where the male-specific minor histocompatibility antigen H-Y is responsible for rejection.
  • skin grafts from ALPP transgenic donors transplanted into tg (ALPP E429G ) mice showed strong ALPP staining, 6 months post-transplantation
  • R26 Promoter was amplified via PCR from genomic DNA extracted from tg(hPLAP) F344 rats (Kisseberth et al., Developmental Biology 214, 128-138 (1999) and genomic ALPP sequence including 3 ' UTR was amplified from human whole blood genomic DNA.
  • PCR products fori 5 ' -GTCGACTAGATGAAGGAGAGC-3 ' and revl 5 ' - GAGCCACATATGGGAAGCGGT-3 ' , for2 5 ' -ATGCCCAGAATTCCTGCCTCG-3 ' and rev2 5 ' -GAAAGGAGCCTGCCTGGTACC-3 ' ; for3 5 ' -GGTACCAGGCAGGCTCCTTTC- 3 ' and rev3 5 ' -GAGGCAGAATCTCGCTCTGTC-3 ' ) where cloned into pCR2.1 or pCR-4-TOPO® (Invitrogen, Carlsbad, USA) and sequenced to verify mutation-free amplification.
  • pCR2.1 or pCR-4-TOPO® Invitrogen, Carlsbad, USA
  • R26P and ALPP genomic sequence segments (wt for ALPP and SOE-mutated for ALPP E429G ) were eventually assembled and cloned into a modified pEGFP-N3 vector, where CMV promoter was removed by ASCI and Nhel digest, and EGFP was removed by Kpnl and Xbal digest.
  • Complete R26P-ALPP and R26P- ALPP cassettes where sequenced to verify sequences. Both cassettes were then excised from vector backbone with Sail and Aflll digest, separated by agarose gel electrophoresis, isolated from gel using gel extraction kit (Qiagen, Hilden, Germany) following the manufacturers instructions.
  • Modified pEGFP-N3 vectors containing either R26P-ALPP or R26P-ALPP E429G cassettes where transfected in mouse 3T3 fibroblasts using Effectene (Qiagen, Hilden, Germany) following the manufacturers instruction.
  • Stable transfected cells where selected by G418 (300 pg/ml and 600 pg/ml; Sigma, Kunststoff, Germany) treatment und subsequently single cell clones where obtained by limiting dilution, plating 1 cell per well in 96 well plates. Clones were expanded in RPMI 1640 medium supplemented with 10% FCS and Pen/Strep.
  • the purified DNA constructs were injected into a pronucleus at a concentration of 2.0 ng/ ⁇ injection buffer. Injected zygotes were transferred at the same day into pseudo-pregnant surrogate mothers. To identify transgenic founder animals the genotype of the offspring was tested by Southern blot and PCR analysis using primers R26f 5 ' -TGAATTCCTGCCTCGCCACTGT-3 ' and E2r 5 ' - AAGGCCTGGCTCACTCACCATC-3 ' and U3f 5 ' -GATGGAGACCATCCTGGCTAAC-3 ' and U4r 5 ' -GATCTAGTAACGGCCGCCAGTG-3 ' amplifying products of 350 bp and 210 bp, respectively, for the transgene allele.
  • genomic DNA was. isolated from tail biopsies by phenol/chloroform extraction and isopropa- nol precipitation and 10 g was digested by restriction enzyme BamHI, and separated by agarose gel electrophoresis, wt genomic DNA was used as negative control. DNA was transferred to Nylon membrane (Hybond N+; Amersham Pharmacia Biotech, Uppsala, Sweden) and hybridized with two different probes. Probes where gained by restriction end nuclease digest of R26P-ALPP cassette and subsequent isolation of agarose gel. Probe A by Blnl digest
  • Probe labeling was performed using PRIME-It-II-Random Labeling Kit
  • mice where sacrificed by exsanguination from V. cava under Ketamin/Xylazin anaesthesia (70/7 mg/kg i.p.) and tissue samples from liver, kidney, lung, spleen, heart, brain, duodenum, jejunum, intestine, stomach, pancreas, thymus, uterus, over, testis, aorta, lymph node, gallbladder, skin, muscle, tibia, femur and calvaria where harvested and fixed in 40% EtOH, dehydrated and embedded in paraffin and where subject to histological analysis.
  • Ketamin/Xylazin anaesthesia 70/7 mg/kg i.p.
  • Frozen lung samples were homogenized in 0.25 M Tris-HCl pH 6.8 + 0.4% SDS for 2 x 60 seconds at 6500 rpm and cooling at - 20 °C for 2 min between the runs in the MagNA Lyser Instrument (Roche) and homogenates were diluted 1:1 with 6% SDS. 1.25 pg for tg(ALPP) and 50 ⁇ iq protein for tg (ALPP E429G ) and wild-type derived samples were separated by SDS-polyacrylamide gel electrophoresis and immunoblotting was performed. For immunological detection of ALPP and ALPP E 29G rabbit monoclonal-anti-hPLAP Clone SP15 (Thermo Fisher Scientific) and ECL Plus (GE Healthcare) was used.
  • RNA isolation tissue was harvested, immediately shock frozen in liquid nitrogen, and stored at -80°C until RNA isolation. Frozen tissue was homogenized in TRI Reagent (Ambion) for 60 sec at 6500 rpm in the agNA Lyser Instrument (Roche) . RNA was extracted with l-Bromo-3-chloropropane (Sigma) and precipitated using isopropanol. RNA purity and quality was determined spectrophotometrically (BioPhotometer ; Eppendorf) as the A260/A280 ratio showed expected values between 1.8 and 2 and the A260/A230 ratio values were 1.8 or greater. Additionally the absence of RNA degradation was verified via Agarose gel. Reverse transcription of RNA was performed using iScriptTM cDNA Synthesis Kit (Bio-Rad) following the instructions of the manufacturer.
  • Recipient mice C57BL/6, tg(ALPP) and tg (ALPP E429G ) were either immunised four weeks pre surgery i.p. or not with 100 ⁇ freshly harvested whole blood of donor mice (C57BL/6 or tg(ALPP)). Coagulation of whole blood was prevented by adding 10 ⁇ trisodium-citrate-dihydrate-solution (0,136 molar) per 100 ⁇ blood.
  • mice were anaesthetised with 2-4% isoflurane. 0,5 x 0,5 cm full skin allografts were transplanted from donors to recipients. One donor mouse was used for 3-10 recipients of different groups. In any case an auto-graft served as control. Grafts were protected from self-destruction by bandaging for the first week and with applying a ruff for another week. Oral met- amizol application was used for analgesia administered pre- surgery and every 6 hours post-surgery for 24h. For post-surgery infection control Enrofoxacin (Baytril®; 10 mg/kg) was administered s.c. daily for 3 days.
  • Photographs were taken after one week and subsequently from the 3 rd week at an interval of 3 weeks until mice were sacrificed 24 weeks post surgery by exsanguina- tion from V. cava under Ketamin/Xylazin anesthesia (70/7 mg/kg i.p.) For evaluation of immune mediated rejection sections where stained with anti-CD54R and number of positive cells where counted per tissue section.
  • TN buffer 0.1 Tris-HCl, pH 9.5, 0.1 M NaCl, 5 mM MgC12
  • TN buffer 0.17 mg/ml of the substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma) and nitrotetrazolium blue chloride at room temperature overnight.
  • BCIP 5-bromo-4-chloro-3-indolyl phosphate
  • nitrotetrazolium blue chloride nitrotetrazolium blue chloride

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Abstract

La présente invention concerne un système de modèle de poursuite cellulaire comprenant - un donneur abritant un marqueur (« marqueur de donneur ») ou pouvant exprimer ledit marqueur de donneur, ledit donneur abritant ledit marqueur de donneur ou les cellules dudit donneur pouvant exprimer le marqueur de donneur étant transférable(s) à un animal récepteur ; et - un animal récepteur tolérant vis-à-vis d'un marqueur qui est un variant immunologiquement neutre du marqueur de donneur (« marqueur de récepteur »), ledit marqueur de récepteur étant codé dans le génome de l'animal récepteur sous une forme pouvant exprimer ledit marqueur de récepteur et ledit marqueur de récepteur étant d'un point de vue physicochimique ou biologique différentiable du marqueur de donneur.
PCT/EP2010/006115 2009-10-08 2010-10-07 Système de modèle de poursuite cellulaire, transgénique pour variants marqueurs Ceased WO2011042181A1 (fr)

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CN103421793A (zh) * 2013-07-28 2013-12-04 华中农业大学 猪肝脏特异性表达基因ttr启动子的克隆及应用

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CN103421793A (zh) * 2013-07-28 2013-12-04 华中农业大学 猪肝脏特异性表达基因ttr启动子的克隆及应用

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