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WO2000032804A1 - Procedes permettant l'introduction stable en vrac et l'expression de genes etrangers dans des parasites eucaryotes - Google Patents

Procedes permettant l'introduction stable en vrac et l'expression de genes etrangers dans des parasites eucaryotes Download PDF

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Publication number
WO2000032804A1
WO2000032804A1 PCT/IL1999/000651 IL9900651W WO0032804A1 WO 2000032804 A1 WO2000032804 A1 WO 2000032804A1 IL 9900651 W IL9900651 W IL 9900651W WO 0032804 A1 WO0032804 A1 WO 0032804A1
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Prior art keywords
schistosoma
parasite
schistosome
host
gfp
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Inventor
Joseph Hamburger
Avraham Laban
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Yissum Research Development Co of Hebrew University of Jerusalem
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Yissum Research Development Co of Hebrew University of Jerusalem
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Priority to NZ512325A priority Critical patent/NZ512325A/en
Priority to IL14345599A priority patent/IL143455A0/xx
Priority to AU14076/00A priority patent/AU764284B2/en
Publication of WO2000032804A1 publication Critical patent/WO2000032804A1/fr
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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
    • A01K67/60—New or modified breeds of invertebrates
    • A01K67/61—Genetically modified invertebrates, e.g. transgenic or polyploid
    • A01K67/63—Genetically modified worms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to transgenic eukaryotic parasites such as parasitic worms and, more particularly, to the use of transgenic eukaryotic parasites, such as parasitic worms as universal grafts for in vivo delivery of beneficial gene products in humans and in animals.
  • the present invention relates to methods for stable transformation of Bilharzia parasites (Schistosoma Spp.) and to the use of the resulting stably transformed parasites as universal grafts for in vivo delivery of beneficial gene products in humans and in animals.
  • Genetic engineering for producing desired gene products is presently the sole means available for producing, rather than extracting or isolating from natural sources, moderate to large size proteins in quantity.
  • proteins of therapeutic importance such as, for example, hormones, enzymes, receptors, antigens and cytokines.
  • the quantity required as well as the required antigenic compatibility to the recipient of these products dictates the use of recombinant DNA technologies.
  • Introducing the desired genes to host cells in vitro with appropriate upstream control elements has been used for the production of medically important proteins.
  • Recombinant therapeutic proteins initially prepared in bacteria are increasingly being produced in yeasts, and even more so in mammalian cells for obtaining suitable post-translational glycosylation where it is required for a fully functional product! ->2, 3 _
  • a number of drug delivery systems are being developed to overcome this problem, including implantable devices that release medication over a prolonged period of time in, what is known in the art as, a slow release regime.
  • Gene transfer has gained a special importance in this context since genes can be inserted into the cells of animals or human patients where they could function, as therapeutic agents. This has led to experimenting the use of several viral vectors for introducing functional genes into cells extracted from patients, and subsequent implantation of these transformed cells for in vivo delivery of the desired therapeutic product, in what is known in the art as ex vivo gene therapy UM.
  • the use of gene transfer, for a variety of research, medical and veterinary purposes, will be greatly facilitated if a universal transplant exhibiting long survival, and bearing no risk of malignant transformation, can be easily tailored for in vivo production of a desired gene product, and can be easily introduced, removed and reintroduced. Eukaryotic parasites exhibiting these features are the subject of the present invention.
  • Parasitism has evolved as an adaptation to life in a specialized ecological niche offered by another organism, the host, and co-evolution made parasites highly adapted to life in their host. Parasites can be found within every life form, from bacteria and plants to farm animals, laboratory animals and humans.
  • murine retroviral vectors which are stably integrated into the hosts' genome, have been used in human gene therapy trials, but since they integrate with the host genome at random they can potentially induce malignancy or deleterious insertional mutations ⁇ .
  • Adeno-associated virus vectors AAV
  • AAV Adeno-associated virus vectors
  • parasitic worms for this purpose are of particular interest because of their following advantageous features: (i) they exist in their host as separate genetic entities with independent capacity for synthesis of natural gene products, and potentially also of products of newly introduced genes; (ii) they have the qualities of universal transplants and sometime of a prolonged survival in their definitive hosts; (iii) they can be found in a variety of locations in the body where therapeutic gene products can exert their beneficial effect locally or systemically; (iv) they do not multiply as adults in their definitive host (in order to multiply their eggs have to reach the environment or intermediate hosts for further development), and therefore it is possible to control their burden in the definitive host by controlling the number of the infective stages to which it is exposed, and hence to control the amount of gene products secreted; (v) some species of parasitic worms multiply clonally as larvae in their intermediate host, which makes possible easy propagation of transgenic forms which have incorporated genes expressing desired products; (vi) elimination of the pathogenic qualities of parasitic worms is, in some cases, readily possible; (ii
  • An infected snail may release cercariae from about 5 weeks after infection (depending on water temperature) throughout the snail's life time (several additional weeks) 8.
  • Schistosomes were selected as a model parasite of choice for gene transfer because of their multiple advantages. For this purpose the relevant information required for evaluating the feasibility of stable transformation and expression of foreign proteins by schistosomes and for developing, accordingly, the construct vectors and gene transfer conditions required for this purpose was integrated.
  • the transgenic schistosomes were developed to serve as a model platform intended for the delivery and stable expression of a variety of desirable gene products in their intended host.
  • the present invention provides transgenic eukaryotic parasites for use as universal grafts for in vivo delivery of beneficial gene products in humans and in animals.
  • a eukaryotic diploid multicellular parasite transformed with a transgene there is provided a eukaryotic diploid multicellular parasite transformed with a transgene.
  • a method of providing a eukaryotic host with a protein or polypeptide comprising the step of infecting the eukaryotic host with a eukaryotic diploid parasite transformed with a polynucleotide sequence encoding the protein or polypeptide.
  • a method of genetically modifying a eukaryotic diploid parasite comprising the step of transforming the eukaryotic diploid parasite using a group transformation method.
  • the group transformation method is selected from the group consisting of electroporation, chemical transformation, lipofection and biolistic bombardment.
  • polynucleotide sequence is a transgene.
  • the protein or polypeptide is secreted from the parasite.
  • the infection is by a plurality of individuals of the parasite, all of the individuals are of a single sex.
  • the single sex is selected from the group consisting of male and female.
  • the parasite is a worm.
  • the worm is a flat worm.
  • the flat worm is a trematode.
  • the trematode is a schistosome.
  • the schistosome is selected from the group consisting of Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Schistosoma bovis, Schistosoma mattheei, Schistosoma rhodhaini, Schistosoma magrebowiei, Schistosoma intercalatum, Schistosoma curasoni, Schistosoma mekongi, Schistosoma spindale, Schistosoma leipere, Schistosoma turkestanicuin, Schistosoma inidiciim, Schistosoma nasalis and Schistosoma suis.
  • the host is human or animal and the parasite is infective to the human or animal.
  • the parasite is sterile.
  • the parasite is sensitive to a known drug, the drug is therefore effective in removing the parasite from the host.
  • the polynucleotide sequence is integrated in the parasite's genome.
  • the integration is by homologous recombination into a selected genomic locus.
  • the selected genomic locus is a repetitive sequence.
  • the selected genomic locus is a unique sequence.
  • the parasite has distinguishable sexes, whereby a single sex of the sexes is used for the infection.
  • the present invention successfully addresses the shortcomings of the presently known configurations by providing a transgenic eukaryotic parasite for use as universal grafts for in vivo delivery of beneficial gene products in humans and in animals.
  • the transgenic eukaryotic parasites and their use as universal grafts for in vivo delivery of beneficial gene products in humans and in animals are enabled by the development of the first operable transformation procedure for multicellular eukaryotic parasites.
  • GST glutathion-S- transferase
  • FIG. 2 is a schematic representation of a GST-GFP fusion construct, wherein TATA indicated the location of the GST promoter, ex
  • FIG 3. is a schematic representation of an GFP-Sml-7 construct which includes the GST promoter region (TATA);
  • FIG. 4 is a full sequence of the GFP-GST recombinant vector including the GST-GFP fusion construct of Figure 2 (SEQ ID NOJ);
  • FIG. 5 is a map of the GFP-GST recombinant vector including the
  • FIG. 6 is a full sequence of the recombinant GFP-Sml-7 vector including the GFP-Sml-7 fusion construct of Figure 3 (SEQ ID NO:2);
  • FIG. 7 is a map of the GFP-Sml-7 recombinant vector GFP-Sml- 7 fusion construct of Figure 3;
  • WT wild type cercariae
  • P the GFP-GST recombinant vector of Figure 5;
  • FIGs. 9a-d are confocal microscopy images of cercariae with incorporated GFP-GST vector, and with control, wherein Figures 9a and 9b show GFP-positive cercariae, fluorescence (yellow-white) is seen over a red background, Figure 9c shows positive signals in transgenic cercariae fluorescence (red) in combination with Nomarski microscopy, whereas, Figure 9d shows several wild type cercariae with negative signal (only the red background is visible); FIGs. lOa-d are confocal microscopy images of cercariae with incorporated GFP-Sml-7 vector ( Figures lOa-c) and controls (Figure 10d);
  • FIGs. l l a-i arc confocal microscopy of transgenic adult worms and of controls, wherein Figures 1 l a-d show s worm from an experiment in which GST-GFP (inverse) was employed, Figure l i e shows a wild type worm, and Figures l l f-i show a worm from an experiment where the GFP was at the direct position.
  • FIGs. 12a-f are confocal microscopy of transgenic adult worms and of controls, wherein Figure 12a shows a strongly positive male- female pair from a GFP-GST transgenesis, Figures 12b-c show a positive male worm from an Sml-7 transgenesis experiment, Figures 12d-e shows two negative male-female wild type worms, whereas Figure 12f shows a female wild type worm; and FIGs.
  • FIGS. 13a-d show confocal microscopy images obtained after staining with anti-GFP fluorescent antibodies, wherein Figures 13a-b show bodies of GFP-GST transgenic cercariae, one with Cy-5 fluorescent antibody staining (red over blue, Figure 9b), and the other with both GFP (green) and Cy-5 fluorescent antibody (red) staining ( Figure 9a), and Figures 13c-d show cercariae from an experiment with GFP-SMl -7 transgenesis, stained by both GFP fluorescence and CY-5 fluorescence.
  • FIG. 14 is a graph demonstrating shedding of cercariae from snail exposed to post electroporation of free swimming miracidia by a GST- GFP construct. Five independent experiments are shown.
  • FIG. 15 show PCR amplification results obtained with free swimming cercaria from various electroporation transformation experiments.
  • 16 show PCR amplification results obtained with DNA extracted from adult worms. Free swimming miracidia were transformed by electroporation and served to infect snails. Cercaria obtained from transfected snails were used to infect mice, from which adult worms were recovered an analyzed. Lanes: 1 - Sm-GFP, 5 males; 2 - GST-GFP (inverse), 5 males; 3 - GST-GFP (inverse) 5 females; 4 - GST-GFP, 5 males; 5 - GST-GFP, 5 females; 6 - wild type, 5 females; 7 - wild type, five males; 8 - GST-GFP plasmid (positive control); 9 - no DNA (negative control).
  • the present invention is of transgenic eukaryotic parasites such as parasitic worms, e.g., schistosomes, which can be used as universal grafts for in vivo delivery of beneficial gene products in humans and in animals.
  • transgenic eukaryotic parasites such as parasitic worms, e.g., schistosomes
  • schistosomes can be used as universal grafts for in vivo delivery of beneficial gene products in humans and in animals.
  • a eukaryotic diploid multicellular parasite transformed with a transgene there is provided a eukaryotic diploid multicellular parasite transformed with a transgene.
  • eukaryotic diploid refers to organisms having a diploid set of chromosomes. It will be appreciated that, by definition, all eukaryotes are diploid in at least a part of their life cycle, typically throughout their life cycle.
  • multicellular refers to organisms having differentiated cell types interacting there amongst to form a functional organism.
  • parasite refers to an organism which, in at least a part of its life cycle, lives on or within another species, from which it obtains nutrients and/or shelter.
  • the term “transformed” also means “genetically modified” and refers to the result of a process of inserting heterologous nucleic acids into the genome of a species. Transformation may be effected according to the present invention by group transformation methods, such as electroporation, chemical transformation, lipofection or biolistically via particle bombardment. However, as is further exemplified in the Examples section that follows, transfo ⁇ iiation cannot be effected according to the present invention by microinjection, which is an individual transformation method. As is further exemplified hereiunder electroporation, which is a group transformation method, was successfully employed to stably transform miracidia, the first schistosome larval stage, whether still in ova or after hatching.
  • transgene refers to any polynucleotide sequence which is used to stably transform an organism or cells thereof.
  • a transgene typically forms a part of an expression cassette which includes an expressible polynucleotide sequence which typically encodes a polypeptide or protein, i.e., the transgene.
  • the cassette of the present invention may include one or more of the following genetic elements: a selectable marker, an origin of replication, cis acting control elements such as a transcriptional promoter and an enhancer, a translation start site, a polyadenylation site, a signal sequence for secretion of the protein product and the like.
  • a selectable marker such as a plasminogen, a virus, or a virus.
  • cis acting control elements such as a transcriptional promoter and an enhancer
  • a translation start site such as a polyadenylation site
  • a signal sequence for secretion of the protein product and the like.
  • the appropriate assembly of these elements into an operative expression cassette is within the skills of an ordinary artisan.
  • control elements may direct the expression of the coding region of the transgene.
  • the present invention provides a method of providing a eukaryotic host with a protein or polypeptide.
  • the method according to this aspect of the invention is effected by infecting the eukaryotic host with a eukaryotic diploid parasite transformed with a polynucleotide sequence encoding the protein or polypeptide. Since many parasites are known to have restrictive species specificity, the eukaryotic diploid parasite and the eukaryotic host are selected compatible, in other words, the parasite is selected infectious to the host.
  • the term "eukaryotic host” refers to a eukaryotic organism from which a parasite obtains nutrition and/or shelter.
  • the protein or polypeptide is secreted from the parasite.
  • the polynucleotide sequence preferably includes signal peptides for secretion.
  • non-secreted proteins are also within the scope of the present invention since parasite death can cause release of the protein to the host's circulation.
  • the protein may be any desired protein for which a gene has been isolated. Typically the protein of choice is missing, dysfunctional or ineffective in the host, whereas the parasite serves to replenish the missing, dysfunctional or ineffective protein.
  • the protein may be a hormone, a growth factor, an enzyme, a clotting factor, a cytokine, an antigen, a receptor, a proteinaceous anti-microbial molecule, a proteinaceous neuro-transmitter, etc.
  • the protein may be insulin for treatment of insulin dependent diabetes.
  • the protein may be a growth hormone or sex hormone for treatment of conditions where any of these hormones is absent, dysfunctional or ineffective, or any other hormone of impaired expression in the host.
  • the parasite burden is maintained un-increased by infection with sterile (non-reproducing) parasite individuals.
  • Sterilization l o may be accomplished genetically, for example by crossing heterozygotes for a recessive sterility gene, physically by removal of sex and/or reproduction organs or by irradiation or chemical treatment known to damage these organs.
  • Burden selection depends on various factors, including, but not
  • 15 limited to, the amount of gene product secreted by the parasite and the amount of gene product required by the host for reversing symptoms or treating a condition.
  • One ordinarily skilled in the art would know how to adjust the parasite burden by monitoring the host's symptoms.
  • the transgenic parasite is a worm, preferably a flat worm, preferably a trematode, most preferably a schistosome, including the species 25 Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Schistosoma bovis, Schistosoma mattheei, Schistosoma rhodhaini, Schistosoma magrebowiei, Schistosoma intercalatum, Schistosoma curasoni, Schistosoma mekongi, Schistosoma spindale, Schistosoma leipere, Schistosoma turkestanicuin, Schistosoma inidiciim, 30 Schistosoma nasalis and Schistosoma suis.
  • a schistosome including the species 25 Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Schistosoma bo
  • a parasite compatible with that animal is selected, whereas for treatment of man, a parasite compatible with man is selected, all according to the species specificity of the parasite.
  • One 35 ordinarily skilled in the art would know how to select a parasite species which is compatible to treat a given host.
  • the parasite is sensitive to a known drug, the drug is therefore effective in removing the parasite from the host when so desired.
  • schistosomes are sensitive to a variety of schistosomicidial drugs, including, but not limited to, praziquantel, oxammquin, metrifonate, hycanthone, nicrosamide and other.
  • drugs for other parasites are well known in the art.
  • the polynucleotide sequence is integrated in the parasite's genome.
  • the integration may by effected by homologous recombination into a selected genomic locus, such as a repetitive sequence or a unique sequence. Integration by homologous recombination is presently preferred since the chances of hampering genes which are important or crucial for the functionality of the parasite in general and within the host in particular are reduced. Integration into a repetitive sequence is advantageous since multiple harmless integration sites are available, increasing both the chances of integration and the number of integration events, however, these sites are disadvantageous in that in some cases they are located in non-transcribed regions of the genome. Integration by homologous recombination into unique sequences is also within the scope of the present invention.
  • the integration site is preferably selected close to and down stream of strong and effective expression control sequences.
  • the scope of the present invention is not limited to integration by homologous recombination, in other words non-specific or non-targeted integration is also within the scope of the present invention, such that even parasites for which no sequence information is available can be transformed.
  • non integrated or extrachromosomal (e.g., episomal) transgenes are also within the broad scope of the present invention, although at present less favorable due to the possibility of diluting or loosing the transgene during cell divisions.
  • schistosomes served as a choice model system demonstrating the potential use of eukaryotic parasites in general for in vivo delivery of desired gene products in their host.
  • the use of parasitic worms is presently of choice. This may include tapeworms such as Hymenolepis diminuta for introducing gene products into the intestines, and the f ⁇ laria Brugia malayi for introducing gene products into the lymphatics, and into the circulation. Conditions for abrogating pathogenicity and transmission may influence parasite selection.
  • transgenic schistosomes are employed where unisexual infections provide these requirements.
  • the present invention is designed to provide the conditions for stable transformation of schistosomes and a modular system into which desired genes and control elements can be introduced.
  • the foreign genes were reporter genes introduced into the schistosomal genome preferably by homologous recombination. It is demonstrated that control elements of a schistosomal gene such as GST can be employed for expressing the foreign genes, and that the foreign genes can be introduced into an established (unique) schistosomal gene or into highly repeated DNA sequences of satellite DNA.
  • control elements of a schistosomal gene such as GST
  • the foreign genes can be introduced into an established (unique) schistosomal gene or into highly repeated DNA sequences of satellite DNA.
  • the use of alternative strong promoters which have been previously demonstrated to be active in transgenic eukaryotes, such as the SV40 promoter 19 ? but not excluding other promoters, can also be considered for the same purpose.
  • Targeting of the foreign genes into genomic locations other than the GST gene and the Sml-7 repeated sequence can also be considered.
  • the transfected gene may be toxic in higher concentrations, or that its expression will require external regulation.
  • constructs with inducible promoters can be prepared.
  • the most effective inducible promoters that are in use for transformations of eukaryotic cells are promoters with tetracycline regulatory systems ⁇ . Addition or depletion of tetracycline causes these promoters to start or stop transcription of the corresponding downstream gene coding regions.
  • Other triggers of inducible promoters can be incorporated as they become available.
  • the introduction of the construct containing the foreign gene into a satellite DNA region offers a possibility to avoid putative damage that may arise from introduction of the foreign gene within a single copy gene whose function is essential for the survival of the parasite.
  • the stable introduction of foreign DNA into the schistosomal genome enables stable expression throughout its life cycle, depending on the control elements of choice. This, however, does not exclude introduction of the desired genes as episomes including the desired gene in a recombinant vector. It also does not exclude introduction of the desired foreign genes into other locations such as mitochondria ⁇ intra-cytoplasmic locations, and artificial chromosomes ' ⁇ - 1 ⁇ as e ⁇ as i n 0 other possible locations not mentioned here.
  • the non- motile primary sporocyst which can be obtained in vitro directly by transformation from miracidia ' 2 is a very likely stage into which foreign genes can be introduced and which can then be transplanted directly into the snail host as is done with daughter sporocysts taken from infected snails-29. Since schistosomes multiply clonally within their intermediate host (the snail), introduction into miracidia or sporocysts and then infection of the snail (naturally for miracidium or artificially for the sporocyst) should enable expansion of transgenic clones. Infection of the snail by a single miracidium or sporocyst will ensure a single-sex progeny of cercariae for a long period of shedding (weeks).
  • the clone can be expanded to any desired size by transplantation of the transformed sporocysts into naive snails29. In this way "farming" of desired transgenic clones can be accomplished and cercariae thereof can be taken "from the shelf at any time for introduction into a few or into many target animals or humans at the same time.
  • S. mansoni infection in laboratory rodents and the following summary is a generally accepted overview '3.
  • S. mansoni worms develop to maturity (and egg laying capacity) within five weeks. Eggs in the tissues require one week to mature and then survive for three weeks. During the first five weeks of infection Thl cells are dominant in the murine immune response with INF- ⁇ and IL-2 as dominant lymphokines. After egg laying begins, Th2-type responses become dominant with IL-4, IL-5 and IL-10 as dominant lymphokines.
  • IL-4 secretion is associated with IgE response and IL-5 with eosinophilia.
  • IL-10 down regulates the Thl responses. At about 12 weeks of infection immunological down- regulation occurs, accompanied by down regulation of the granulomatous response around S. mansoni eggs and by a reduced collagen synthesis.
  • the present invention is suitable for in vivo release by the parasites of a variety of desired products of transgenes. It will enable preparation of parasites carrying more than one transgene, or introduction of more than one population of transgenic schistosomes into target hosts for multiple effects, when so necessary.
  • transgenic larvae cercariae or schistosomula
  • transplantation of fully developed worms grown in animals for exerting an immediate effect will also be possible, but will be logistically more difficult.
  • gene products that can be delivered by the present invention are the following, not excluding others not mentioned, hormones and growth factors, enzymes, clotting factors, cytokines, antigens, receptors, anti-microbial molecules, neuro-transmitters, etc. These can be used in three main biomedical/animal sciences domains as follows.
  • Over-expression of desired gene products For veterinary purposes: Over-expression of desired gene products; expression of inhibitors of natural gene products; correction (total or graded) of genetic or acquired deficiencies; altering the rate of development and of gaining body mass; altering fecundity including sperm count and oestral cycle; altering efficiency of milk production; altering the rate of production of transgene products in transgenic farm animals (e.g., in milk) and in clones thereof; altering susceptibility to disease agents such as microbes, but not excluding other agents; etc.
  • adenosine deaminase (ADA) deficiency For medical purposes: Restoration of deficiencies whether genetic 1 or acquired, such as, but not limited to, hormonal deficiencies (for example, deficiencies in growth hormone and insulin), metabolic deficiencies (for example, deficiency in metabolic enzymes), hematological deficiencies (for example, deficiencies in clotting factors), immunological deficiencies (for example, adenosine deaminase (ADA) deficiency), etc.; immunotherapy (including vaccination requiring long term antigenic stimulation, and over production of cytokines); anti- microbial therapy (including the anti-viral action of interferon, and microbe-binding soluble receptors for eliminating disease agents such as HIV); anti-cancer therapy; treatment of drug addiction; treatment of a variety of poisoning conditions; amelioration of geriatric conditions; etc. Since S. mansoni is a parasite of man this embodiment of the present invention is suitable for immediate clinical practice.
  • hormonal deficiencies for example, deficiencies in growth hormone and insulin
  • metabolic deficiencies for example, defici
  • the procedure disclosed avoids genotypic alteration of patients, thus avoiding the accompanying risks of mutagenesis and malignant transformation.
  • Schistosomes are separate genetic entities within their host. Thus making them transgenic for expressing desired genes within the host, should be much safer than introducing foreign genes directly into the host's genome, a manipulation which may result in malignant transformation or insertional mutations harmful to the hostl .
  • Gene transfer into worms has so far been successful only in the free living non-parasitic nematode Caenorhabditis elegans ⁇ . The success in this case is due to the simple and rapid (a few days) life cycle of this organism.
  • the miracidium is a ciliated multicellular organism with 4 epidermal plates arranged in 4 tiers and covered with cilia and apical musculature and glandular structure suitable for penetrating the snail host.
  • Germinal cells undergo multiplication shortly after the miracidium penetrates into the snail and transforms into a mother sporocyst°M, which is the start of asexual multiplication to form daughter sporocysts and subsequently and are located in highly perforated ova 12 capable of allowing passage of macromoleculesl3.
  • Schistosome developmental stage suitable for transformation The maintenance of schistosome cultures in the laboratory is a common practice (see experimental section below) and a variety of life cycle stages can be harvested. Foreign genes are introduced into the schistosome germ cells, which are responsible for larval development and multiplication. Germ cells are present in miracidia, sporocyst, cercariae, and young worm (schistosomula) and in sexual organs of adult worms. When transformation into germ cells takes place before clonal multiplication within the snail host, any one transfected larva serves as a source for numerous transgenic cercariae, each of which can develop to a transgenic adult worm.
  • Miracidia schistosome larvae, which are released from fully developed eggs and infect snails by active penetration, are the main proposed target for transgenesis. The reason for this choice is explained below. Miracidia are taken for transformation when still within eggs or, alternatively, after they are released from the eggs by hatching under hypotonic conditions. The selection of miracidia for transformation is based both on their position in the life cycle prior to clonal multiplication, as well as on their anatomy which favors introduction of foreign genes into germ cell as explained below. Large numbers of miracidia can transform in vitro to large number of primary sporocysts which are also candidates for transformation as is further detailed below.
  • the miracidium is a ciliated multicellular organism with four epidermal plates arranged in four tiers and covered with cilia and apical musculature and glandular structure suitable for penetrating the snail host. Most of the posterior third portion of the organism is filled with a cluster of germinal cells, which are interconnected and are also connected to the surface of the organisml l . Germ cells undergo multiplication shortly after the miracidium penetrates into the snail and transforms into a primary (mother) sporocyst (Pan CT. Studies on host-parasite relationship between Schistosoma mansoni and the snail Australorbis glabratus. Am. J. Trop. Med. Hyg.
  • These features combined, and in particular the connection between germ cells among themselves and with the surface of the organism, as well as their acidophilicity, are favorable for introducing nucleic acids into miracidia.
  • Mature eggs containing fully developed miracidia are suitable for transformation because of the anatomy of the fully developed miracidium within them as described above.
  • Hitting miracidia mature eggs and miracidia, and also primary sporocysts, in group with a wide scatter of DNA is shown herein to be the sole approach for successful introduction of foreign DNA into their germ cells.
  • a confluent distribution of DNA molecules is proposed as a preferred choice in order to ensure entrance of the DNA through the micropores of embrionated eggs, and into the extensions of genii cells which reach the surface of the miracidium (see above). This approach enables the simultaneous production of numerous transformed miracidia at once.
  • transgenesis can be effected, again by group transformation, using particle bombardment as described below.
  • the transformation approach presented herein increases the chances of a successful transgenesis of schistosomes, and therefore a higher chance that sufficient numbers will withstand the damages caused by the transformation procedure and subsequent developmental restrictions. Microinjection for introducing foreign DNA into schistosome eggs is not only not attractive, it also fails to yield transformants.
  • Particle bombardment of eggs and miracidia is considered possible but less efficient as is compared to electroporation, chemical transfection and lipofection because the latter three methods, and in particular, electroporation, involve milder driving forces and confluence of high concentrations of DNA around the target.
  • Particle bombardment delivers DNA attached to relatively large particles.
  • 1.6 mm gold micro-carriers were recently used for delivering nucleic acids into adult schistosomes for transient expression thereof (Davis RE et al. Transient expression of DNA and RNA in parasitic helminths by using particle bombardment. Proc Natl Acad Sci, USA 96:8687-92, 1999).
  • Particles of this size cannot pass through the micro-pores in the schistosome eggshell and are therefore likely to require more drastic bombardment energies in order to penetrate the hard shell and enter the miracidium within the egg.
  • a multitude of particle passing the miracidial ciliated epithelium are likely to reduce the mobility of the miracidium and therefore its ability to penetrate the snail for survival.
  • such particles covered with the DNA do not offer a confluent distribution of DNA molecules in order to enable efficient targeting to germ cells. It is therefore propose herein to employ particle bombardment primarily for transfection of primary sporocysts with the added possibility of transforming miracidia (free or within eggs).
  • sporocysts can be obtained in large numbers from miracidia and maintained in vitro by available methods (Lodes MJ & Yoshino T. Characterization of excretory-secretory proteins synthesized in vitro by Schistosoma mansoni primary sporocysts. J Parasitol 75:853-62, 1989). They are devoid of vigorous movement and of the capacity to penetrate into snails. Therefore, following particle bombardment, sporocysts can be implanted into snails (Jourdane J & Therone A. Schistosoma mansoni: Cloning by microsurgical transplantation of sporocysts. Exp Parasitol 50:349-57, 1980).
  • the control of gene expression in schistosomes has not been sufficiently defined and a system for introducing foreign DNA into the parasite has not yet been developed.
  • control elements responsible for high degree of expression and secretion will operate in the environment of the schistosomal cell.
  • Many of the control elements generally employed are species specific for the target organism of transformation, so that elements working properly in a mammalian system may not be suitable for lower organisms like schistosomes.
  • LTRs from the promoter of retroviruses or SV40 can, when active, enable high levels of gene expressionl ⁇ . These may indeed be suitable for gene transfer in schistosomes too.
  • a more specific approach is to combine the foreign gene with control elements of the target organism (schistosomes in our case) and introduce the construct into the selected location in the target genome by homologous recombination20>21
  • the expression of the foreign gene which was integrated into the target genome will be under the control of the target gene into which it was integrated. Expression will therefore be stable and the newly introduced gene will be replicated stably with the recipient genome.
  • schistosomal genes have been cloned and characterized, and all the genes which express a protein which is secreted, such as Glutathion-S-transferase22,23 s m 31/32 4 an d others25,26, 27 ⁇ see m suitable for the purpose of the present invention.
  • Glutathion-S-transferase22,23 s m 31/32 4 an d others25,26, 27 ⁇ see m suitable for the purpose of the present invention.
  • An alternative site for introducing foreign genes into schistosomes is an abundant repeated sequence of 121 bp units tandemly arranged which was found in the genome of S. mansonfi% .
  • transgenic organisms When introducing foreign genes into schistosomes the selection of transgenic organisms by selective markers seems less suitable than by reporter genes because selection may be hampered by the slow life cycle and the multicellular nature of the organism. Suitable reporter genes may actually enable identification of transgenic sporocysts in a dissected snail for transfer into naive snail for propagation and maintenance29 (see below).
  • reporter genes like the genes for chloramphenicol acetyl transferase (G47)30, ⁇ galactosidase ( ⁇ -Gal > ⁇ , luciferase (luc) ⁇ , or green fluorescent protein of jellyfish (GFP)33,34 ⁇ ne latter, although less sensitive than luc by 3 orders of magnitude, has recently been widely used in transgenesis because its expression in a transgenic animal indicates that expression is efficient far beyond the detection limit of the expression products. GFP was hence selected as a reporter gene in this work.
  • Schistosome males are preferably selected as carriers of transgenes for in vivo delivery of desired gene products.
  • Males are larger than females, they have a larger tegumental area and their tegument is directly exposed to the exterior environment for release of tegumental sloughing or secretion products containing transgene products.
  • the females on the other hand are slender, they have a smaller tegumental area and they are encircled in permanent copulation within a canal (gynecophoric canal) within the males.
  • Furthe ⁇ nore the female of schistosomes requires continuous contact within the gynecophoric canal of the male in order to complete its growth and maintain sexual maturity (Clough ER. Morphology and reproductive organs and oogenesis in bisexual and unisexual transplants of mature Schistosoma mansoni females. J Parasitol 67:535-9, 1981).
  • the contact with the male involves passage of nutrients and developmental stimuli from male to female (see, e.g., Atkinson KH & Atkinson BG. Biochemical basis for the continuous copulation of female Schistosoma mansoni. Nature 283:478-479, 1980).
  • Schistosomes multiply clonally in their intermediate host, and this is expected to greatly facilitate expansion and "farming" of desired transgenic clones: Schistosomes multiply clonally in freshwater snails in a way that presents an important advantage for propagation of transgenic larvae.
  • the miracidium transforms at the penetration site into a mother sporocyte within which germ balls are formed which develop into daughter sporocytes.
  • Daughter sporocysts then emerge and migrate to the snails digestive gland, the hepatopancreas, and germ-balls within them continuously develop into thousands of cercariae (the stages infective to man or other hosts) which continue to be shed from the snail throughout his life, generally for many weeks ⁇ 'H .
  • Schistosomes reside in the mesenteric-portal veins or in the vesical plexus (depending on the species). They are capable of secreting macromolecules36 which should be accessible into the blood, with subsequent systemic distribution. With regard to their residence in veins they are unique among parasitic worms, although access of materials secreted by parasitic worms to the hosts' vasculature also occurs among nematodes causing lymphatic filariasis which reside in the lymphatics37.
  • schistosomes may occasionally be found in ectopic locations38 5 and may be theoretically transplanted in blood vessels of additional organs where the substances they secrete may be required for therapy. In adult schistosomes ongoing turnover of the outer membrane regularly releases membrane constituents into the blood stream39. Other constituents are secreted as has been described above22-27.
  • Schistosomes are about 1 cm long, but the biomass of the male is several times larger than that of the slender female, and its maturation is independent of the presence of the female.
  • the slender female requires continuous contact with the male in order to complete its growth and maintain sexual maturity.
  • the female is permanently earned by the male in a special "gynecophoric" canal and studies have shown the passage of nutrients and developmental stimuli from male to female40 5 41 ,42 These differential features make the schistosome male a prefened candidate for in vivo production of desired gene products.
  • schistosome pathogenicity depends mainly on their capacity to lay eggs, a proportion of which do not succeed to be passed out with excreta, but become trapped in the host's tissues (of the intestines and liver or of the bladder-depending on the schistosome species).
  • Antigens secreted from the trapped eggs induce a vigorous immune granulomatous response and subsequent fibrosis43.
  • T-dependent immune responses lead to the structural and hemodynamic changes typical of the disease (schistosomiasis or Bilharzia)*.
  • eggs are an important factors in causing acute disease in humans, as is the case in murine schistosomiasis mansoni ⁇ o and in S. mansoni-infected baboons ⁇ will be discussed.
  • Eggs deposited in the intestines and the liver of animals and humans, infected with S. mansoni induce pathology in the intestines and in the liver.
  • chronic hepatosplenic schistosomiasis mansoni porto-systemic anastomoses may develop due to portal hypertension which develops as a result of the cumulative egg- related hepatic pathology.
  • eggs and occasionally worms can be found in the lungs and to a lesser extent a variety of ectopic anatomic locations.
  • Schistosomes have a long life span suitable for a prolonged expression of transgenic gene products in vivo: S. mansoni can live for decades as it was found in Yemeni immigrants to Israel ⁇ . Their prolonged survival in the definitive host points to the existence of mechanisms of immune evasion M Even when an immune response develops against these parasites it will affect the young worms (schistosomula) at various stages after penetration and migration within the definitive host, and even then, mechanisms of immune attrition can affect only a fraction of the re-infecting larvae ⁇ O. ⁇ n man, immunity against schistosomes can be detected during puberty after a prolonged infection51 >5 ?
  • Schistosomes do not multiply in their vertebrate host and they can be easily introduced into their hosts or removed from it, to enable control of the worm burden and hence of the quantity of transgene products: Cercariae of schistosomes penetrate actively through the skin of target host when it becomes exposed to waterbome cercariae by dipping ⁇ ' 1 1. Cercariae can alternatively be injected into the skin directly or following artificial transformation to schistosomula, which can even be cryopreserved for long term storageM Following penetration cercariae transform rapidly into schistosomula which migrate via the blood stream to the lungs and then to the liver where they mature, and subsequently migrate to their target location.
  • telomere length can be determined by the size of the infective dose, and that replenishing is possible until a desired therapeutic effect is achieved.
  • removal of schistosomes is possible by a number of new and effective drugs (notably praziquantel) which are being widely used for individual and community-based chemotherapy.
  • Prazyquantel is very effective by a single oral dose (sometimes divided) and yields a high percentage of cure ⁇ O. This should potentially enable removal of transgenic worms from treated individuals at any desired time.
  • the monitoring of schistosome worm burden is possibly by examining circulating antigens in their hostel . This should make possible control of schistosome worm burden.
  • transgenic schistosomes Prevention of environmental dissemination of transgenic schistosomes can be expected to be straightforward: The capacity of schistosomes to be transmitted in nature depends on presence of specific infected snails shedding cercariae in sites where definitive hosts (humans or animals) contact the water. Egg-bearing excreta are the source of snail infection and therefore introducing unisexual transgenic cercariae into target hosts is not expected to result in environmental contamination with eggs.
  • schistosome species infecting man there are those which infect farm animals, thus the new technology should be suitable for animal experimentation for basic science purposes as well as for veterinary/animal husbandry purposes:
  • the hosts of various schistosome species occurring in Africa are well knownM Among them S. mansoni and S. haematobium are essentially human parasites, but other species of African schistosomes can infect cattle, sheep, goat, and other ruminants of economic importance.
  • S. japonicum which infects people in the Far East also infects 31 species of wild mammals and 13 species of domestic animals including dog, pig, cow, water buffalo and goat- . All species of schistosomes can be used for the proposed technology.
  • GST genes of schistosomes other than S. mansonfi have been identified ⁇ and may be used as optional sites for inserting desired genes.
  • highly repeated sequences similar to the one described to S. mansonfl° are also present in the genome of other schistosome species including Schistosoma haematobium (see SEQ ID NO: 9, disclosing a Oral repeat sequence in the genome of Schistosoma haematobium, estimated to present in 50,000 copies per genome), and are potential sites of gene insertion.
  • the new technology presented here for in vivo expression of desired gene product by the parasite can expand the use of the mouse, and of other laboratory animals compatible for schistosome infection, for a variety of basic and applicable research purposes.
  • EXAMPLE 2 Materials and Methods Parasite strain, maintenance of its life cycle and harvesting of parasites: Schistosoma mansoni, an Egyptian strain, was originally obtained from Wellcome Laboratories 35 years ago and its life cycle maintained by standard methods in outbred albino mice and in its snail host Biomphalaria glabrata. Briefly, livers were collected from infected mice about 9 weeks after subcutaneous injection with 350 cercariae. Numerous ova surrounded by granulomata were embedded in the liver tissue. The livers were homogenized and the eggs washed several times in 1.7 % NaCl solution (to avoid miracidial hatching under inadvertent hypotonic conditions).
  • Miracidia were collected from the egg suspension in a dark room and under a narrow beam of light (which attracts these phototrophic larvae). Snails were exposed in separate small containers to 5-10 miracidia each for 16 hours under fluorescent light source. At least 24 snails were infected as a batch. Six or more weeks later, after completion of asexual multiplication of larvae within the snails, cercariae were shed from the snails under light in a beaker containing dechlorinated water, and 7 week old female mice were injected subcutaneoulsy with 350 cercariae. S.
  • Electroporated eggs were left at room temperature for 20 minutes then 0.8 ml double distilled water was added for hypotonicity and the quvettes left for 0.5 hours under light, for enabling hatching of miracidia. Snails were exposed individually to 5-10 miracidia/snail for 16 hours under light. Hatching and viability (motility) were subsequently determined in search of electroporation conditions that will yield about 50 % viability of miracidia. For determining hatching rate, the content of each cuvette was transferred to a well of a 24 well culture plate, lugol was added for killing the miracidia and for staining, and about 100 ova and miracidia in the same area were screened.
  • the number of free miracidia and of empty ova (eggshells) were determined and the average between them provided the hatching rate (% hatching).
  • the contents of each cuvette was centrifuged at 1000 rpm for 2 minutes to sediment eggs and dead miracidia and motile miracidia in the supernatant were collected, immobilized and stained with lugol, and counted. Control counts were carried out with eggs kept under similar conditions but without pulsing.
  • Miracidia from eggs in intestines of infected mice Harvesting of eggs from intestines yielded many more eggs (ca. 500,000/10 mice) than from livers. Following harvesting of eggs and washing with RPMI as described above, 7 ml of phosphate buffered saline diluted 1/6 was added. The egg were then distributed in a small Petri dish (3 cm diameter), and placed under light for hatching for 45 minutes. Miracidia (80-120/400 ⁇ l) were placed in a cuvette and electroporated as described above.
  • Cercariae harvested from snails infected with transformed miracidia underwent analysis confocal microscopy (see below) or were employed to infect mice, which in turn were a source of adult wonns, collected 7-10 weeks later by perfusion ⁇ .
  • Isolation of cercarial genomic DNA Cercariae were shed from infected snails infected by wild type (WT) or electroporated miracidia from the sixth week after exposure to miracidia. Genomic DNA was isolated from cercaria by lysing cells in OJ % Triton x 100 + TEN (10 mM Tris-HCl pH 8.0, 10 mM EDTA and 10 mM NaCl).
  • the lysate was treated with RNase A (0J mg/ml) in 37 °C for 30 minutes and then with Proteinase K (0J mg/ml) in 37 °C for 30 minutes. Residual proteins were removed from the lysate by phenol extraction followed by phenol- chloroform extraction. Genomic DNA was recovered by ethanol precipitation.
  • Plasmids construction All the plasmids employed were derived from the commercially available pBluescript plasmids (Stratagene). Into the EcoRY restriction enzyme site of these plasmids introduced was a 1900 bp long fragment from the 5' end of genomic copy of the GST gene23 isolated from genomic DNA of Schistosoma mansoni by PCR reaction using specific primers GST-1 and GST-2 (see paragraph on primers design and PCR conditions, below).
  • Figure 1 provides a schematic representation of the GST gene.
  • the GFP gene which encodes the green fluorescence protein from the medusa Aequorea victoria ⁇ - -5 * 34 was than inserted into the Pstl site located in the second exon of the GST gene23 ("GST-GFP fusion gene").
  • the GFP coding sequence that was used was obtained from plasmid pGFP by PCR reaction with specific primers covering most of the entire gene (see paragraph on primer design, below).
  • the size of the GFP fragment was designed to fit the reading frame of the GST protein in exon 2.
  • the resulting const ct contains a fusion of the GST-GFP proteins in the same reading frame. This construct is expected to produce the fluorescent protein under the regulation of gene expression of GST from Schistosoma.
  • This fusion protein also contains the peptide sequences that are targeting this protein to cellular compartments were the native GST protein resides, namely passage through the tegument of the parasite. Due to the long homology between the plasmid harboring the GFP and the GST genomic sequences, the cassette of GFP-GST can be incorporated by homologous recombination and stably expressed as part of the genome. Alternatively, GFP protein with the GST leader and under regulation of the GST promoter sequence may be transiently expressed by the vector positioned as an episome. The GFP was also introduced inversely for obtaining a control construct in which expression is not expected.
  • Figure 2 provides a schematic representation of the GST-GFP fusion gene.
  • the long homology of the GST gene was replaced with shorter homology of the sequence SM1-7.
  • This sequence is found in the genome in high copy numbers (10 %) 8 therefore the vector may target many sites in the genome.
  • the regulatory sequences that were used in this type of vector included the promoter of GST.
  • the major part of the 5' end of the GST sequence was deleted by digestion with Clall and Afllll restriction enzymes, as well as all of the 3' end of this gene in the GST-GFP fusion vector, leaving only the promoter region (the first 200 nucleotides of the GST gene).
  • FIG. 3 provides a schematic representation of this constrict.
  • Primer design and PCR conditions Two sets of primers were used in plasmids construction, the GST primers were used for amplification of the GST gene from the genome of Schistosoma and the second set of primers was used to amplify the GFP coding region from plasmid pGFP.
  • the computer program 01igo4 was used to select the optimal and unique primers for both genes.
  • the PCR for amplification of the GST gene was performed under the following conditions: 30 cycles of: denaturation - 94 °C, 60 sec; annealing - 55 °C, 60 sec; and elongation - 72 °C, 3 min.
  • Primers to amplify the GFP gene were: uGFP: 5'- ATGAGTAAAGGAGAAGAACTTTTC-3' (SEQ ID NO:5); and 1GFP: 5'- TTTGTATAGTTCATCCATGCC-3' (SEQ ID NO:6).
  • GFP1 5'-TCTCCCATGATGTATACATTATGT-3' (SEQ ID NO:7)
  • GFP2 5'-TCTCCATCGAAGGGTCATCACG-3' (SEQ ID NO:8).
  • the PCR for amplification of the GFP gene and fragments was performed under the following conditions: 25 cycles of: denaturation - 94 °C, 30 sec; annealing - 51 °C, 30 sec; and elongation - 72 °C 60 sec.
  • Cercariae shed from snails infected with WT miracidia or with miracidia that underwent electroporation with recombinant plasmids were analyzed directly after they were shed, or following fixation with 3 % paraformaldehyde, treatment with 50 mM NH4CI, and washing with PBS.
  • Adult worms were collected from infected mice and examined directly or following fixation.
  • Scanning laser confocal microscope (Zeiss 410) was employed to examine cercariae and adults by laser excitation at 488 nm, employing an excitation filter FT 510 and an emission filter LT 515. Contrast level used was 310-320.
  • Permeabilization was canied out by incubation at room temperature for 0.5 hours with a solution containing 0J % triton X-100 and 1 % BSA in PBS, then washing with PBS. Blocking was canied out by incubation for 0.5 hours at room temperature in 5 % Goat serum.
  • the first antibody (diluted 1 :500) was reacted with the parasites at 37 °C for 0.5 hours or overnight at 4 °C and following four washes in PBS the second antibody (diluted 1 :400) was reacted with the parasites for 0.5 hours at 37 °C.
  • the parasites were examined by confocal microscopy after mounting in a solution containing 86 % glycerol, 10 % PBS, 0J % NaN , and 3-4 % DABCO (1 ,4 Diaza Bicyclo (22.2) Octane.
  • the scanning laser confocal microscope (Zeiss 410) was employed with excitation by a Helium-Neon Laser at 650 nm, and emission at 680 nm.
  • Figure 4 presents the sequence of the vector canying the GFP-GST fusion coding infonnation (SEQ ID NOJ).
  • Figure 5 presents the map of this recombinant plasmid.
  • Figure 6 presents the sequence of the vector in which the GFP was inserted within the SM1-7 repeated sequence (SEQ ID NO:2).
  • Figure 4 presents the map of this recombinant plasmid
  • Electroporation conditions Percent hatching of miracidia from ova which underwent electroporation was higher than in control unpulsed ova in 6 out of 10 electroporation conditions tested. Percent hatching was similar to control in 2 electroporation conditions (25 ⁇ F/1500 V and 0.25 ⁇ F/ 2000V), and lower than control values in 2 other electroporation conditions (25 ⁇ F/ 500V, and 0.25 ⁇ F/1500 V). The results of one experiment (out of two) are presented in the Table 1 below.
  • motility was measured, the selected electroporation conditions (25 ⁇ F/500 V) were employed for introducing] foreign DNA (5-10 mg/cuvette). The count of motile miracidia was quarter to half of the control.
  • Rate of cercariae development Cercariae were shed in a pool of snails (at least 10 snails/pool but usually more at the time of shedding). Cercarial shedding in normal schistosome/snail combination typically starts 4 weeks after exposure to 5-10 miracidia, it usually reaches a maximal rate ranging between 1000 and 2800 cercariae/snail, and the peaks of cercarial shedding is between days 60 and 90 following exposure.
  • Figure 8 presents a representative PCR experiment demonstrating amplification of the GFP region by corresponding primers, when PCR was carried out with total genomic DNA prepared from cercariae. Amplification signals were demonstrated whether the GSP sequence was introduced directly (B), or inversely (A) within the schistosomal genome. Introduction of the transgenes into the schistosomal genome was thus demonstrated. However, simultaneous presence of the recombinant vector as an episome was not excluded. Demonstration by PCR of the incorporation of the GFP-SMl-7 transgene into the schistosomal genome is still required (although expression was clearly demonstrated by confocal microscopy as described below).
  • GFP GFP was expressed by most of the cercariae examined (1-10/group depending on availability) in some experiments only part of the cercariae (20-60 %) were positive. Although a statistical analysis was not done due to the small number of cercariae examined in each experimental group, these results suggest a very efficient transgenesis. When more than one miracidium is used for infecting a snail a mixture of positive and negative cercariae is not surprising considering that not all of the miracidia became effectively transfected. Positive signals were diffuse or concentrated in discrete foci. Representative results of confocal microscopy with cercariae are presented in Figures 9a-d and l Oa-d.
  • Figures 9a-d demonstrates a gallery of 4 photographs of experiments where the GFP-GST construct was employed. GFP-positive cercariae are demonstrated in Figures 9a-c, whereas Figure 9d is of a group of wild type cercariae.
  • Figures 9a-b fluorescence (yellow-white) is seen over a red background.
  • Figure 9c fluorescence (red) is seen in combination with Nomarski microscopy.
  • Figure 9d shows several cercariae with negative signal (only the red background is visible).
  • Figures lOa-d present a gallery of photographs, three ( Figures lOa-c) with positive signals from experiments where the GFP-Sml-7 construct was introduced, and one ( Figure lOd) wild type as control.
  • Figures l la-i present a gallery of nine photographs as follows: Figures l la-d show a worm from an experiment in which the GST-GFP (inverse) was employed, Figure l ie shows a wild type worm, and Figures 11 f-i show a wo ⁇ n from an experiment in which the GST-GFP was employed. Strong expression typified the male worm in Figures 1 lf-i. Focal fluorescence (autofluorescence) at the tips (mouth and tail) of the male worm of Figures l la-d was also observed.
  • FIG. 12a-f present a gallery of six photographs of adult worms, in which Figure 12a shows a strongly positive male-female pair from a GFP-GST transgenesis, Figures 12a-b show two positive male worms from an GFP-Sml-7 transgenesis experiment, Figures 12d-e show two negative male-female wild type worms, and Figure 12f show a negative female wild type worm showing non specific fluorescence of the ovarium (non specific fluorescence of the gut is also found when pigment resulting from digestion of hemoglobin is present).
  • Figures 13a-d present a gallery of four photographs taken with transgenic cercariae which underwent fluorescent antibody staining.
  • the photographs of Figures 13a-b were taken with bodies of GFP-GST transgenic cercariae, one of them (13b) with fluorescent Cy-5 labeled antibody staining (red over blue) staining, and the other with both GFP (green) and Cy-5 fluorescent antibody (red) staining.
  • the other 2 cercariae shown in Figures 13c-d were from an experiment with GFP-SMl-7 transgenesis. They were viewed by both GFP and CY-5 staining. Differences in intensity can be observed. Examining adult worms is particularly important because of the non specific fluorescence observed in some cases at the anterior and posterior ends and at the position of the ovarium.
  • Schistosoma mansoni Chromosomal localization of DNA repeat elements by in- situ hybridization using biotinylated DNA probes. Exp. Parasitol. 69:175-188.

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Abstract

L'invention concerne un parasite multicellulaire diploïde eucaryote, transformé par un transgène. L'invention traite d'un procédé permettant de fournir un hôte eucaryote avec une protéine ou un polypeptide comprenant l'étape consistant à infecter l'hôte eucaryote avec un parasite diploïde eucaryote transformé par une séquence de polynucléotides codant la protéine ou le polypeptide.
PCT/IL1999/000651 1998-12-01 1999-12-01 Procedes permettant l'introduction stable en vrac et l'expression de genes etrangers dans des parasites eucaryotes Ceased WO2000032804A1 (fr)

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US8734807B1 (en) 2013-04-06 2014-05-27 Gabriel Langlois-Rahme Preventing and curing Schistosomiasis mansoni by inhibiting Trk receptors on female Schistosoma

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WO1997011191A1 (fr) * 1995-09-21 1997-03-27 Ira Miller Procede d'expression et de secretion de transgenes dans des schistosomes

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WO1997011191A1 (fr) * 1995-09-21 1997-03-27 Ira Miller Procede d'expression et de secretion de transgenes dans des schistosomes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KAPPEL ET AL.: "Regulating gene expression in transgenic animals", CURRENT OPINION IN BIOTECHNOLOGY,, vol. 3, 1992, pages 548 - 553, XP002924757 *
MUDGETT ET AL.: "Electroporation of embryonic stem cells for generating transgenic mice and studying in vitro differentiation", METHODS IN MOLECULAR BIOLOGY,, vol. 48, November 1995 (1995-11-01), pages 167 - 184, XP002924756 *
PALMITTER ET AL.: "Metallothionein-human GH fusion genes stimulate growth of mice", SCIENCE,, vol. 222, 18 November 1983 (1983-11-18), pages 809 - 814, XP002924759 *
PURSEL ET AL.: "Expression and performance in transgenic pigs", J. REPROD. FERT.,, 1990, pages 235 - 245, SUPPL. 40, XP002924758 *

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US8734807B1 (en) 2013-04-06 2014-05-27 Gabriel Langlois-Rahme Preventing and curing Schistosomiasis mansoni by inhibiting Trk receptors on female Schistosoma

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