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WO1997011191A1 - Procede d'expression et de secretion de transgenes dans des schistosomes - Google Patents

Procede d'expression et de secretion de transgenes dans des schistosomes Download PDF

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Publication number
WO1997011191A1
WO1997011191A1 PCT/US1996/015083 US9615083W WO9711191A1 WO 1997011191 A1 WO1997011191 A1 WO 1997011191A1 US 9615083 W US9615083 W US 9615083W WO 9711191 A1 WO9711191 A1 WO 9711191A1
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schistosome
clones
gene
transgenic
transgene
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PCT/US1996/015083
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English (en)
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Ira Miller
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Ira Miller
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Priority to EP96933832A priority Critical patent/EP0851936A1/fr
Priority to AU72411/96A priority patent/AU7241196A/en
Publication of WO1997011191A1 publication Critical patent/WO1997011191A1/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
    • 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

Definitions

  • This invention relates to the field of gene therapy.
  • Most current strategies of gene therapy employ mechanisms to alter the patient's own cells to produce the desired gene product, through viral and non-viral vectors that introduce DNA that encodes the desired product.
  • Some of the major pitfalls of such methods are: low efficiency of introduction and expression, potential for viral infection by contaminating replication competent virus, potential for recombination with host DNA and for promoting malignant transformation, irrever- sibility of the process, and the need for labor-intensive individualized treatment.
  • the method described here avoids these problems because it uses an intermediate vector for gene expression in the patient, a vector that can be mass- produced and batch-characterized, that can be eliminated at will and that does not alter the DNA of the patient's own cells.
  • This patent describes a method of creating genetically engineered schistosomes as a vector for secretion of therapeutic proteins into the bloodstream of humans and other susceptible hosts. This process will result in a sustained in vivo protein expression system. This system avoids the need for large scale protein purification and for repeated injections of therapeutic proteins that must be administered parenterally, such as insulin or erythropoietin.
  • This mode of protein expression is a form of "gene therapy” applicable in situations where the gene introduced, hereafter referred to as the "transgene”, does not require expression in the cells of the patient but rather can be functionally expressed in an intermediate, symbiotic vector.
  • this patent deals with the creation of transgenic schistosomes which lay soft, degradable eggs or which lay eggs with reduced sclerotin content.
  • transgene products for insertion into the vector.
  • An exhaustive list of potential products for expression in this system is not intended.
  • the system described herein is suitable for expression of any protein that is active in the plasma or that can be targeted from the bloodstream to its appropriate extracellular or intracellular location.
  • proteins suitable for this therapeutic system are noted below, with examples given for each.
  • insulin Although blood-glucose-level-regulated- expression of insulin is required for proper glucose control, a constant, low-level baseline expression of insulin may prove to be extremely valuable for preventing hyperglycemic episodes leading to ketoacidosis. In addition, low level baseline expression may reduce the number of daily injections needed for many insulin-dependent diabetics.
  • leptin This newly discovered adipocyte hormone is sure to play a role in body fat regulation for many individuals. Probably, only low levels of expression are required for therapeutic benefit, and it does not require a timed expression pattern.
  • calcitonin Osteoporosis is a major cause of morbidity and mortality among post-menopausal women, the elderly and steroid-dependent individuals. Calcitonin injections are one mode of therapy used for such individuals. A boost in baseline calcitonin levels using this vector may replace that mode of therapy.
  • non-hormonal circulating proteins such as: alpha-1-anti-trypsin: Deficiency of this plasma protein causes significant disease in 1/3500 individuals, leading to cirrhosis as well as emphysema. Constant low levels of expression are required to prevent tissue destruction. factor VIII: Deficiency of this protein causes hemophilia in 1/10,000 males. Constant low serum levels are required to prevent morbidity. cholesterol ester transfer protein inhibitor: Aberrant lipoprotein profiles are a significant cause of morbidity from atherosclerosis. Agents acting to increase HDL/LDL cholesterol, such as a peptide designed to inhibit this enzyme, may have a tremendous effect on disease in individuals at risk.
  • human immunodeficiency virus co-receptor ligands Recently, co-receptors for HIV on T-cells (the SDF-1 chemokine receptor, LESTR/fusin) and macrophages (the beta-chemokine receptor, CC-CKR) have been identified. Individuals with elevated levels of beta-chemokines are resistant to HIV infection. Artificially raising serum levels of the ligands for these receptors may protect against infection with HIV or slow disease progression.
  • this method is desirable for this method to be used against lysosomal storage diseases, for example, for the insertion of beta-glucocerebrosidase, deficiency of which causes Gaucher's disease, most common in Ashkenazic Jews.
  • Exogenous administration of the purified enzyme from placenta is potentially curative, as the protein is targeted to the lysosomal compartment .
  • Therapy is presently limited by availability of enzyme, which must be repetitively injected.
  • collagen vascular diseases triggered by immune complexes e.g.
  • erythrocyte complement receptor levels are reduced, leading to delayed clearance of circulating immune complexes and deposition in tissues, with ensuing glomerulonephritis or vasculitis.
  • Expression of a soluble CRl receptor might facilitate clearance of immune complexes by the reticuloendothelial system and prevent relapses.
  • cytosolic, mitochondrial intracellular non-lysoso al
  • Methods may be developed in the future to allow postranslational transmem- brane passage of desired proteins, possibly based upon the paradigm of the dimeric ricin and diphtheria toxins.
  • the schistosome expression vector could be used to deliver proteins to treat glycogen storage diseases, hormone receptor defects, and many metabolic disorders requiring replacement of a cytosolic or even possibly subcellularly localized (not only lysosomal) protein.
  • the invention is a transgenic schistosome, male or female depending on the particular embodiment, whose genome has been stably transformed by DNA encoding a transgene within appropriate regulatory contexts .
  • the transgenic schistosome secretes the transgene product into the blood ⁇ stream of its human or other definitive host.
  • the invention takes advantage of developed methods for propagating schistosomes in snails at the sporocyst life cycle stage in order to obtain large clonal populations of recombinant schistosomes.
  • the method adapts existing technology that has been developed for microinjection of eggs of other species for use in the injection of schistosome eggs, based upon known aspects of schistosome biology.
  • the invention includes methods to use male schistosomes as vectors in unisexual infections and to use female schistosomes in bisexual infections. In female infections, methods to decrease egg production and eggshell maturation are described.
  • the invention uses specific types of DNA constructs encoding antisense RNAs and ribozymes to interfere with schistosome eggshell protein production or maturation either directly or by interfering with the action of tissue-specific transcription factors, and the invention describes methods to clone these transcription factors and eggshell tanning enzymes.
  • the invention employs use of schistosome genomic DNA locus control region-like elements to confer high level tissue-specific expression of the transgene, based upon work done in the mouse, and it describes how to identify and utilize these regulatory regions for creation of the transgene construct.
  • the invention utilizes DNA constructs encoding various mRNA regulatory sequences and signal peptides based upon published schistosome and non-schistosome sequences.
  • the invention describes strategies for mating recombinant schistosomes to obtain the most effective transgenic schistosome vector.
  • Figure 1 is a flow-chart of the method for obtaining clones of recombinant schistosome clones
  • Figure 2 is a diagrammatic representation of how to adapt- endogenous schistosome gene transcriptional regulatory sequences for use in the transgene vector
  • Figure 3 is a diagrammatic flow-chart of how to obtain female transgenic clones on an eggshell knockout background
  • Figure 4 is a diagram of the transcriptional regulatory regions to accompany the various transcripts of the multiple DNA constructs to be used in 2-step and 3-step schistosome genetic modifications.
  • the protein expression is targeted to the worm's integument.
  • the integument is not specialized for protein secretion, it nonetheless has a tremendous metabolic capacity for surface membrane protein production, a pathway to which exogenous proteins are targeted in this invention.
  • the schistosome integument is a multi-laminate membrane (Silk MH et al.
  • Proteins targeted for secretion in this location should eventually find their way to the worm exterior, either after fusion of secretory vesicles with the exterior leaflet or after sloughing of the exterior leaflet, with release of material from the interme branous space.
  • Transgene expression targeting to schistosome tissues other than the integument may, in fact, prove to be more efficacious .
  • Structures that normally actively secrete soluble proteins are obvious targets for expression. These structures include the gut, the vitelline gland, the Mehlis gland and the ootype.
  • hermaphrodites have been found in unisexual nale infections and never result in egg formation in schistosome species that infect humans (Shaw MK and Erasmus DA, Schisxzosoma mansoni : The Presence and Ultrastructure of Vitelline Cells in Adult Males, Journal of Helminthology 56:51-53, 1982; reviewed in Hermaphraditism in Male Schistosomes, pp 162-164, in Ch. 4, sexual and Conjugal Biology, in Schistosomes, PF Basch, 1991) .
  • hermaphrodite males full utilization of the powerful secretory system of the female reproductive system most likely requires expression in females.
  • Isolates of schistosomes including S. mansoni , S. haema tobium and S. japonicum are obtained from stool (or urine for S. Haematobium) from infected humans or from established laboratory strains, and passaged in susceptible Biomphalaria glabrata (or Bulinus for S. Haematobium) snails and susceptible mouse strains or hamsters (see table 2-3 in Basch, 1991, and references therein) as described (Hacket F, The culture of Schistosoma mansoni and production of life cycle stages, in Methods in Molecular Biology, vol. 21: Protocols in Molecular Parasitology, JEH Hyde ed.
  • Freshly laid (stage I) eggs containing oocytes undergoing meiosis and male pronuclei, are harvested by microdissection from the intestines of schistosome-infected laboratory animals (Box 1) , such as mouse, hamster or guinea pig (Pellegrino et al . , 1962, Pelligrino and Faria, 1965) . Infected animals are sacrificed in the week prior to the forty-fourth day pos -infection, when immature eggs predominate (Pelligrino J et al.
  • stage I Those in stage I are dissected away fro ⁇ intestinal tissue and placed sterily into a chamber for microinjection containing cell culture medium (see below) with 25 mM HEPES (pH 7.4) in place of bicarbonate. Collaginase treatment is performed at this point, if necessary, to remove adherent mouse tissue. Approximately 50 stage I eggs are removed from each maximally infected animal.
  • Older eggs have a dense eggshell coat that may prevent easy introduction of a microinjection needle.
  • nuclear or cytoplasmic enzymes required for recombination may be present exclusively during early zygotic period. Identifying this early egg stage is also necessary because later stages, passed in the stool, contain multicellular developing miracidial organisms, injection of which may be more difficult and may lead to a non-clonal distribution of DNA integration in progeny, complicating the analysis of transgenics.
  • An alternative method for obtaining early stage eggs for microinjection is to isolate eight-week old adult schistosomes, culture them in vi tro, and recover the earliest laid eggs from the culture medium as soon as possible.
  • the technique for recovering adult schistosomes and the optimized parameters for culture oE eggs to reach maturity in vi tro have been assessed in detail (Newport G and Weller TH, 1982.
  • mice 3 Conditions for DNA microinjection (Box 3) are based upon optimized protocols from mouse egg microinjection (Brinster RL et al. , Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs, Proceedings of the National Academy of Sciences, 82:4438-42, 1985), for which an integration efficiency of 27.1% has been achieved.
  • the schistosome zygotic nuclei are approximately 6 to 8 microns ir diameter (Nez MM and Short RB, Gametogenesis in Schistosomatium douthitti (Cort) (Schistos atidae: Trematoda) , Journal of Parasitology 43: 167-82, 1957; Neill PJ et al.
  • Microinjection is performed with eggs placed under silicone oil on commercially-available depression slides or laboratory-prepared dried agarose coated glass cover slips, using standard techniques. Nuclear injection is confirmed in parallel control samples by using the nondiffusible dye FITC-dextran to follow injections with fluorescence microscopy as has proven useful in microinjection of other helminths (Fire A, Integrative Transformation of Caenorhabdi tis elegans, The EMBO Journal 5:2673-80, 1986) .
  • injected eggs are maintained at 37°C in 5% C0 2 in bicarbonate buffered cell culture medium (see Newport and Weller, 1982, Parasitology, referenced above, for schistosome egg culture technique) with addition of fetal calf serum and casein hydrolysate for approximately six days, until the miracidia reach maturity.
  • table 1 casein hydrolysate and mouse r.b.c.
  • table 2 8% fetal calf serum in DSMH
  • Miracidial hatching and infection (Box 5) are performed by placing individual mature eggs in small beakers containing a small amount of spring water and a single snail and exposing to bright light for five minutes as described (Maclnnis AJ, 1970) . Infected snails are then reared together until miracidia are produced (Box 6) . Propagation and analysis of recombinant clones (Boxes 7-11) is done preferably as follows: Because the released miracidia are used to infect susceptible snails on a one miracidium per snail basis, each snail releases thousands of genetically identical cercaria, which constitute a schistoso- mal clone. Analysis of infected snails is conducted weekly after the third week to determine which are productively infected.
  • Extrachromosomal arrays are identified by the absence of higher molecular weight bands that correspond to segments of schistoso-rte genomic DNA flanking the integra ⁇ tion site revealed by Southern blotting after digestion with a unique restriction site within the transgene, and by fast migration during electrophoresis of undigested DNA prepara- tions (Box 7, A) .
  • Karyotyping of recombinant schistosome strains is performed on interphase chromosomes from cercaria.
  • PCR directed to the repetitive pWl element can be used for Schistosom mansoni , (Webster P, Mansour TE, Bieber D, Isola ⁇ tion of a female-specific highly repeated Schistosoma mansoni DNA probe and its use in an assay of cercarial sex, Molecular and Biochemical Parasitology 36:217-22, 1989; Gasser RB, Morahan G and Mitchell GF, Sexing single larval stages of Schistosoma mansoni by polymerase chain reaction, Molecular and Biochemical Parasitology 47:255-58, 1991, both incor- porated herein by reference) (Box 7, B) .
  • Pilot studies utilize human growth hormone as a reporter gene, which has a well- characterized and sensitive assay system (Selden RF et al. , Molecular and Cellular Biology 6:3173-79, 1986) . Immunohisto-- chemistry and in si tu hybridization are also performed in order to confirm the location of expression within the worms.
  • breeding of transgenic clones may be necessary to ultimately obtain high-copy number clones of the appro ⁇ priate sex.
  • male cercaria are desired for human subject infection and are easily obtained in bulk from the appropriate male transgenic schistosome clones maintained in snails by sporocyst transfer. If the primary clone is female and fertile, then, to take advantage of this recombinant, a male transgenic clone must be selected from among the offspring of this clone after breeding with normal males by co- infection of the laboratory animal host.
  • the injected transgene vector contains the cDNA sequence of the desired transgene within the DNA context required to direct a high level of t:.ssue-specific expression, and the cDNA contains the signal sequences necessary to specify protein secretion.
  • Figure 2A shows how genomic sequences from a model schistosome gene (I) are used to produce the plasmid DNA construct containing the transgene (II) . In this strategy, genomic sequences are incorporated en bloc .
  • the upstream (a) and downstream (b) schistosome genomic fragments adopted to flank the transgene are each approximately 3.5 kb long, to include local promoter and enhancer sequences.
  • the model schistosome gene chosen has the desired pattern of tissue-specific expression in the integument, in the vitelline cells, in the Mehlis gland or in the ootype. To confirm that no other schistosomal genes are contained within these flanking sequences, Northern blot analysis of schistosome RNA . from all tissues and life cycle stages is performed using the flanking sequences as a probes, and open reading frames are found within the flanking sequences by DNA sequencing. Also adopted into the transgene construct are 5' (c) and 3' (d) untranslated regions of the model gene, to promote proper post-transcriptional and post-translational processing within the schistosome target location.
  • trans-splicing (Rajkovic A et al. , A spliced leader is present on a subset of mRNAs from the human parasite Schistosoma mansoni , Proceedings of the National Academy of Sciences (USA) 87:8879-83, 1990), and intracellular trafficking to secretory pathways.
  • the model gene coding sequence (e) is replaced by the transgene cDNA coding region (f) .
  • No intron is included in the transgene transcript (g) since most schistosome genes are intronless .
  • the amino-terminal signal peptide is derived from either the model gene or from the transgene.
  • the DNA vector is propagated within a bacterial plasmid. Plasmid sequences are not microinjected into schistosome eggs and are removed from the transgene vector portion of the plasmid construct by restriction digestion at rare cutting endonudease cloning sites (h, h' ) engineered into the plasmid. Following digestion at these sites, the DNA fragments are separated by agarose gel electrophoresis or gel filtration and purified by standard techniques.
  • Appropriate schistosomal model genes are those of highly- expressed tissue-specific genes.
  • the regulatory regions of tegmental antigen genes such as Sml5.9 (Abath F GC et al. , Structure of the gene encoding a putative Schistosoma mansoni tegumental antigen precursor, Molecular and Biochemical Parasitology 60:81-92, 1993), Sm21.7 (Francis P and Bickle Q, Cloning of a 21.7 kDa vaccine-dominant antigen gene of
  • Schistosoma mansoni reveals an EF hand-like motif, Molecular and Biochemical Parasitology 50:215-24, 1992) , Sm22.6 (Jeffs SA et al. , Molecular cloning and characterisation of the 22-kilodalton adult Schis tosoma mansoni antigen recognised by antibodies from mice protectively vaccinated with isolated tegumental surface membranes, Molecular and Biochemical Parasitology 46:159-68, 1991), or of the glucose transporter genes, the SGTP's (Skelly PJ et al.
  • flanking sequences from genes encoding eggshell proteins such as pl4 (Kunz WK et al., Sequences of two genomic fragments containing identical coding region for a putative eggshell precursor protein of
  • Schistosoma mansoni Nucleic Acids Research 15:5894, 1987; Koster B et al. , Identification of a putative eggshell precursor gene in the vitellarium of Schistosoma mansoni, Molecular and Biochemical Parasitology, 31:183-98, 1988) and p48 (Chen L, Rekosh DM, LoVerde PT, 1992) are used.
  • Targeting expression to the Mehlis gland and ootype is likewise per ⁇ formed with genomic sequences from Mehlis gland and ootype- specific genes, such as the those coding for Mehlis gland and ootype secretory products, cloned by tissue-specific differen- tial expression or subtractive hybridization approaches.
  • Figure 2B shows how analysis of the model gene promoter/ enhancer is used to increase the ability of the transgene to compete with the endogenous gene for transcription factors, thus reducing expression of the model gene while increasing expression of the transgene.
  • This approach is applied to the - eggshell model gene in particular, to reduce production of granuloma-provoking eggs.
  • the construct is graphically identical to the one in Figure 2A, except for the addition of tissue-specific core promoter elements (i) .
  • tissue-specific core promoter elements i
  • These sequences are identified in the model gene (I) promoter/enhancer region using standard DNAse protection and gel shift analyses and by sequence analysis for sequence motifs conserved among promoters of different eggshell genes as well as among different species or strains.
  • a core element (i) (about 10 base pairs long) , with its immediately-neighboring upstream and downstream sequences (about 80 base pairs long, in total) which likely contains binding sites for interacting transcrip ⁇ tion factors, is multimerized and reinserted into the promoter of the transgene expression construct (II) in its original location (e.g. in the proximal promoter) to increase tissue- specific expression of the transgene. It is also placed near the ends of the DNA construct to sop up tissue-specific transcription factors and reduce expression of the model (eggshell) gene.
  • the embodiment of this invention depicted in Figure 2B (a transgene expression construct with extra tissue-specific core promoter elements) , can be created without any preliminary experimentation.
  • the vitelline gland eggshell gene p48 is the source of upstream and downstream promoter/ enhancer regions. Based upon their presence in several similarly regulated vitelline-specific genes and upon evolutionary conservation to drosophila and silkmoth eggshell genes, several putative core promoter elements have already been identified (Chen et al. , 1992) . One could use the 80 base pair region from -335 to -255 as the repeated segment (i) . This sequence contains two putative core elements. One of these, "TCAGCT" (-278 to -273) is also found within the proximal promoters of the S.
  • flanking genomic sequences incorporated into the transgene vector are sorretimes sufficient to target gene expression appropriately, "position effect" can alter the expression pattern of a transgene. That is to say, depending upon the site of integration into genomic DNA, the transgene might be appropriately expressed, inappropriately expressed in undesired tissues, or not expressed at all. Usually, screening large numbers of recombinant organisms is sufficient to find a clone with the appropriate tissue-specific expression. Built-in higher order regulatory elements in the transgene vector can reduce the relevance of the site of transgene integration into the schistosome genome thus reducing the work to obtain the desired clones. Therefore, in one version of the transgene construct used in this invention, in addition to the nearby promoter and other flanking regulatory elements of the model gene, two types of distant cis-acting elements derived from the model gene are incorporated into the vector.
  • the first type is a locus control region (LCR) .
  • LCR locus control region
  • mansoni pl4 eggshell genes are clustered with two head-to-tail copies of one gene separated by a 7.5 kb region (Bobek LA, Rekosh DM, LoVerde PT, Small Gene Family encoding an eggshell (chorion) protein of the human parasite Schistosoma mansoni , Molecular and Cellular Biology 1988 8:3008-16, 1988), localized to chromosome 2 (Harai H, Tanaka M and LoVerde PT, Schistosoma mansoni : chromosomal localization of female-specific genes and a female-specific DNA element,
  • LCRs are important in their activation. LCR elements that regulate the model gene are identified based upon tissue-specific DNAse hypersensitivity pattern (Tuan D and London IM, Mapping of DNase I-hypersensitive sites in the upstream DNA of human embryonic epsilon-globin gene in K562 leukemia cells, Proceedings of the National Academy of Sciences (USA) 81:2718-22, 1984; Tuan D et al. , The "beta-like-globin" gene domain in human erythroid cells, Proceedings of the National Academy of Sciences (USA) 82:6384-88, 1985) . These are then incorporated into the transgene construct to further refine and support transgene expression.
  • LCR sequences Analysis of the genomic structure of the model gene is required to find long range cis-acting LCR sequences.
  • the search for these sequences begins at the proximal promoter of the model gene and proceeds in both the 5' and the 3' direction, to initially span up to lOOkb of DNA in either direction.
  • the search is confined to the region of the genome expressed in the tissue of interest, so each DNA segment is first used as a probe of Northern blots of schistosome RNA from the various tissues and life cycle stages. If, for example, a transcript that is expressed in the gut is identified 20 kb downstream of a model vitelline gland gene, then the LCR cannot lie beyond 20 kb downstream of the model gene.
  • the region to be assayed for DNAse hypersensitive sites can be delimited.
  • probes are generated near convenient restriction endonudease sites spaced every 2 to 5kb, and DNAse hypersensitivity assays are performed as described (Tuan et al PNAS, 1985, Tuan and London, PNAS, 1984) .
  • control chromatin from a region of the worm not expressing the model gene or from the opposite sex (for female-specific transcripts) is used to determine if the hypersensitivity is tissue-specific.
  • Non-tissue-specific DNAse hypersensitivity sites are putative boundary elements and serve to delimit the region searched for long range cis-acting sequences.
  • Figure 2C shows how a possible result of a search for an LCR is used to increase the activity of a transgene construct.
  • Boundary elements recognized by their non-tissue-specific pattern of DNAse hypersensitivity and by their ability to interact with specific protein factors (Zhao K, Hart CM and Laemmli UK, Visualization of chromosomal domains with boundary element-associated factor BEAF-32, Cell 81:879-89, 1995) , are sequences scattered throughout the genome which isolate genomic units, preventing an activated genomic region from affecting neighboring transcription units (Kellum R and Schedl P, A position-effect assay for boundaries of higher order chromoscmal domains, Cell 64:941-50, 1991.
  • Boundary elements flanking the model gene are identified as non-tissue-specific DNA hypersensitivity regions or they are identified on the basis of ability to function as insulator elements in drosophila (Kellum and Schedl, 1991) . Even copies of drosophila insulator/boundary elements (Udvardy A, Maine E, and Schedly P, The 87A7 chromomere: Identification of novel chromatin structures flanking the heat shock locus that may define the boundaries of higher order domains, Journal of Molecular Biology 185:341-358, 1985; Farkas G and Udvardy A Sequence of ses and ses' Drosophila DNA fragments with boundary function in the control of gene expression, Nucleic Acids Research 20:2604, 1992, incorporated herein by reference) could be used for this purpose, as the sequences display high evolutionary conservation, being functional in distantly related species (Chung JH, Whiteley M and Felsenfeld G, A 5' element of the chicken beta-globin domain serves as an insulator in human
  • boundary elements (1) from drosophila or identified during the search for LCR sequences
  • This step is to promote regulated transcriptional control by preventing inappropriate activation of schistosome genes near the site of transgene integration and by functionally isolating the transgene from its surrounding host chromatin.
  • LCR boundary elements
  • proximal promoter and flanking elements incorporated into the vector.
  • all of the sequences sufficient to confer integration-site- independent, copy-number-dependent and tissue-specific expression of the transgene are utilized. Not all of these elements are incorporated into each version of the vector, and simple vectors lacking distant LCR and boundary elements may prove to function adequately when numerous recombinants can be obtained.
  • the identification of LCRs should not be considered undue experimentation, although it may involve considerable work, because the techniques involved (genomic DNA subcloning and mapping, Northern blotting and DNAse hypersensitivity assays) are routine. Isolation of these regions is simply a matter of iteration. The ultimate determinant of vector adequacy rests upon assay of protein expression by transgenic adult worms.
  • the transgene vector construct can accommodate several kilobases of coding sequence with about 7 kb of flanking regulatory DNA.
  • the length of the regulatory region of the transgene vector is minimized in order to save room for transgene coding regions and in order to maximize the number of molecules of DNA tha" can be injected into the egg, to increase the probability of recombination.
  • Biochemical and functional assays are performed to obtain the smallest sufficient regulatory regions, retaining only minimally sufficient LCR and promoter elements. In biochemical assays the functional sequences of the regulatory cis-acting regions are highlighted by tissue-specific DNAse hypersensitivity patterns.
  • the minimally sufficient regions are further defined in vivo by testing their ability to regulate a reporter gene.
  • the cDNA for betagalactosidase could be incorporated into the vector, and the effectiveness of the regulatory sequences assessed by staining the adult worms with X-gal.
  • nuclease hypersensitivity studies require relatively pure cell preparations in order to identify the LCR and boundary elements. Vitelline gland cells are the predominant cell type in posterior female worm segments and in early eggs. Dissected tissue from these regions provides convenient source material for characterization of the eggshell genes.
  • Genomic sequences corresponding to tissue-specific genes of interest are obtained from a schistosome genomic DNA library (lambda phage, cosmid or phage Pl) using standard DNA hybridization techniques.
  • Hybridization probes include cDNA and genomic DNA obtained from cooperating investigators, generated via PCR using oligonucleotides based on published sequences and obtained from schistosome libraries. Some of the relevant cDNAs are identified by subtractive cDNA hybridization.
  • Northern blot hybridization is used initially.
  • Female worms are sectioned into anterior (Mehlis gland and ootype) region and posterior (vitelline gland) region, guided by in si tu fluorescent histochemical identification of the vitelline gland region (Bennet et al, 1978) .
  • mRNA from immature female worms from unisexual female infections and from eggs is isolated.
  • the subtracted cDNAs are sorted by cross-hybridization pattern to identify clones of the same gene and by Northern blot hybridization to the various schistosome mRNA preparations mentioned.
  • Probes from clones abundantly and specifically expressed in the vitelline gland, Mehlis gland and ootype regions are used for in si tu hybridization tc confirm the cell type of expression.
  • Such shell-deficient eggs could be broken down by the host without provoking granuloma formation, as evidenced by the effect of vitamin C deficiency, which prevents eggshell hardening and granuloma formation in infected animals (Krakower C, Hoffman WA, Axtmayer JH, Defective granular eggshell formation by Schistosoma mansoni in experimentally infected guinea pigs on a vitamin C deficient diet, Journal of Infectious Diseases 74:178-83, 1944) .
  • two types of genetic modifications of the schistosome are required: the first one interferes with eggshell production, the second introduces the transgene. This sequential task is accomplished by performing the first modification in male worms (which become asymptomatic carriers) and the second one in their female, affected progeny.
  • FIG. 3 shows a flowchart of the technique for creating transgenic schistosomes harboring a knockout construct for an eggshell gene or eggshell maturation enzyme.
  • Female worms paired with males in infected mouse intestines (A) , lay stage I eggs (B) in vivo or in vitro.
  • the stage I eggs are identified by a central fertilized egg that contains male and female pronuclei (i) , surrounded by vitelline cells (ii) and a thin eggshell (iii) .
  • Both male and female stage I eggs are microinjected with a knockout DNA construct because they cannot be distinguished at the time of microinjection.
  • This construct is designed to knock out an eggshell gene, an eggshell maturation enzyme (tyrosine hydroxylase or phenolase (see below) ) or a vitelline specific transcription factor (see below) .
  • Karyotyping is performed on the clones at the sporocyst stage in infected snails (C) , and female clones are discarded.
  • Male cercaria (D) harboring the knockout construct are used to infect mice (E) in co-infections with wild type female cercaria.
  • Normal female and male eggs harboring the eggshell knockout construct (F) are laid, because the vitelline cells are contributed by the wild type female parent.
  • transgenic female lines are obtained on a wild type strain background. If they are fertile, then they can simply be crossed with the male knockout construct-carrying line (D) by co-infection of mice, with selection of progeny female sporocyst clones harboring both the transgene and the knockout constructs.
  • RNA Knockout Vectors can be specifically designed. Antisense RNA is used naturally in diverse organisms to mediate destruction of complementary RNA strands, presumably by annealing with it and activating its digestion by RNAses (reviewed by Delihas N, Regulation of gene expression by trans-encoded antisense RNAs, Molecular Microbiology 15:411-14, 1995) . Hundreds of experiments have utilized this concept to down regulate specific messages, and the technique has proven highly useful for reducing biological activity of dozens of transcription factors in cell lines (e.g.
  • Reis LF et al., Critical role of a common transcription factor, IRF-1, in the regulation of IFN-beta and IFN-inducible genes, EMBO Journal 11:185-93, 1992) and in transgenic animals (e.g. Matsumoto K et al. , Evaluation of an antisense RNA transgene for inhibiting growth hormone gene expression in transgenic rats, Developmental Genetics, 16:273-77, 1995) and plants (e.g. Kuipers AG et al. , Factors affecting the inhibition by antisense RNA of granule-bound starch synthase gene expression in potato, Molecular and General Genetics 246:745-55, 1995) .
  • Antisense constructs are generated by incorporating the cDNAs to targeted transcription factor, tanning enzyme or eggshell protein genes in reverse orientation into the expression construct, as described below. Ribozyme constructs have also been shown to have biological utility, although it is unclear whether in practice, they are more effective than antisense RNA (James W and Al-Shamkhani A, RNA enzymes as tools for gene ablation, Current Opinion in Biotechnology, 1995 6:44-49, 1995) .
  • Ribozyme expression constructs have been used in transgenic animals to knockout targeted gene function (Zhao JJ and Pick L, Generating loss-of-function phenotypes of the fushi tarazu gene with a targeted ribozyme in Drosophila, Nature 365:448-51, 1993) . Ribozyme constructs are created with the hammerhead ribozyme incorporated into short regions homologous to targeted schistosome mRNA sequence, akin to antisense RNA constructs.
  • high level expression of the transgene in the vitelline cell directed by regulatory- sequences of an eggshell gene may reduce tannable eggshell protein below levels needed to make functional tannable eggshells.
  • the gravid female releases the fertilized ova in a sea of transgene product.
  • This high level expression is accomplished by the use of the most developed form of the vector, including boundary domain elements to flank the construct, and incorporation of relevant LCR-like and local enhancer and promoter elements to obtain position-independent, copy-number dependent expression, as outlined above in Figure 2D.
  • the endogenous eggshell genes are present in the genome in multiple copies but they are not amplified as are the chorion genes of drosophila.
  • the pl4 gene may only be present at about three copies per haploid genome of S. mansoni (Bobek et al, 1988 (see above)), and p48 is probably only present in a single copy, based on its low frequency in an unamplified genomic library (1 per 140,000 for p48 versus 6/100,000 for pl4, Bobek, 1988, Chen, 1992 (see above)) .
  • At least two of the pl4 genes are contiguous, separated by 7.5 kb of intergenic DNA, and arranged tail to head.
  • each transgene construct copy is transcribed at the same level as an endogenous pl4 or p48 eggshell gene, then it would require 80 copies of the transgene construct to reduce eggshell protein production by 90%.
  • This copy number is attainable by cross-breeding independent transgene integrants, considering that low transgene copy number clones are likely to remain fertile, because ipso facto they have preserved vitelline function. If an average transgenic schistosome carries five tandem copies of the transgene, then four generations of cross-breeding three independent clones and selection of progeny achieves this goal.
  • RNA-mediated "knockout" constructs of eggshell genes are unlikely to significantly interfere with expression of these genes in the wild type schistosome, since the target genes are expressed at high levels.
  • expression of such knockout constructs may act synergistically with the above strategy to reduce already low eggshell gene transcripts to even lower levels.
  • the third strategy for interfering with eggshell production involves targeted interference with eggshell maturation. The rationale for this is that the schistosome egg shell requires enzymatic tanning (cross-linking) in order to harden. This outer shell is produced from secretory products of the vitelline cells which envelop the egg in the worm uterus.
  • the main component of the eggshell is sclerotin, a proteinaceous material that has undergone a quinone-dependent tanning process (Nollen PM, Digenetic trematodes: Quinone tanning system in eggshells, Experimental Parasitology, 30:64-67,1971; Wharton DA, The production and functional morphology of helminth eggs shells, Parasitology, 86 Suppl :85-97, 1983 ; Smyth JD and Halton DW, The physiology of Trematodes, 2nd Ed.
  • the result of interfering with the eggshell tanning process is to prevent sclerotin hardening.
  • the residual non-polymerized eggshell protein is not expected to provoke disease, as mice treated with inhibitors of egg tanning do not develop hepatosplenomegaly (Bennett JL and Gianutsos G, Disulfuram: a compound that selectively induces abnormal egg production and lowers norepinephrine leveles in S. Mansoni , Biochemical Pharmacology 27:817-20, 1978) .
  • An additional benefit is that for transgene products secreted by vitelline cells, softening the eggshell can be expected to facilitate diffusion of the transgene product into the host bloodstream instead of potentially becoming entrapped in the eggshell matrix.
  • Cloning eggshell maturation genes The schistosome enzymes involved in this process have not yet been isolated. They are a putative tyrosine hydroxylase and a phenol oxidase.
  • the tyrosine hydroxylase gene is to be cloned based on the extensive evolutionary sequence conservation. For example, the tyrosine hydroxylase genes of the fruit fly Drosophila melanogaster (Nechameyer WS and Quinn WG, Neuron 2:1167-75, 1989) and of the cow (Saadat S et al. , J. Neurochemistry, 51:572-78, 1988) are 76% identical between drosophila amino acid number 291 and 451.
  • the drosophila sequence is used to probe at low stringency cDNA libraries made from female schistosomes or from early stage eggs, sources of vitelline mRNA.
  • degenerate oligonucleotide primers based on the most conserved regions are used to generate an RT-PCR product for subsequent high stringency cDNA library screening (see Appendix) .
  • a phenolase from Drosophila melanogaster involved in sclerotinization of the exoskeleton has been cloned by genetic means and its sequence has been published (Pentz ES and Wright TRF, Drosophila melanogaster diphenol oxidase A2 : gene struc ⁇ ture and homology with the mast-cell tum(-) transplantation antigen, P91A Gene 103:239-42, 1991) .
  • low stringency screening of a female schistosome cDNA library may identify the clone of interest.
  • expression cloning is used, with selection based upon enzymatic activity.
  • a bacteriophage cDNA expression library is constructed (e.g.
  • the phenolase and tyrosine hydroxylase are identified by subtractive hybridization, in which case schistosome tyrosine hydroxylase is recognized based upon predicted homology to other members of the family, and phenolase is recognized based upon homology to known oxidases.
  • the rDNAs encoding both of these enzymes must show high level expression in the vitelline cells. Tyrosine hydroxylase is likely to be expressed in neurons as well.
  • a third potential target in this pathway is an activator of tyrosine hydroxylase, which has been found in mammals as well as drosophila, and has been cloned (Swanson KD and Ganguly R, Characterization of a Drosophila melanogaster gene similar to the mammalian genes encoding the tyrosine/trypto ⁇ phan hydroxylase activator and protein, kinase C inhibitor proteins, Gene 113:183-90, 1992) . It is not presently known if a similar activator is required for schistosome tyrosine hydroxylase activity. In vivo interference with the mRNAs encoding eggshell maturation enzymes is to be performed via antisense RNA or ribozyme constructs.
  • the fourth strategy for interfering with eggshell production involves blocking vitelline cell transcription factors.
  • the most efficient way to reprogram vitelline cells to make the transgene product in place of eggshell proteins would be by manipulating expression of differentiated vitelline cell-specific transcription factors that coordinate expression of the gene products of differentiated vitelline cells. Typical transcription factors are expressed at levels orders of magnitude below many other gene products.
  • RNA-mediated knockout are most successful when the targeted moiety is expressed at low levels. (Cameron and Jennings, Antisense Res and Dev, 4:887-94, 1994) Therefore, RNA-mediated knockout strategies are expected to be more successful when applied to these regulatory genes than when directed towards the gene products they regulate.
  • vitelline droplets appearing in the last stage (Erasmus DA, Schistosoma mansoni : development of the vitelline cell, its role in drug sequestration and changes induced by Astiban, Experimental Parasitology 38:240-56, 1975) .
  • the orderly pattern of cellular differentiation suggests the existence of stage-specific transcription factors to coordinate gene expression, with eggshell and tanning mRNAs expressed as a result of activation of the transcription factors of the terminally differentiated cell.
  • the homologous promoter elements within the silkmoth and drosophila chorion genes and the Schistosoma mansoni eggshell genes Choen et al.
  • the fifth eggshell production interference strategy is dominant-negative interference with transcription factor activity.
  • Current understanding of functional domains of many transcription factors as predicted their by primary amino acid sequence suggests a way to interfere with their function at the protein level.
  • the transcriptional activation domain is identifiable as a negatively charged amphipathic helix or a proline and/or serine/threonine rich domain.
  • expression of a truncated version that lacks the essential transcriptional activation domain has been shown to inhibit the function of the endogenous wild-type factor by competition for target DNA elements (Langer SJ et al.
  • DNA-binding portion of the protein has been shown to inhibit the function of the wild-type molecule in a dominant-negative fashion (Logeat F et al., Inhibition of transcription factors belonging to the rel/NF- appa B family by a transdominant negative mutant, EMBO Journal 10:1827-32, 1991; Beckman H and Kadesch T, The leucine zipper of TFE3 dictates helix-loop- helix dimerization specificity, Genes and Development 5:1057-66, 1991), similar to the naturally found Id family (Benezra R et al.
  • the protein Id a negative regulator of helix-loop-helix DNA binding proteins, Cell 61:49-59, 1990) and to CHOP (Ron D, Habener JF, CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transc iption, Genes and Development 6:439-53, 1992) factors.
  • CHOP Non D, Habener JF, CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transc iption, Genes and Development 6:439-53, 1992
  • knockout of a vitelline cell transcription factor complicates the strategy for transgene expression in strategies 4 and 5 above.
  • the regulatory regions of the knockout construct are derived from a highly expressed, ubiquitous housekeeping gene.
  • the promoter of such a gene does not require a vitelline cell- specific transcription factor for activity.
  • Such a model gene could be one encoding a ubiquitous ion transporter (e.g. Na/K ATPase) or a glycolytic enzyme (e.g. triosephosphate isomerase (dos Reis MG et al.
  • step (1) eggs are microinjected with DNA from a plasmid construct containing a vitelline transcription factor knockout transcript (a) (i.e. ribozyme, antisense RNA, or dominant negative transcription factor) .
  • a vitelline transcription factor knockout transcript
  • This transcript is under control of promoter/enhancer and LCR regions (b and b' ) taken from a housekeeping gene.
  • step F a transgene whose expression is targeted to the Mehlis gland or ootype is microinjected, using DNA from a plasmid construct containing the transgene (c) , flanked by regulatory promoter/enhancer and LCR sequences (d and d' ) taken from a Mehlis gland or ootype-specific model gene.
  • a surrogate factor is designed to activate the transgene, and eggshell protein expression is usurped by transgene expression.
  • This is illustrated as a three step strategy of schistosome genomic modification in Figure 4B.
  • Step B schistosome eggs are microinjected with DNA from a knockout construct encoding a transcript (a) targeting a vitelline gland tissue-specific transcription factor (TFA) .
  • the knockout constructs designed to antagonize TFA could be activated by non-tissue-specific "housekeeping gene” regulatory sequences (b and b' in construct la) or by tissue-specific regulatory sequences (constructs lb and lc) .
  • the TFA knockout transcript (a) would be expressed in all cell types due to "housekeeping gene” promoter/enhancer sequences (b and b') , but it would be active only in mature vitelline cells, where it would encounter and inactivate TFA.
  • tissue-specific expression of the TFA knockout RNA (a) could be achieved by utilizing the promoter context of TFA itself (c and c' in construct lb) , which would assure synchronous expression, but not high levels of expression.
  • tissue-specific expression of the TFA knockout RNA (a) in the context of regulatory sequences from a downstream eggshell gene (d and d' ) would enable high level expression of the knockout construct, but it would also allow transient expression of the eggshell genes.
  • the cDNA for a transcript (e.i encoding a replacement transcription factor B (TFB) is inserted into a construct in the second step (2) utilizing the vitelline cell-specific regulatory sequences of either TFA itself (c and c' in construct 2a) or of an eggshell gene (d and d' in construct 2b) .
  • TFB is a chimeric protein consisting of TFA with its DNA- binding domain replaced by a DNA-binding domain with a different and known specificity, choosing a replacement DNA- binding domain of the same class (e.g. basic, zinc finger, helix-turn-helix, homeobox.) However, if a dominant-negative knockout is used, only the transcriptional activation domain of TFA is incorporated into TFB, to avoid heterodimerization with the dominant-negative protein. As shown in the shaded inset, in the transgene expression vector (construct 3) , all binding sites (e) for TFA within endogenous eggshell gene regulatory regions (exemplified by (f) ) are functionally replaced by adding adjacent TFB binding sites (g) .
  • construct 3 all binding sites (e) for TFA within endogenous eggshell gene regulatory regions (exemplified by (f) ) are functionally replaced by adding adjacent TFB binding sites (g) .
  • TFA sites within the regulatory region of the TFA knockout construct (lb and lc) and of the TFB expression construct (2a, 2b) are likewise replaced by TFB binding sites, as indicated by the grey and the black boxes in elements (c) and (d) in Figure 4B.
  • This step facilitates positive feedback regulation of the surrogate transcription factor TFB to mimic a likely aspect of TFA biology and stabilizes the TFA knockout expression.
  • This three step strategy is theoretically feasible for knockout of any terminal vitelline cell-specific transcription factor, barring the possibility that two or more necessary factors are both coordinately expressed and stabilize one another's expression.
  • the TFB expression construct can be injected at step F (see Figure 3) .
  • the double transgenic males (harboring both the TFA knockout and the TFB expression construct) are mated with normal females by co-infection of mice (step E, Figure 3) .
  • the transgene construct is injected into the ensuing eggs, and female clones harboring all three constructs are selected for use.
  • Upstream primers (based on drosophila TY3H amino acids 368-373)
  • Downstream primers (based on drosophila TY3H amino acids 428-433)

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Abstract

L'invention porte sur un procédé d'obtention de schistosomes servant de vecteurs intermédiaires de transgènes en vue de la sécrétion de produits géniques désirés. Lesdits produits sont sécrétés dans la circulation sanguine d'un patient par des schistosomes élaborés via la lignée germinale avec de l'ADN codant pour les transgènes. Le recours aux schistosomes comme vecteurs intermédiaires favorise la production en masse, le contrôle de qualité, l'arrêt de la thérapie quand on le veut, et le titrage des doses. Le procédé s'applique à des situations dans lesquelles la protéine ainsi obtenue exerce son activité fonctionnelle dans le plasma ou dans les vésicules endocytotiques.
PCT/US1996/015083 1995-09-21 1996-09-20 Procede d'expression et de secretion de transgenes dans des schistosomes WO1997011191A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000009731A1 (fr) * 1998-08-14 2000-02-24 Haldane Research Limited Parasites transgeniques utilises comme agents de therapie genique
WO2000032804A1 (fr) * 1998-12-01 2000-06-08 Yissum Research Development Company Of The Hebrew University Of Jerusalem Procedes permettant l'introduction stable en vrac et l'expression de genes etrangers dans des parasites eucaryotes
WO2002038752A3 (fr) * 2000-11-13 2002-09-26 Jonathan Kurtis Dispositif de liberation prolongee d'un agent bioactif et procedes de preparation et d'utilisation de ce dispositif
US8734807B1 (en) 2013-04-06 2014-05-27 Gabriel Langlois-Rahme Preventing and curing Schistosomiasis mansoni by inhibiting Trk receptors on female Schistosoma
WO2015172147A1 (fr) * 2014-05-09 2015-11-12 Research Foundation Of The City University Of New York Cassettes de régulation génique issues de la région tcr(alpha)-lcr

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0554064A1 (fr) * 1992-01-28 1993-08-04 Yeda Research And Development Co. Ltd. Vaccin contre la schistosomose (bilharziose

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0554064A1 (fr) * 1992-01-28 1993-08-04 Yeda Research And Development Co. Ltd. Vaccin contre la schistosomose (bilharziose

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000009731A1 (fr) * 1998-08-14 2000-02-24 Haldane Research Limited Parasites transgeniques utilises comme agents de therapie genique
WO2000032804A1 (fr) * 1998-12-01 2000-06-08 Yissum Research Development Company Of The Hebrew University Of Jerusalem Procedes permettant l'introduction stable en vrac et l'expression de genes etrangers dans des parasites eucaryotes
AU764284B2 (en) * 1998-12-01 2003-08-14 Yissum Research Development Company Of The Hebrew University Of Jerusalem Methods for bulk stable introduction and expression of foreign genes into eukaryotic parasites
WO2002038752A3 (fr) * 2000-11-13 2002-09-26 Jonathan Kurtis Dispositif de liberation prolongee d'un agent bioactif et procedes de preparation et d'utilisation de ce dispositif
US8734807B1 (en) 2013-04-06 2014-05-27 Gabriel Langlois-Rahme Preventing and curing Schistosomiasis mansoni by inhibiting Trk receptors on female Schistosoma
WO2015172147A1 (fr) * 2014-05-09 2015-11-12 Research Foundation Of The City University Of New York Cassettes de régulation génique issues de la région tcr(alpha)-lcr
EP3140407A4 (fr) * 2014-05-09 2017-11-08 Research Foundation Of The City University Of New York Cassettes de régulation génique issues de la région tcr(alpha)-lcr
US10053708B2 (en) 2014-05-09 2018-08-21 Research Foundation Of The City University Of New York TCR(alpha)-LCR-derived gene regulatory cassettes

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