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WO1997048810A1 - Procede de traitement de l'intolerance au lactose - Google Patents

Procede de traitement de l'intolerance au lactose Download PDF

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Publication number
WO1997048810A1
WO1997048810A1 PCT/US1996/010571 US9610571W WO9748810A1 WO 1997048810 A1 WO1997048810 A1 WO 1997048810A1 US 9610571 W US9610571 W US 9610571W WO 9748810 A1 WO9748810 A1 WO 9748810A1
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WIPO (PCT)
Prior art keywords
plant
plants
galactosidase
enzyme
transcriptional
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PCT/US1996/010571
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English (en)
Inventor
John A. Howard
Kenneth W. Culver
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Prodigene
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Priority to PCT/US1996/010571 priority Critical patent/WO1997048810A1/fr
Priority to AU63876/96A priority patent/AU6387696A/en
Publication of WO1997048810A1 publication Critical patent/WO1997048810A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • 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
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention involves the treatment of lactose intolerance in mammals. Specifically, the present invention involves the treatment of lactose intolerance in mammals by feeding directly to the mammal a composition comprising a transgenic plant having ⁇ -galatosidase.
  • Lactose (milk sugar) is a key nutrient in mammalian milk, comprising the major carbohydrate source during the neonatal period. Lactose is, from an evolutionary as well as from a biological viewpoint, a unique sugar as only in milk does it exist as a free molecule. It is synthesized by lactose synthetase, exclusively in the mammary gland of virtually all placental mammals during late pregnancy and lactation. Lactose concentration in milk is inversely related to the content of fat and protein; human milk contains the highest concentration (7%) of lactose.
  • Lactose is hydrolyzed to glucose and galactose by lactase, or more precisely, lactase-phlorizin hydrolase (LPH) , an intrinsic microvillus membrane glycoprotein with at least three characteristic enzyme activities: lactase ( ⁇ -D-galactoside galactohydrolase) , phlorizin hydrolase (phlorizin glucohydrolase) and glycosylceramidase.
  • LPH is one of the three enterocyte enzymes with ⁇ -galactosidase activity.
  • lysosomal acid ⁇ -galactosidase which probably does not contribute to dietary lactose hydrolysis; and a cytosolic ⁇ -galactosidase, reportedly with no specificity for lactose.
  • lactase activity is rate-limiting in the absorption of lactose.
  • the location of the enzyme on the villus-crypt axis makes it particularly sensitive to villus injury.
  • lactose activity is not directly regulated by availability of substrate.
  • human LPH differs from that in other mammals in which lactose-specific activity has been reported to increase in response to augmentation of the carbohydrate content of the diet. In such experiments, lactase-specific activity is thought to rise as a consequence of hyperphagia and reduced enzyme degradation.
  • Lactose intolerance is characterized by symptoms, as described above, after the ingestion of a test dose of lactose in water or milk.
  • lactose malabsorption is reserved for those patients in whom the intestinal malabsorption of lactose has been confirmed using an appropriate test of lactose absorption (lactose absorption test) or malabsorption (lactose breath hydrogen test). Lactase deficiency is defined only when a low level ( ⁇ 2 standard deviations below the mean) , or, very rarely, no level of lactase activity is found in a small intestinal biopsy sample appropriately assayed.
  • Lactase deficiency can be either a primary or secondary event.
  • Primary lactase deficiency occurs as a developmental process in premature infants, or as a rare clinical syndrome. It also appears as "late onset lactase deficiency" in the majority of the world's population around the age of five years.
  • Secondary lactase deficiency is found following mucosal injury.
  • the treatment of lactose intolerance has included four general principles: reduction or restriction of dietary lactose, substitution of alternative nutrient sources to avoid reduction in energy intake, regulation of calcium intake, and use of a commercially available enzyme substitute.
  • Calcium may be supplemented in the form of calcium carbonate. Turns® and OsCal® are somewhat effective. In infants, liquid calcium gluconate is readily tolerated. Live-culture yogurt, which contains endogeneous ⁇ - galactosidase, is sometimes used as a useful alternative source of both calcium and calories.
  • lactose preparations are actually bacterial or fungal ⁇ -galactosidases.
  • ⁇ - galactosidase will also hydrolyze lactose to glucose and galactose as the native lactase enzyme.
  • lactose-containing food or ingested with meals containing lactose these are somewhat effective in reducing symptoms. However, they are not capable of completely hydrolyzing all dietary lactose.
  • the efficacy of the commercially available products depends on the dose of lactose and enzyme consumed as well as the activity and survival of the microbial enzyme in the gastrointestinal tract. Thus the degradation of the carrier
  • the present invention is directed to a method of treating lactose intolerance in mammals which involves feeding a composition comprising a transgenic plant having ⁇ -galactosidase directly to the patient.
  • the enzyme in the transgenic plant can effectively metabolize lactose and has potency comparable to commercially available ⁇ -galactosidase products.
  • the present invention is also directed to a method of producing ⁇ -galactosidase in transgenic plants.
  • FIG. 1 represents plasmid pPHl7872.
  • FIG. 2 represents plasmid pPHI3667.
  • FIG. 3 represents plasmid pPHl7836.
  • FIG. 4 represents plasmid pPHI3630.
  • Lactose is hydrolyzed to D-glucose and D-galactose in the presence of the enzyme ⁇ -galactosidase and water.
  • An inability to convert lactose in the gut results in intestinal discomfort due to the conversion of the lactose by microorganisms to lactic acid and short chain fatty acids.
  • the present invention involves a method for treating mammals with lactose intolerance by feeding the mammal a transgenic plant that expresses the ⁇ -galactosidase gene.
  • Transgenic plants have been used to produce heterologous or foreign proteins. Some examples to date include the production of interferon in tobacco (Goodman, et al., 1987), enkephalins in tobacco, Brassica napus and Ababidopsis thaliana (Vendekerchove, et al., 1989), human serum albumin in tobacco and potato (Sijmons, et al. , 1990) antibodies in tobacco (Hiatt, et al., 1990) and hepatitis B antigen (Mason, et al., 1992).
  • transgenic plants or plant organs, preferably seeds are obtained in which ⁇ - galactosidase ("lacZ”) is produced.
  • an expression construct comprising a DNA sequence encoding the desired enzyme and regulatory sequences capable of directing the expression of the protein in the plant or seeds.
  • the expression construct provides for the stable transformation of the plant.
  • the transgenic plant or plant organ containing the desired enzyme is used as a practical oral delivery system of the enzyme to the patient.
  • the lacZ gene is operably linked to a regulatory sequence (capable of expression in the plant chosen) to obtain a vector, the plant is transformed by introducing the vector into the plant, the plants exhibiting successful transformation are selected, and those plants are regenerated. The regenerated plants are propagated, and plant tissue is extracted after selecting for fractions high in lacZ protein content.
  • the preferred embodiment of the present invention is a composition comprising transgenic plants or plant organs having an amount of the enzyme effective to provide protection against lactose intolerance.
  • protecting against lactose intolerance includes reducing or eliminating gastro-intestinal symptoms related to lactose ingestion such as bloating, vomiting, cramps, distention, flatulence, abdominal discomfort, nausea and diarrhea.
  • transgenic plants comprising lacZ can effectively be used to treat lactose intolerance in mammals via the oral route.
  • An expression cassette according to the present invention comprises a DNA sequence encoding lacZ operably linked to transcriptional and translational control regions functional in a plant cell.
  • DNA sequences coding for lacZ can be identified by referring to the published literature or searching a data base of DNA sequences, such as GenBank and the like. Once a DNA sequence coding for a selected enzyme is known, it can be used to design primers and/or probes that are useful in the specific isolation of a DNA or cDNA sequence coding for the enzyme. If a DNA sequence is known, primers and probes can be designed using commercially available software and synthesized by automated synthesis.
  • DNA sequence coding for lacZ can be operably linked to transcriptional and translational control regions by subcloning into an expression vector.
  • Transcriptional and translational control regions include promoters, enhancers, cis regulatory elements, polyadenylation sequences, transcriptional and translational initiation regions and transcriptional termination sequences.
  • the promoters are preferably those that provide for a sufficient level of expression of a heterologous gene to provide for a sufficient quantity of lacZ to treat a patient orally.
  • the preferred promoters are those that provide for a level of gene expression of about .01% to about 10% of the total cell protein. More preferred promoters provide about 0.1% to about 10% gene expression. Even more preferred promoters provide about 1% to about 10% gene expression.
  • Promoters can be inducible (such as heat shock promoters) , constitutive (such as 35s) or tissue specific
  • Transcriptional and translational control regions are typically present in expression vectors.
  • expression vectors are selected for compatibility and stability in the type of plant cell to be transformed.
  • Some expression vectors including promoters and the 3' regulatory regions are commercially available.
  • an expression cassette is formed and subcloned into an appropriate vector system, it can be transformed into suitable host cells.
  • suitable host cells include bacteria such as E ⁇ Coli, Agrobacterium tumefasciens, and plant cells or tissue such as corn suspension cultures, wheat callus suspension cultures, rice protoplasm, soybean tissue, sunflower tissue, alfalfa tissue, and other edible plant cells and tissue.
  • the expression system and vector selected is one that is compatible and stable in the selected host cell.
  • vectors are preferably selected to maximize stable integration of the foreign DNA into the plant cell genome.
  • Methods of transforming cells depend on the type of host cells selected.
  • methods of transformation include the freeze/thaw method, calcium phosphate precipitation, protoplast transformation, liposome mediated transformation and electroporation.
  • preferred methods of transformation include the freeze/thaw method, calcium phosphate precipitation, protoplast transformation, liposome mediated transformation and electroporation.
  • Agrobacterium mediated transformation direct transformation of protoplast using electroporation, or direct transfer into protoplast or plant tissue using microparticle bombardment, or combinations of these methods. These methods are well known to those skilled in the art.
  • Plant cells and tissues to be transformed include those plants useful as food such as alfalfa (Medicago sativa) , barley (Hordeum vulgare), beans (Phaseolus spp.), maize (Zea mays) , flax (Linum usitatissimum) , kapock (Ceiba pentandra) ,
  • Lentil (Lens culinaris), lespedeza (Lespedeza spp.), Lupine
  • the preferred plant species are maize, soybeans, sunflower, rapeseed, and alfalfa because these represent the major components of food and/or animal feed.
  • the protein is expressed in the seed of seed-producing plants such as sunflower. In those plants where the leaves are used as food or feed, leaf specific expression is preferred.
  • transgenic plants Once transgenic plants are obtained, they can be grown under appropriate field conditions until they produce seed. Presence of the DNA sequence coding for lacZ and expression of lacZ in the transgenic plant can be determined and quantitated.
  • An expression cassette encoding lacZ is preferably stably integrated into plant cell genome. An expression cassette is stably integrated when the gene can be detected repeatedly in future generations. The presence of the DNA sequence coding for the enzyme in the plant genome or chromosomal material can be verified and copy number can be quantitated using hybridization methods known to those of skill in the art. The level of gene expression can be quantitated using quantitative Western Blots, Elisas or enzymatic assays. Transgenic plants that are expressing the most enzyme as a percentage of the total plant cell protein are preferably selected for further propagation.
  • Transgenic plants can be selfed or crossed and the progeny plants evaluated for the presence of a DNA sequence encoding the enzyme and/or expression of the enzyme.
  • the especially preferred transgenic plants of the invention are those that can transmit the DNA sequence encoding the enzyme to the next generation of plants.
  • Transgenic seed can be collected from transgenic plants and the level of gene expression of the enzyme in the seed can be determined as described previously.
  • the level of gene expression of the enzyme in the seed is preferably that amount that provides for treatment of a mammal.
  • Transgenic seeds that express or contain the enzyme at about 0.01% to about 10% of the seed protein are preferably selected.
  • Transgenic plants, plant organs, and seeds can be combined into food or animal feed using methods and components known to those of skill in the art.
  • the amount of the transgenic plant, plant organ or seed material added to the food or feed material is that amount that provides sufficient lacZ to a patient to treat for lactose intolerance.
  • the amount of enzyme administered will vary depending upon the patient, the frequency of administration, and the condition of the patient.
  • Transgenic plant, plant organ or seeds containing lacZ can provide a low cost enzyme composition that is easy to administer and distribute.
  • the composition is administered orally to animals, preferably to domestic animals including but not limited to the cow, pig, horse, sheep, goat, and to humans.
  • the appropriate range or dose of the transgenic plant material and seed can be determined using standard methodology.
  • the range of dosages of the enzyme is preferably from about 3,000 to about 20,000 FCC units per dose, more preferably from about 4,000 to about 18,000 FCC units per dose, most preferably from about 6,000 to about 12,000 FCC units per dose.
  • the FCC unit is defined as l ⁇ M lactose cleaved per minute at 37°C and a pH of 4.5.
  • the transgenic plants or seeds can be administered by feeding to mammals in one or more discrete doses with meals or with pre-incubation in lactose-containing food.
  • An expression cassette for expression of lacZ in soybean is formed as follows.
  • the plasmid pPHI7872 as shown in Figure 1, is prepared.
  • This plasmid contains the phaseolin promoter and termination sequences.
  • lacZ that is engineered to contain a transit sequence from Brazil nut protein ("BNP") at the 5' end.
  • BNP Brazil nut protein
  • the cDNA of lacZ is used as a template for PCR.
  • the 5' primer includes the transitional sequence of BNP.
  • the resulting product is verified by DNA sequencing and cloned into the phaseolin expression cassette.
  • the open reading frame can be removed by cutting
  • DNA sequences coding for lacZ are well known. See e.g. Kalnins, A., et al., "Sequence of the lacZ Gene of Escherichia Coli,” EMBO J ⁇ ; Vol. 2; pp. 593-597; (1983); incorporated herein in its entirety by reference. Such DNA sequences are also obtained using standard techniques, as described in Maniatis, et al., A Guide to Molecular Cloning, Cold Spring Harbor, New York (1989); incorporated herein in its entirety by reference. Plasmids including the DNA sequence encoding lacZ are selected by examining the restriction digest patterns from plasmids that are isolated from cells growing on ampicillin.
  • Soybean (Glycine max) seed of Pioneer variety 9341, is surface sterilized by exposure to chlorine gas evolved in a glass bell jar. The gas is produced by adding 3.5 ml hydrochloric acid (34 to 37% w/w) to 100 ml sodium hypochlorite (5.25% w/w) . The seed is exposed for 16 to 20 hours in a container approximately 1 cubic ft in volume.
  • Overnight cultures of Agrobacterium tumefaciens strain LBA4404 (harboring the modified binary plasmid, 1680) are grown to log phase in minimal A medium containing tetracycline, 1 ⁇ g/ml. The cultures are pooled and an optical density measurement at 550 nanometers is taken. Sufficient volume of the culture is placed in 15 ⁇ iL conical centrifuge tubes, such that upon sedimentation between 1 and 2 x 10 10 cells are collected in each tube with 10 9 cells/ml. Sedimentation is performed by centrifugation at 6,000 x g for 10 min. After centrifugation, the supernatant is decanted and the tubes are held at room temperature.
  • Inoculations are conducted in batches such that each plate of seed is treated with a newly resuspended pellet of Agrobacterium. One at a time, the bacterial pellets are resuspended in 20 ml inoculation medium.
  • the inoculation medium consists of B5 salts (G5893) , 3.2 g/L; sucrose, 2.0% w/v; 6-benzylaminopurine (BAP) , 45 ⁇ m; indolebutyric-acid
  • IBA 0.5 ⁇ M
  • AS acetosyringone
  • IBA 0.5 ⁇ M
  • AS acetosyringone
  • the inoculum is then poured into a petri dish containing prepared seed and the cotyledonary nodes are mascerated with a surgical blade. This is accomplished by dividing seed in half by longitudinal section through the shoot apex preserving the 2 whole cotyledons. The two halves of the shoot apex are then broken off their respective cotyledons by prying them away with a surgical blade. The cotyledonary node is then mascerated with a surgical blade by repeated scoring along the axis of symmetry.
  • Explants are prepared in roughly 5 minutes and then incubated for 30 minutes at room temperature, with bacteria but without agitation. After 30 minutes, the explants are transferred into plates of the same medium solidified with Gelrite (Merck & Company Inc.), 0.2% w/v. Explants are embedded with the adaxial side up, leveled with the surface of the medium and cultured at 22°C for 3 days under cool white fluorescent light, approximately 20 ⁇ Em "2 S "1 .
  • the counterselection medium consists of B5 salts (G5893) , 3.2 g/1; sucrose, 2% w/v; BAP,
  • the explants are then picked to agarose-solidified selection medium.
  • the selection medium consists of B5 salts
  • the explants are again washed with the liquid medium on the gyratory shaker.
  • the wash is conducted overnight in the counterselection medium containing kanamycin sulfate, 50 ⁇ g/ml.
  • the explants are embedded in the agarose-solidified selection medium, adaxial side down, and cultured for another two week period.
  • the elongation medium consists of B5 salts (G5893) , 3.2 g/1; sucrose, 2% w/v; IBA, 3.3 ⁇ M; gibberellic acid, 1.7 ⁇ M; vancomycin, 100 ⁇ g/ml; cefotaxime,
  • the elongation medium is solidified with Gelrite, 0.2% w/v.
  • the green sectors are embedded, adaxial side up, and cultured as before. The culture is continued on this medium with transfers to fresh plates every two weeks.
  • the shoots are 0.5 cm in length, they are excised at the base and placed in rooting medium in 13 x 100 ml test tubes.
  • the rooting medium consists of B5 salts
  • the rooting medium is buffered to pH 5.7 with MES, 3 mM, and solidified with Gelrite at 0.2% w/v. After 10 days, the shoots are transferred to the same medium without IBA or PGA. The shoots are rooted and held in these tubes under the same environmental conditions as before.
  • the plantlet When a root system is well established, the plantlet is transferred to sterile soil mixed in plantcons. The temperature, photoperiod and light intensity remain the same as before.
  • An expression cassette encoding lacZ is used to generate transgenic sunflower plants and seeds.
  • the DNA sequence coding for lacZ is inserted into an expression cassette under control of the napin promoter for seed specific expression.
  • the expression cassette is present in a vector, such as plasmid pPHI3667, as shown in Figure 2.
  • the plasmid pPHI3667 has Ncol and Hpal cloning sites that provide for seed specific expression under control of the napin promoter.
  • a DNA sequence encoding lacZ is obtained as described in Example 1.
  • the DNA sequence is directionally subcloned into the Ncol and Hpal in pPHI3667. Plasmids having a DNA sequence encoding lacZ are selected, amplified and isolated from E. coli by DNA minipreps. See e.g. Maniatis, 1982.
  • This expression cassette is then subcloned into a binary vector, such as PHI 5765, using the EcoRI site. The binary vector is then transferred into an Agrobacterium tumefaciens helper strain.
  • Sunflower plants are transformed with Agrobacterium strain LBA4404 after microparticle bombardment, as described by Bidney, et al., Plant Mol. Bio.; Vol. 18; p. 301; 1992; incorporated herein in its entirety by reference. Briefly, seeds of Pioneer Sunflower Line SMF-3 are dehulled and surface sterilized. The seeds are imbibed in the dark at
  • the explants are bombarded twice with tungsten particles suspended in TE buffer, as described above, or with particles associated with plasmid pPHl3268. Some of the TE/particle bombardment explants are further treated with a Agrobacterium tumefaciens strain carrying pPHI3268 in 3667 by placing a droplet of bacteria suspended in the inoculation medium, OD 600-2.00, directly onto the meristem. The meristem explants are co-cultured on 374BGA medium in the light at 26°C for an additional 72 hours of co-culture.
  • Agrobacterium-treated meristems are transferred following the 72 hour co-culture period to medium 374 (374BGA with 1% sucrose and no BAP, IAA or GA 3 ) and supplemented with 250 ⁇ g/ml cefotaxime.
  • the plantlets are allowed to develop for an additional 2 weeks under 16 hour day and 26°C incubation conditions to green or bleach. Plantlets are transferred to Kan and grown until they develop seed. The presence of ⁇ -galactosidase in plants and seeds are confirmed and quantitated as described in Example 1.
  • a vector carrying a DNA sequence coding for the lacZ protein under control of a promoter functional in the plant is used to form transgenic maize.
  • a vector carrying a DNA sequence coding for lacZ is introduced into maize tissue or suspension cells by microparticle bombardment. This is a promoter/terminator selected for corn, well known to one skilled in the art.
  • a selectable marker such as BAR
  • BAR a selectable marker
  • pPHI7836 containing the PAT gene driven by the ubiquitin promoter and followed by the NOS terminator sequence, can be used.
  • germ cells are used including those derived from a meristem of immature embryos.
  • Suspension cell lines are used generate embryogenic suspension cultures.
  • embryogenic suspension cultures are derived from type II embryogenic culture according to the method of Green, et al., Molecular Genetics of Plants and Animals, editors Downey, et al., Academic Press, NY; Vol. 20; p. 147; (1983); incorporated herein in its entirety by reference.
  • the callus is initiated from maize inbreds designated R21 and B73 x G35. Both R21 and G35 are proprietary inbred lines developed by Pioneer Hi-Bred International, Inc., Johnston, Iowa. Suspension cultures of the cultivar "Black Mexican Sweet” (“BMS”) are obtained from Stanford University. The cultures are maintained in Murashige and Skoog (“MS”) medium as described in Murashige, et al., Physio. Plant; Vol. 15; pp. 453-497; (1962); incorporated herein in its entirety by reference; and supplemented with 2,4-dicholorophenoxyacidic acid (2,4-D) at 2 mg/L and sucrose at 30 g/L.
  • BMS Black Mexican Sweet
  • the suspension cultures are passed through a 710 micron sieve 7 days prior to the experiment and the filtrate maintained in MS medium.
  • cells are harvested from the suspension culture by vacuum filtration on a Buchner funnel (Whatman No. 614) .
  • the microparticles have a equal mixture of 3528 (selectable) and lacZ (non-selectable plasmid) DNA loading, applied by adding 1 ⁇ g per 10-100 ⁇ l of volume of DNA in TE buffer (at pH 7.7) to 25 ⁇ l of a suspension of 50 mg of tungsten particles per ml distilled water in a 1.5 ml Eppendorf tube. The particles become agglomerated and settle.
  • Embryo formation is then induced from the embryogenic cultures and germinated into plants.
  • a two culture medium sequence is used to germinate somatic embryos observed on callus maintenance medium.
  • Callus is transferred first to a culture medium (maturation medium) which, instead of a 0.75 mg/L, 2,4-D, has 5.0 mg/L indoleacetic acid (IAA) .
  • the callus culture remains on this medium for 10 to 14 days, while callus proliferation continues at a slower rate.
  • observation under a dissecting microscope indicates that somatic embryos have begun germinating, although they are white in color, because this culture phase is done in darkness.
  • the callus is transferred from the maturation medium to a second culture medium which further promotes germination of the somatic embryos into a plantlet.
  • This culture medium has a reduced level of IAA versus the first culture medium, preferably a concentration of about 1 mg/L.
  • the cultures are placed in the light. Germinating somatic embryos are characterized by a green shoot which elongates, often with a connecting root access. Somatic embryos germinate in about
  • the plants are about 7-10 cm tall, and are of sufficient size and vigor to be hardened to greenhouse conditions.
  • plants to be transferred to the growth chamber are removed from the sterile containers and solidified agar medium is rinsed off the roots.
  • the plantlets are placed in a commercial potting mix in a growth chamber with a misting device which maintains the relative humidity near 100% without excessively wetting the plant roots. Approximately 3 or 4 weeks are required in the misting chamber before the plants are robust enough for transplantation into pots or into field conditions. At this point, many plantlets, especially those regenerated from short term callus cultures, grow at a rate similar to seed derived plants.
  • Ten to fourteen days after pollination the plants are checked for seed set. If there is seed, the plants are then placed in a holding area in the green house to mature and dry down. Harvesting is typically performed 6 to 8 weeks after pollination.
  • This methodology is used successfully to regenerate corn plants expressing the chloramphenicol acetyltransferase gene under control of the 35S cauliflower mosaic virus (35S CaMV) promoter.
  • Direct introduction of foreign DNA into suspension culture or tissues of monocot plants are used successfully for regenerating transgenic monocot plants such as maize, wheat, rice and other grasses.
  • the DNA sequence coding for the lacZ is inserted into an expression cassette under control of the gamma zein promoter for seed specific expression.
  • the expression cassette is present in a vector such as a plasmid pPHI3630 as shown in Figure 4.
  • Plasmid pPHI3630 has a gamma zein promoter and termination sequences. Heterogeneous gene coding sequences are inserted between the Ncol and Hpal sites.
  • a DNA sequence coding for the lacZ protein is obtained as described in Example 1. This DNA sequence is inserted into the multiple cloning site at Ncol and Hpal in plasmid pPHI3630, using standard methods. Plasmid including a DNA sequence coding for the lacZ protein, under control of a seed specific promoter, is selected and isolated by examining the restriction patterns of the resulting plasmid and sequencing. Maize cells are transformed by microparticle bombardment as described above. Transformed cells containing a DNA sequence coding for the lacZ protein are identified and selected by PCR. Transgenic corn plants are regenerated and seeds obtained as described previously. Expression of lacZ protein in seeds is confirmed and quantitated by Elisa or Western Blot analysis. Stability of the expression of the lacZ protein is evaluated by these same methods over successive generations.
  • Lactose malabsorbers Healthy male and female subjects ranging in age from 25-60 years are classified as lactose malabsorbers on the basis of their increase in breath hydrogen concentration to >20 ppm after ingestion of 400 ml of 20 gms of lactose and/or an increase in serum glucose of >20 mg/dl within 120 minutes after ingestion of the lactose test dose. See generally: Collares, E.F., Belangero, V.M.S. and Da-Silva, P.E.M.R., "Gastric Emptying of Maltose, Sucrose, Lactose and Lactulose in Rats with Ontogenic Lactase Deficiency, " Brazilian J ⁇ Med. Biol. Res.; Vol. 24; pp.
  • Subjects are excluded if they are pregnant or lactating, had prior gastrointestinal surgery, had a recent illness, or had antibiotics within the last month.
  • the individuals are given each of six different treatments in random order in a double-blind fashion. All subjects receive 400 ml of 2% low fat milk containing 20 grams of lactose or glucose plus Group A, two soft-gel Vitamin E capsules and Group B, two soft-gel Lactaid® capsules and Group C, two caplets containing transgenic material.
  • the doses are based on optimal safety: efficacy ratios.
  • the subjects undergo an overnight 12-hour fast before receiving test material.
  • a baseline breath hydrogen level (end alveolar breath sample) and a blood glucose sample are obtained.
  • eight hourly breath samples are obtained following the consumption of the experimental material.
  • Blood samples are obtained 30,60,90 and 120 minutes after administration of lactose. Only water is allowed during the 8-hour breath collection period.
  • the changes in hydrogen concentration (DPPM) are calculated by subtracting the fasting hydrogen values from subsequent test values.
  • the subjects maintain a diary of symptoms of gas, stomach pain, cramps, diarrhea and loose stools on a zero to five scale (from no symptoms to severe symptoms) for each hour for each of eight hours following the test meals. The results indicate that the transgenic plant material is efficacious relative to standard therapy.

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Abstract

L'invention concerne des procédés de traitement de l'intolérance au lactose chez les humains, qui consiste à administrer directement au patient une composition comprenant une plante transgénique à β-galactosidase. L'invention concerne également des procédés de production de β-galactosidase dans des plantes transgéniques.
PCT/US1996/010571 1996-06-19 1996-06-19 Procede de traitement de l'intolerance au lactose WO1997048810A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US1996/010571 WO1997048810A1 (fr) 1996-06-19 1996-06-19 Procede de traitement de l'intolerance au lactose
AU63876/96A AU6387696A (en) 1996-06-19 1996-06-19 Method of treating lactose intolerance

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Application Number Priority Date Filing Date Title
PCT/US1996/010571 WO1997048810A1 (fr) 1996-06-19 1996-06-19 Procede de traitement de l'intolerance au lactose

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WO1997048810A1 true WO1997048810A1 (fr) 1997-12-24

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3718739A (en) * 1971-06-15 1973-02-27 Baxter Laboratories Inc Treating lactase deficiency with an active lactase
EP0449376A2 (fr) * 1990-03-23 1991-10-02 Gist-Brocades N.V. Production d'enzymes dans des semences et leur utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3718739A (en) * 1971-06-15 1973-02-27 Baxter Laboratories Inc Treating lactase deficiency with an active lactase
EP0449376A2 (fr) * 1990-03-23 1991-10-02 Gist-Brocades N.V. Production d'enzymes dans des semences et leur utilisation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
PEN, J., ET AL.: "Phytase-containing transgenic seeds as a novel feed additive for improved phosphorous utilization", BIOTECHNOLOGY, vol. 11, no. 7, July 1993 (1993-07-01), pages 811, XP002026203 *
SOLOMONS, N.W., ET AL.: "Dietry manipulation of postprandial colonic lactose fermentation: II. Additipn of exogenous, microbial beta-galactosidase at mealtime", AMERICAN JOURNAL OF CLINICAL NUTRITION, vol. 41, 1985, pages 209 - 221, XP000645072 *
SUNTIO, T. M. ET AL: "A new bifunctional reporter gene for in vivo tagging of plant promoters", PLANT MOLECULAR BIOLOGY REPORTER, (1994) VOL. 12, NO. 1, PP. 43-57. 31, XP000618865 *
TEERI, T.H., ET AL.: "Gene fusions to lacZ reveal new expression patterns of chimeric genes in transgenic plants", EMBO JOURNAL, vol. 8, no. 2, 1989, pages 343 - 350, XP002026202 *

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