Transgenic Livestock as Bioreactors: Stable Expression of Human Alpha-1-Antitrypsin by a Flock of Sheep

Carver, Andrew S.; Dalrymple, Michael; Wright, Gordon; Cottom, D.; Reeves, D.; Gibson, Yvonne; Keenan, Jayne L.; Barrass, J. David; Scott, Ann R.; Colman, Alan; Garner, Ian

doi:10.1038/nbt1193-1263

Cited by 49 publications

(31 citation statements)

References 19 publications

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“…Three recombinant proteins from milk are being tested in clinical trials: antithrombin III from goats, ␣-1-antitrypsin from sheep, and ␣-glucosidase from rabbits. [8][9][10] We have focused on the production of recombinant human ␣-glucosidase (rhAGLU) for the treatment of Pompe disease, an autosomal recessive muscle disorder. The classic infantile form of the disease leads to death at a median age of 6 to 8 months 11 and is diagnosed by absence of ␣-glucosidase activity and presence of fully deleterious mutations in the ␣-glucosidase gene.…”

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confidence: 99%

Long-Term Intravenous Treatment of Pompe Disease With Recombinant Human α-Glucosidase From Milk

et al. 2004

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ABSTRACT. Objective. Recent reports warn that the worldwide cell culture capacity is insufficient to fulfill the increasing demand for human protein drugs. Production in milk of transgenic animals is an attractive alternative. Kilogram quantities of product per year can be obtained at relatively low costs, even in small animals such as rabbits. We tested the long-term safety and efficacy of recombinant human ␣-glucosidase (rhAGLU) from rabbit milk for the treatment of the lysosomal storage disorder Pompe disease. The disease occurs with an estimated frequency of 1 in 40 000 and is designated as orphan disease. The classic infantile form leads to death at a median age of 6 to 8 months and is diagnosed by absence of ␣-glucosidase activity and presence of fully deleterious mutations in the ␣-glucosidase gene. Cardiac hypertrophy is characteristically present. Loss of muscle strength prevents infants from achieving developmental milestones such as sitting, standing, and walking. Milder forms of the disease are associated with less severe mutations and partial deficiency of ␣-glucosidase.Methods. In the beginning of 1999, 4 critically ill patients with infantile Pompe disease (2.5-8 months of age) were enrolled in a single-center open-label study and treated intravenously with rhAGLU in a dose of 15 to 40 mg/kg/week.Results. Genotypes of patients were consistent with the most severe form of Pompe disease. Additional molecular analysis failed to detect processed forms of ␣-glucosidase (95, 76, and 70 kDa) in 3 of the 4 patients and revealed only a trace amount of the 95-kDa biosynthetic intermediate form in the fourth (patient 1). With the more sensitive detection method, 35 S-methionine incorporation, we could detect low-level synthesis of ␣-glucosidase in 3 of the 4 patients (patients 1, 2, and 4) with some posttranslation modification from 110 kDa to 95 kDa in 1 of them (patient 1). One patient (patient 3) remained totally deficient with both detection methods (negative for cross-reactive immunologic material [CRIM negative]). The ␣-glucosidase activity in skeletal muscle and fibroblasts of all 4 patients was below the lower limit of detection (<2% of normal). The rhAGLU was tolerated well by the patients during >3 years of treatment. AntirhAGLU immunoglobulin G titers initially increased during the first 20 to 48 weeks of therapy but declined thereafter. There was no consistent difference in antibody formation comparing CRIM-negative with CRIMpositive patients. Muscle ␣-glucosidase activity increased from <2% to 10% to 20% of normal in all patients during the first 12 weeks of treatment with 15 to 20 mg/kg/week. For optimizing the effect, the dose was increased to 40 mg/kg/week. This resulted, 12 weeks later, in normal ␣-glucosidase activity levels, which were maintained until the last measurement in week 72. Importantly, all 4 patients, including the patient without any endogenous ␣-glucosidase (CRIM negative), revealed mature 76-and 70-kDa forms of ␣-glucosidase on Western blot. Conversion of the 110-kDa precurs...

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confidence: 99%

Long-Term Intravenous Treatment of Pompe Disease With Recombinant Human α-Glucosidase From Milk

et al. 2004

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“…These promoters have behaved differently across animal species. The most stable expression patterns have been observed throughout multiple lactations in transgenic animals that contain a single integration locus Carver et al, 1993). In most cases, the biosynthesis of both the recombinant and endogenous proteins has been compatible with normal mammary gland physiology.…”

Section: Transgenic Livestock As An Alternative Source Of Recombinantmentioning

confidence: 83%

“…Mosaicism and multiple integration sites frequently occur in founder animals and this complicates analysis of founder animals (Velander et al, 1992b). Thus, phenotype and genotype can not be reliably defined in transgenic animals until successive generations of offspring obtained from outbreeding with nontransgenic animals are analyzed Velander et al, 1992b;Carver et al, 1993). A production lineage must be shown to reproducibly secrete the recombinant protein at a level and functionality which can be associated with a given trans-genotypic signature.…”

Section: Transgenic Livestock As An Alternative Source Of Recombinantmentioning

confidence: 99%

Production and secretion of recombinant human fibrinogen by the transgenic murine mammary gland

Butler¹,

O'Sickey²,

Lord³

et al. 1999

Theriogenology

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The mammary gland of lactating transgenic animals has several advantages for production of heterologous proteins including a high cell density that results in high concentrations of secreted protein and the ability to perform several types of post-translational modifications. Transgenes were constructed from the 4.1 kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the A", B$ and ( fibrinogen chains to evaluate the requirements of the transgenic murine mammary gland for high level secretion of fully assembled human fibrinogen. After introducing the constructs into the murine zygotes by microinjection, secretion of fully assembled fibrinogen into milk was measured at concentrations between 10 :g/ml to 200 :g/ml.In one line of mice the total secretion of fibrinogen and unassembled subunits approached 700 :g/ml in milk. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the B$ and ( chains were rate limiting. Also, the subunit complexes ( 2 , A"( 2 and the individual subunits A", B$ and ( were found as secretion products. This is the first time that secretion of individual B$-subunits by any cell type has been reported and suggests the organization of the secretion pathway in mammary epithelia is different from that in liver. Glycosylated forms of individual B$-chain contained a complex saccharide with low mannose. Glycosylation of the (-chain was also observed. These results suggest the 4.1 mWAP promoter can drive expression of fibrinogen cDNAs to high levels and that the amount of fully assembled fibrinogen secreted is equal to the level of the lowest expressing chain.iii

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“…The possibility that our transgenic line had multiple insertion sites can be excluded because the offsprings had essentially same copy numbers as their founder mice. It was also reported that genetic background of the normal mouse mated with the transgenic animals affected the expression level [24]. We used only F1 hybrid (C57BL/6 × DBA) to get transgenic offspring.…”

Section: Discussionmentioning

confidence: 99%

Expression Analysis of a Bovine .BETA.-Casein/Human Lactoferrin Hybrid Gene in Transgenic Mice.

Kim

Lee

et al. 1997

Journal of Reproduction and Development

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Abstract. In our previous study, using a bovine β-casein/human lactoferrin hybrid gene, transgenic mice were generated to target the expression of human lactoferrin (hLF) into the mammary glands. Here, we report the fidelity of transgene expression by eleven transgenic mice and their progeny. Expression of hLF in lactating mice was demonstrated by northern and western blots, ELISA, and immunohistochemical staining. Northern blot analysis in five different tissues revealed that the transgene was expressed in the mammary gland. hLF was expressed in five of eight transgenic mouse lines analyzed by ELISA. The expression level of hLF in milk at day 10 of lactation ranged from 2.5 to 200 µg/ml. The apparent molecular weight of the recombinant hLF was indistinguishable from the native form upon immunoblotting. Immunohistochemical staining revealed that hLF in transgenic mice was exclusively expressed in the epithelial cells of mammary gland. The transgenic mice produced in this study provide a model for the eventual generation of transgenic farm animals producing high levels of hLF in their milk.

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Transgenic Livestock as Bioreactors: Stable Expression of Human Alpha-1-Antitrypsin by a Flock of Sheep

Cited by 49 publications

References 19 publications

Long-Term Intravenous Treatment of Pompe Disease With Recombinant Human α-Glucosidase From Milk

Long-Term Intravenous Treatment of Pompe Disease With Recombinant Human α-Glucosidase From Milk

Production and secretion of recombinant human fibrinogen by the transgenic murine mammary gland

Expression Analysis of a Bovine .BETA.-Casein/Human Lactoferrin Hybrid Gene in Transgenic Mice.

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