Milk Of Transgenic Rabbits Research Articles

Isolation and some effects of functional, low-phenylalanine κ-casein expressed in the milk of transgenic rabbits

Patients suffering certain metabolic diseases (e.g. phenylketonuria) need a low-phenylalanine diet throughout their lives. Transgenic rabbits were created to express low-phenylalanine kappa-casein in their milk. The aim was to demonstrate for the first time the feasibility of producing a modified milk protein in addition to normal milk proteins. A gene construct containing the coding region of the rabbit kappa-casein gene was modified by site-specific oligonucleotide directed mutagenesis. Four of the five phenylalanine amino acids present in the mature protein were mutated and the gene construct was used to create two transgenic rabbit lines. The transgenic rabbits produced the recombinant kappa-casein at a high level in their milk causing a reduction in the average size of the casein micelles. The low-phenylalanine kappa-casein was digestible with chymosin and it was separated from its native counterpart and from the other milk proteins by a one-step HPLC method on a reversed-phase column. In the future, low-phenylalanine casein produced in transgenic animals could be used as dietary replacements to meet the special requirements of certain consumer groups.

High Level Expression of Tissue-Nonspecific Alkaline Phosphatase in the Milk of Transgenic Rabbits

Alkaline phosphatase is a promising therapeutic agent in the Gram-negative bacterial lipopolysaccharide (LPS) mediated acute and chronic diseases. Contrary to other alkaline phosphatase isozymes, purified tissue-nonspecific alkaline phosphatase (TNAP) is not available in large quantities from tissue sources, which would enable to analyse its efficacy in animal sepsis models. Two transgenic rabbit lines were created by pronuclear microinjection with the whey acidic protein promoter-humanTNAP minigene (WAP-hTNAP). Lactating females of both lines produced biologically active human TNAP. As indicated by fractionation of milk samples the recombinant alkaline phosphatase was associated with the membrane of milk fat globules. Alkaline phosphatase enzymatic activity was two orders of magnitude higher compared to normal human serum levels. The demonstration that this TNAP is physiologically active would provide the clue to use transgenic animals as bioreactor for bulk production of the human tissue-nonspecific alkaline phosphatase in milk. This may be a valuable and possibly viable option with important implication in attenuating LPS mediated inflammatory responses.

Production of Two Vaccinating Recombinant Rotavirus Proteins in the Milk of Transgenic Rabbits

Rotaviruses are the main cause of infantile viral gastroenteritis worldwide leading to approximately 500,000 deaths each year mostly in the developing world. For unknown reasons, live attenuated viruses used in classical vaccine strategies were shown to be responsible for intussusception (a bowel obstruction). New strategies allowing production of safe recombinant non-replicating rotavirus candidate vaccine are thus clearly needed. In this study we utilized transgenic rabbit milk as a source of rotavirus antigens. Individual transgenic rabbit lines were able to produce several hundreds of micrograms per ml of secreted recombinant VP2 and VP6 proteins in their milk. Viral proteins expressed in our model were immunogenic and were shown to induce a significant reduction in viral antigen shedding after challenge with virulent rotavirus in the adult mouse model. To our knowledge, this is the first report of transgenic mammal bioreactors allowing the rapid co-production of two recombinant viral proteins in milk to be used as a vaccine.

Production of Recombinant Human Protein C in the Milk of Transgenic Rabbits from the F3 Generation

The stability of transgene transmission, milk production and milk content of recombinant human protein C (rhPC) in transgenic rabbits of the F3 generation was determined. Transgenic rabbits carrying the 4.2kb mouse whey acid protein promoter and 9.4kb genomic human protein C were produced after mating transgenic (F2 generation) and non-transgenic rabbits. PCR analysis of F3 samples showed that the transgene was transmitted. Milk production, obtained at the 10th, 15th, 20th and 30th day of the first lactation by the weight-suckle-weight method showed no significant difference between transgenic and non-transgenic does. Concerning rhPC secretion, Western blotting detected a light chain (21 kDa) of rhPC in the milk and an ELISA test confirmed rhPC at the level of 0.109-0.301 microg/ml. A milk sample from a non-transgenic rabbit was hPC negative. In conclusion, the stability of hPC transgene transmission and production of rhPC protein C was confirmed in generation F3 of transgenic rabbits, with similar efficiency as in F1 and F2 generations, without reduction of milk production.

A phase I study of recombinant human C1 inhibitor in asymptomatic patients with hereditary angioedema

Hereditary angioedema (HAE) is a congenital disorder with recurrent attacks of localized swelling of submucosal tissue, subcutaneous tissue, or both caused by a deficiency of the plasma protein C1 inhibitor (C1 esterase inhibitor [C1INH]). We sought to evaluate the effects of recombinant human C1INH (rhC1INH) isolated from the milk of transgenic rabbits in 12 asymptomatic patients with HAE. rhC1INH was intravenously administered at doses of 6.25 to 100 U/kg on 2 occasions. rhC1INH appeared safe and was well tolerated. The course of functional C1INH in plasma showed a full initial recovery (dose-normalized maximum concentration of about 0.02 U/mL/U/kg) and a dose-dependent clearance of rhC1INH. After infusion of rhC1INH at 100 U/kg, a clearance of approximately 13 mL/min, a half-life of approximately 3 hours, and a volume of distribution of approximately 3 L were observed. Infusion at this dose led to functional C1INH levels in plasma of at least twice the normal level for about 2 hours and greater than 0.4 U/mL for about 9 hours. rhC1INH displayed dose-dependent biologic activity by increasing the C4 level, which was about 2-fold at 12 hours after rhC1INH at 100 U/kg, and decreasing levels of cleaved C4. The observed safety profile and biologic activity of rhC1INH warrants further clinical studies to assess its efficacy in treating HAE attacks.

Clinical studies of recombinant human C1 inhibitor in subjects with hereditary angioedema

RATIONALE: Recurrent attacks of subcutaneous and/or submucosal angioedema in subjects with hereditary angioedema (HAE) results from uncontrolled activation of the contact and complement systems due to a congenital deficiency of functional C1 inhibitor in plasma. Recombinant human C1 inhibitor (rhC1INH) was isolated from the milk of transgenic rabbits for replacement therapy in subjects with HAE. RhC1INH was highly similar to plasma-derived C1INH and appeared safe in preclinical studies.

Safety and efficacy of recombinant acid alpha-glucosidase (rhGAA) in patients with classical infantile Pompe disease: results of a phase II clinical trial

Pompe disease is an autosomal recessive muscle-wasting disorder caused by the deficiency of the lysosomal enzyme acid alpha-glucosidase. Due to virtual absence of acid alpha-glucosidase, patients with classical infantile Pompe disease develop progressive cardiomyopathy, skeletal muscle weakness and respiratory insufficiency leading to death in early infancy. We report on the results of a phase II clinical trial including two patients with classical infantile Pompe disease receiving enzyme replacement therapy over a period of 48 weeks by weekly infusions. Recombinant acid alpha-glucosidase was derived from the milk of transgenic rabbits. Safety was evaluated by recording adverse events while clinical efficacy was evaluated by ventilator-free survival, left ventricular mass index, motor development as well as histologic and biochemical analysis of muscle biopsies. This therapy was in general well-tolerated. There was an overall improvement in left ventricular mass, cardiac function, skeletal muscle function and histological appearance of skeletal muscle.

Influence of lactation parameters on the N-glycosylation of recombinant human C1 inhibitor isolated from the milk of transgenic rabbits.

The large-scale production of recombinant biopharmaceutical glycoproteins in the milk of transgenic animals is becoming more widespread. However, in comparison with bacterial, plant cell, or cell culture production systems, little is known about the glycosylation machinery of the mammary gland, and hence on the glycosylation of recombinant glycoproteins produced in transgenic animals. Here the influence is presented of several lactation parameters on the N-glycosylation of recombinant C1 inhibitor (rhC1INH), a human serum glycoprotein, expressed in the milk of transgenic rabbits. Enzymatically released N-glycans of series of rhC1INH samples were fluorescently labeled and fractionated by HPLC. The major N-glycan structures on rhC1INH of pooled rabbit milk were similar to those on native human C1 inhibitor and recombinant human C1 inhibitor produced in transgenic mouse milk, with only the degree of sialylation and core fucosylation being lower. Analyses of individual animals furthermore showed slight interindividual differences; a decrease in the extent of sialylation, core fucosylation, and oligomannose-type glycosylation with the progress of lactation; and a positive correlation between expression level and oligomannose-type N-glycan content. However, when large quantities of rhC1INH were isolated for preclinical and clinical studies, highly consistent N-linked glycan profiles and monosaccharide compositions were found.

A phase I study of recombinant human C1 inhibitor (RHC1INH) in asymptomatic patients with hereditary angioedema (HAE)

HAE patients experience recurrent episodes of localized swelling of submucosal and/or subcutaneous tissue. An attack is life-threatening when occurring in the larynx. HAE is caused by deficiency of functional C1INH. IV administration of human plasma-derived C1INH is the preferred treatment of severe abdominal, facial and laryngeal attacks. However, this product has limited availability for reasons relating to its origin, supply and cost. A preparation of recombinant human C1INH (rhC1INH) was developed from the milk of transgenic rabbits. In an open study, the tolerability, PK and biological effects of ascending iv doses of rhC1INH (range 6.25–100 U/kg) were investigated in 12 asymptomatic HAE patients, who received 2 infusions of rhC1INH after a washout of at least 5 weeks. Modelling of the rhC1INH concentration-time profile taking into account the endogenous production for 100 U/kg dose showed a clearance of 13 mL/min, a half-life of 3 h, a volume of distribution of 3 L, and an endogenous 'infusion rate' of 2 U/min. Clearance decreased dose-dependently indicating a saturable mechanism of elimination. At 100 U/kg the functional C1INH level was above the presumed protective level of 0.4 U/ml for about 9 h. The effects of rhC1inh were assessed by the inhibition of plasma C4bc formation that results from C4 cleavage. The magnitude and duration of the decrease in C4bc was dose-dependent. There was no indication that anti-C1 inh and anti-HRI increased or formation of neutralizing antibodies. rhC1inh was well tolerated (no allergic reactions) and showed the desired biological activity (C4 increases and inhibition of C4b/c formation) and will be tested in phase II studies in patients with severe attacks of angioedema. Clinical Pharmacology & Therapeutics (2004) 75, P52–P52; doi: 10.1016/j.clpt.2003.11.198

N- and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits.

Human C1 inhibitor (hC1INH) is a therapeutic N, O-glycoprotein with a growing number of clinical applications, but the current natural supplies are not likely to meet the clinical demands. Therefore, recombinant approaches are of interest, whereby specific attention has to be paid to the generated glycosylation patterns. Here, the N,O-glycoprotein was expressed in the mammary gland of transgenic rabbits and subjected to glycan analysis. After release of the N-glycans of recombinant-rabbit human C1 inhibitor (rhC1INH) by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the O-glycoprotein by centrifugal filtration, then fractionated by a combination of anion-exchange, normal-phase, and high-pH anion-exchange liquid chromatography. The O-glycans, released from the O-glycoprotein by alkaline borohydride treatment, were fractionated by anion-exchange high-performance liquid chromatography (HPLC). The structures of individual components were analysed by 500 MHz 1H NMR spectroscopy, in most cases combined with MALDI-TOF MS. In contrast to the structural data reported for native serum hC1INH, rhC1INH contained a broad array of different N-glycans, made up of oligomannose-, hybrid-, and complex-type structures. In the case of complex-type N-glycans (partially) (alpha2-6)-sialylated (N-acetylneuraminic acid only), mono- and diantennary chains were found; part of the diantennary structures were (alpha1-6)-core-fucosylated or (alpha1-3)-fucosylated in the lower or upper antenna (Lewis x). The manno-oligosaccharide pattern of part of the hybrid- and oligomannose-type structures indicates that besides the usual N-glycan processing route, also the alternative endo-mannosidase pathway is followed. The small core 1-type O-glycans showed the usual (alpha2-3)- and (alpha2-6)-sialylation pattern of O-glycoproteins of nonmucinous origin.

Expression and characterization of functional recombinant bovine follicle-stimulating hormone (boFSHalpha/beta) produced in the milk of transgenic rabbits.

Bovine follicle-stimulating hormone (boFSH) is a heterodimeric glycoprotein that belongs to the pituitary gonadotropins. Bioactive FSH is composed of alpha and beta subunits which require extensive N-glycosylation and sialylation. The mammary gland of transgenic livestock is an attractive source for the synthesis of post-translationally modified proteins. Two mammary gland-specific gene constructs with the cDNA for the boFSH alpha (boFSHalpha) and beta (boFSHbeta) subunits controlled by bovine alpha-s1 casein regulatory sequences were co-microinjected into fertilized rabbit oocytes. Two FSHalpha/FSHbeta double transgenic rabbit lines were established. The transgene expression was strictly lactation and mammary gland specific. Protein analysis revealed the presence of the boFSH heterodimer in the milk of transgenic rabbits showing a molecular weight similar to that of purified pituitary gland derived boFSH (boFSH-P). Subunit specific antibodies detected both polypeptides with the expected molecular sizes. Biochemical characterization demonstrated the expected isoelectric points of the recombinant boFSH. The presence of the post-translationally added terminal sialic acid residues was indicated by wheat germ agglutinin (WGA) lectin Western blotting. The biological activity of the recombinant mammary gland produced boFSH was determined using a FSH-dependent reporter cell line. The bioactivity of the recombinant boFSH was comparable to that of purified boFSH-P.

Enzyme therapy for Pompe disease: from science to industrial enterprise.

Pompe disease or glycogen storage disease type II (OMIM 232300) is a metabolic myopathy with a broad clinical spectrum. Generalised muscle weakness combined with cardiomegaly presents within the first 3 months after birth, if the lysosomal alpha-glucosidase (AGLU) deficiency is complete. Residual enzyme activity prevents cardiac involvement and delays onset of muscle weakness. Enzyme therapy, by intravenous administration of acid AGLU, aims to supplement the missing enzyme activity. At the SHS symposium on Glycogen Storage Diseases Type I and II, in Fulda, two interim accounts were given of studies on the efficacy of enzyme therapy for Pompe disease; one with recombinant human acid AGLU produced in Chinese hamster ovary cells and the other with the same enzyme produced in the milk of transgenic rabbits. this review focuses on the latter study, discusses the scientific, technological and commercial aspects of the enterprise, and addresses the prospects and challenges of enzyme therapy for Pompe disease.

Expression of a single βα chain protein of equine LH/CG in milk of transgenic rabbits and its biological activity

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked α- and β-subunits. eCG possesses the particularity to bind to both LH and FSH receptors in species other than horses and to have a prolonged plasma half-life. All these properties make it of utmost interest for livestock fertilization program. Up to now, the only source of eCG is the serum of pregnant mare. Rabbit mammary gland is considered as a system able to produce recombinant glycoproteins in sufficient quantity for pharmaceutical use. Here we described the production of a recombinant single βα chain of eLH/CG in the milk of transgenic rabbit. The construction of a single-chain permits to by-pass the problem of association–dissociation of the subunits. This recombinant hormone is greatly expressed (21.7 mg/l) and presents similar in vitro LH and FSH bioactivities. However, βαeLH/CG shows an extremely rapid clearance (≈10 min), which could explain the absence of in vivo biological activity. So the rabbit mammary gland is not appropriate for the production of a recombinant active eLH/CG.

Human acid alpha-glucosidase from rabbit milk has therapeutic effect in mice with glycogen storage disease type II.

Pompe's disease or glycogen storage disease type II (GSDII) belongs to the family of inherited lysosomal storage diseases. The underlying deficiency of acid alpha-glucosidase leads in different degrees of severity to glycogen storage in heart, skeletal and smooth muscle. There is currently no treatment for this fatal disease, but the applicability of enzyme replacement therapy is under investigation. For this purpose, recombinant human acid alpha-glucosidase has been produced on an industrial scale in the milk of transgenic rabbits. In this paper we demonstrate the therapeutic effect of this enzyme in our knockout mouse model of GSDII. Full correction of acid alpha-glucosidase deficiency was obtained in all tissues except brain after a single dose of i.v. enzyme administration. Weekly enzyme infusions over a period of 6 months resulted in degradation of lysosomal glycogen in heart, skeletal and smooth muscle. The tissue morphology improved substantially despite the advanced state of disease at the start of treatment. The results have led to the start of a Phase II clinical trial of enzyme replacement therapy in patients.

Production of biologically active salmon calcitonin in the milk of transgenic rabbits.

Salmon calcitonin (sCT) is an example of one of the many bioactive peptides that require amidation of the carboxy terminus for full potency. We describe a method for the production of amidated sCT in the mammary gland of transgenic rabbits. Expression of a fusion protein comprising human alpha lactalbumin joined by an enterokinase cleavable linker to sCT was directed to the mammary gland under the control of the ovine beta lactoglobulin promoter. C-terminal amidation in vivo was achieved by extending the sCT by a single glycine residue that provides a substrate for endogenous amidating activity in the mammary gland. Full characterization of the released sCT demonstrated it to be equivalent to synthetic standard in terms of structure, purity, and potency.

Recombinant human extracellular superoxide dismutase produced in milk of transgenic rabbits.

Expression of human extracellular superoxide dismutase (EC-SOD), a glycosylated, tetrameric metalloprotein, was targeted to the lactating mammary gland of transgenic rabbits. Efficient expression of the recombinant whey acidic protein/ec-sod gene was achieved and up to 3 mg ml-1 of the enzyme was secreted into the milk. Rabbit milk-produced recombinant EC-SOD was primarily found in the whey and purified by a two-step chromatographic method. To evaluate the rabbit milk-produced human EC-SOD, comparisons with native and Chinese hamster ovary cell (CHO)-produced EC-SOD were performed. All proteins were tetrameric and N-glycosylated. The behaviour on SDS-PAGE and size-exclusion chromatography indicated that the masses, and thereby the extent of post-translational modification of the proteins was similar. The monosaccharide composition of both recombinant EC-SOD variants was analysed and indicated similarities in the attached N-glycans on the two proteins. Furthermore, the peptide maps of the three EC-SOD variants revealed that all proteins had similar polypeptide backbones.

Expression of synthetic cDNA sequences encoding human insulin-like growth factor-1 (IGF-1) in the mammary gland of transgenic rabbits

We have developed an expression system where foreign proteins are synthesized specifically in the mammary gland of transgenic rabbits and secreted into the milk. Regulatory elements were isolated from the bovine α s1-casein-encoding gene and combined with a synthetic DNA coding for human IGF-1 and for [Gln 58] IGF-1, an IGF-1 analogue. The resulting hybrid DNA constructs were used to generate transgenic rabbits. Females of seven transgenic lines tested were positive for synthesis of IGF-1. Transmission of the transgene to progeny and IGF-1 production in female offspring was observed in all transgenic lines analysed. As expected, expression of transgene mRNA could only be detected in the mammary gland. Production levels oftransgenic protein were as high as 1 g IGF-1 per liter rabbit milk. IGF-1, as well as [Gln 58] IGF-1, when secreted into rabbit milk, was correctly processed and biologically active. IGF-1 was purified from the milk of transgenic rabbits to a nearly homogenous active form.

Rabbit beta-casein promoter directs secretion of human interleukin-2 into the milk of transgenic rabbits.

To test the potential usefulness of transgenic rabbits as production systems for human proteins of pharmaceutical value, we cloned the rabbit beta-casein promoter and fused it to the genomic sequence of the human interleukin-2 (hIL2) gene. Four transgenic female rabbits were tested for expression and biological activity of the foreign protein in their milk. The milk of all four females proved to contain biologically active hIL2. The results show that transgenic rabbits may represent a convenient and economic system for the rapid production of biologically active protein in milk.