Patent Application
20050229261
1. Field of the Invention
The present invention discloses a method for producing biologically active recombinant proteins in the milk of transgenic animals, characterized in which intact human clotting factor VIII gene and B-domain deleted recombinant factor VIII gene are transferred into the mammal by gene microinjection and embryonic implantation to obtain expression and secretion in the milk of transgenic animals and their offsprings.
2. Description of the Prior Art
Hemophilia is the most common human bleeding disorder and affects approximately one in 5,000 males, causing lifelong, repeated, and potentially life-threatening hemorrhagic episodes. It results from a deficiency in functional blood coagulation factors, either in a serine protease called Factor IX (hemophilia B) or in its cofactor Factor VIII (hemophilia A). Because hemophilia is caused by defects in single X-linked genes that encode circulating plasma proteins, the development of therapeutic strategies has focused on finding ways to replace the defective or deficient proteins.
Human FVIII is synthesized primarily in the liver and secreted into the circulation at low plasma concentration, approximately 100-200 ng/ml. Analysis of the cloned cDNA for human FVIII (Gitschier et al., 1984, Nature 312: 326-330; Toole et al., 1984, Nature 312: 342-347.) has provided more information about its gene and protein structure. The full length FVIII cDNA contains an open reading frame coding for a polypeptide of 2351 amino acids. This primary translation product contains a 19 a.a. signal peptides and six homologous domains in the order A1-A2-B-A3-C1-C2 (Vehar et al., 1984, Nature 312: 337-342.). The 256-kDa precursor protein is proteolytically processed intracellularly to a metal ion-linked heterodimer of a 90- to 200-kDa heavy chain (A1-A2-B) and an 80-kDa light chain (A3-C1-C2), which circulates in the plasma bound to the von Willebrand factor (vWF).
For the past two decades hemophiliacs have been treated with whole plasma, and more recently with highly purified FVIII concentrates prepared from plasma. Higher concentrates purity allows the administration of effective doses at lower volumes to patients. This therapy is effective in controlling bleeding episodes, however, hemophiliacs may suffer from other complications, compromised by the discovery that these products were transmitting human viruses such as the hepatitis B virus and human immuno-deficiency virus (HIV) to patients, many of them later developed diseases (Hoyer, 1993, Methods Enzymol. 222: 169-176).
Since then, plasma screening and viral inactivation procedures have greatly improved the safety of these products, although the potential for transmitting diseases such as Creutzfeldt-Jakob disease must be considered and has led to sporadic shortages in these plasma products. Nonetheless, when non-blood source products derived from recombinant DNA biotechnology became available, further reducing the viral risk, they were quickly embraced by caregivers and patients, despite their higher cost (Pipe and Kaufman, 2000, Nature Biotech. 18: 264-265). One of the challenges created by the biotechnology revolution is the development of methods for the economical production of highly purified proteins in large scales. Recent developments indicate that manipulating milk composition using transgenesis has focused mainly on the mammary gland as a bioreactor to produce pharmaceuticals.
The ability to modify animal genomes through microinjection technology has offered new alternatives for the manufacture of recombinant proteins. Targeting the production of human recombinant protein pharmaceuticals in the milk of transgenic animals solved many problems associated with either microbial or animal cell expression systems. Bacteria often improperly fold complex proteins, introducing more involved and expensive processes. Both bacteria and yeast lack adequate post-translational modification. Bioreactors for cell cultures require high capital expenditures, use large volumes of expensive culture media, and often suffer from relatively low yields.
To express a recombinant protein in the milk of a transgenic animal, expression vectors containing a gene encoding the protein of interest fused to milk specific regulatory promoter elements are generally introduced by microinjection into a pronuclear-stage embryo, or alternatively transfecting the expression vector into a cell line suitable for somatic cell nuclear transfer. Most of this work has been carried out with: the ovine β-lactoglobulin, rodent (mouse, rat and rabbit) whey acid protein (WAP) genes, bovine α-lactalbumin and a-s1-casein genes, as well as the caprine β-gene.
Several blood proteins, hormones, and enzymes have been synthesized in transgenic animals. Of these, human FVIII is probably the largest and most complex protein to be expressed. Analysis of the cloned cDNA for human FVIII has provided more information about its gene and protein structure. The full length FVIII cDNA contains an open reading frame coding for a large polypeptide of 2351 amino acids. The 256-kDa precursor hFVIII protein is proteolytically processed intracellularly to a metal ion-linked hetero-dimer of a 90- to 200-kDa heavy chain (A1-A2-B) and an 80-kDa light chain (A3-C1-C2), which circulates in the plasma bound to the von Willebrand factor (vWF). The B-domain, is encoded by a single large exon and is highly glycosylated, harboring 19 of the 25 N-linked glycosylation sites. The B-domain is released upon co-factor activation, so it is not necessary for clotting function.
According to the regulatory sequences employed, variable levels of expression for the recombinant human FVIII were observed. For example, using a construct containing 2.2-kb ovine β-lactoglobulin 5′-flanking sequences to drive the FVIII cDNA with the introns of the murine metallothionein I (β-Lac/hFVIII-MtI), only extremely low level (4-6 ng/ml) of expression was observed in transgenic sheep (Niemann et al., 1999. Transgenic Res 8: 237-247). Whereas the hFVIII cDNA, under the control of the 2.5-kb mouse WAP promoter, was shown to secrete up to 2.7 μg/ml in transgenic pig milk (Paleyanda et al., 1997. Nature Biotech 15: 971-975).
In this invention, we describe a novel recombination of promoter, leader sequence, coding sequences and poly A tail signaling sequence as a expression cassette to generate of transgenic animals expressing the fusion gene in the mammary gland and the presence of recombinant FVIII protein ranged from 7 to 50 μg/ml with over 35- to 200-fold higher than in normal human plasma, with temporal and spatial expression profiles for clotting active rFVIII in the milk of transgenic animals in different stages of lactation.
The invention relates to transgenic milk that can be used to produce therapeutical recombinant hFVIII for hemophilia patent treatment.
Using mammary gland-specific promoters, a wide range of proteins of bio-pharmaceutical interest have been expressed in rodents, pigs, and dairy animals (Echeland, 1996, Current Opinion in Biotechnology 7: 536; Houdebine et al., 2000, Transgenic Research 9:505). An expression vector, comprising a gene encoding the target protein of interest fused to a milk promoter gene, is introduced by microinjection into the pronucleus of a one-cell embryo. Upon germ line integration and expression, the transgene becomes a dominant Mendelian genetic characteristic that is inherited by the progeny of the founder animal. Mammalian mammary epithelial cells have the capacity to carry out complex protein synthesis with a variety of posttranslational modifications and folding. The milk of transgenic livestock presents a starting material from which human diagnostic or pharmaceutically-active and therapeutic proteins may be purified using established technologies.
Milk Specific Promoters
A variety of transcriptional promoters that preferentially activate transcription in mammary epithelial cells are available. These include the promoters that control the genes encoding milk proteins such as caseins (αS1-, αS2-, β-, γ-, and κ-casein), beta-lactoglobulin (Clark et al., 1989, Bio/Technology 7: 487-492), whey acid protein (Gordon et al., 1987, Bio/Technology 5: 1183-1187), and alpha-lactalbumin (Soulier et al., 1992, FEBS Letters 297: 13). Casein promoters may be derived from the alpha, beta, gamma or kappa casein genes of any mammalian species; a preferred promoter is derived from the goat beta casein gene (DiTullio, 1992, Bio/Technology 10:74-77).
The DNA sequences of many these promoters are available, e.g., in GenBank and in scientific publications such as 1) rat alpha-lactalbumin (Richards et al., 1981, J. Biol. Chem. 256: 526-532); 2) rat WAP (Campbell et al., 1984, Nucleic Acids Res. 12: 8685-8697); 3) rat alpha-casein (Jones et al., 1985, J. Biol. Chem. 260: 7042-7050); 4) rat alpha-casein (Lee and Rosen, 1983, J. Biol. Chem. 258: 10794-10804); 5) human alpha-lactalbumin (Hall, 1987, Biochem. J. 242: 735-742); 6) bovine alpha-S1 casein (Stewart, 1984, Nucleic Acids Res. 12: 389); 7) bovine alpha-casein (Gorodetsky et al., 1988, Gene 66: 87-96); 8) bovine alpha-casein (Alexander et al., 1988, Eur. J. Biochem. 178: 395-401); 9) bovine alpha-S2 casein (Brignon et al., 1977, FEBS Letters 188: 48-55); 10) bovine alpha-lactoglobulin (Jamieson et al., 1987, Gene 61: 85-90; Alexander et al., 1989, Nucleic Acids Res. 17: 6739). For additional regulatory control or stringency, other regulatory sequences such as adjacent sequences, can be obtained from these genes or from homologous genes of other mammals using these promoter sequence as probes to screen genomic libraries.
Signal Peptide Sequences
The recombinant gene construction can also include a signal sequences, particularly a signal peptide sequences of a milk specific gene. For the best secretion efficiency of forigen protein in the mammary gland, the milk-specific signal peptide sequence can be the secretional signal sequence which naturally occurs with the selected milk-specific promoter used in the construct, which are described below. For example, signal sequences from genes coding for caseins, e.g., alpha, beta, gamma or kappa caseins, beta lactoglobulin, whey acid protein, and lactalbumin can be used.
Transgenesis Constructions
A 2.0-kb promoter sequence of bovine alpha-lactalbumin (αLA) was generated by PCR amplification using a genomic DNA from high milk-producing Holstein cow, which obtained from National Taiwan University Farm, as the template. This PCR product containing entire αLA promoter and 19-aa leader sequence (SEQ ID: No. 1) or replacment with bovine αS1-casein 15-aa signal peptide sequence (SEQ ID: No.2) which created a restriction enzyme, HpaI, cloning site in the downstream sequence. These two types of promoter and signal peptide sequences were then subsequently inserted into the pCR3 vector (Invitrogene, San Diego, Calif.).
For the full-length hFVIII construction, the resulting 7.0-kb plasmid containing αLA promoter and its intact signal peptide sequence was double digested with MluI and PstI and treated with calf intestinal phosphatase (Boehringer Mannheim, Indianapolis, Ind.). The pCMV5/hFVIII plasmid containing the intact human FVIII coding sequence was also double digested with MluI and PstI. The in frame sequence from the αLA leader peptide through the hFVIII junction was determined using the Dye Terminator sequencing system (Applied Biosystems Inc., Foster, Calif.). The 9.7-kb transgene consisting of 2.0-kb bovine αLA promoter, 7.2-kb hFVIII cDNA, and 0.5-kb bovine GH gene polyadenosine signal sequence was separated from plasmid pCR-αLA/hFVIII-5 using ClaI and XbaI digestion and purified for microinjection on the twice CsCl2 gradient ultra-centrifugation.
For the B domain-deleted hFVIII construction, the resulting 7.0-kb plasmid containing αLA promoter and αS1-casein signal peptide sequence was double digested with HpaI and XhoI and treated with calf intestinal phosphatase (Boehringer Mannheim, Indianapolis, Ind.). The pCMV5/hFVIII plasmid containing the intact human FVIII coding sequence was used as template to generate a 2233-bp rhFVIII A-domain fragment using degenerate PCR, phFVIII-HpaI(+): 5′-GGT TAA CTG CCA CCA GAA GAT A-3′ (SEQ ID: NO.3) and phFVIII-741aa(−): 5′-AAG CTT CTT GGT TCA ATG GC-3′ (SEQ ID: NO.4), and a 2085-bp rhFVIII C-domain fragment amplified by phFVIII-1643aa(+): 5′-AAG CTT GAA ACG CCA TCA ACG GGA A-3′ (SEQ ID: NO.5) and phFVIII-XhoI(−): 5′-CTC GAG CCT CAG TAG AGG TCC TGT-3′ (SEQ ID: NO.6), respectively. Equal molar ratio of A-domain segment, C-domain segment and pCR3-αLA vector were co-ligated and transformed into host competent cells. The in frame sequence from the αS1-casein leader peptide through the hFVIII junction was determined using the Dye Terminator sequencing system (Applied Biosystems Inc., Foster, Calif.). The 6.8-kb transgene consisting of 2.0-kb bovine αLA promoter and αS1-casein leader sequence, 4.3-kb hFVIII cDNA, and 0.5-kb bovine GH gene polyadenosine signal sequence was separated from plasmid pCR-αLA/hFVIII(ΔB) using ClaI and XbaI digestion and purified for microinjection on the twice CsCl2 gradient ultra-centrifugation.
Transgenic Animal Productions
The purified transgene was microinjected into the male pronuclei of fertilized eggs from superovulated female mice of the outbreed ICR strain and transferred to recipient pseudo-pregnant females as previously described (Chen et al., Transgenic Res 4: 52-59; 1995). For transgenic goat production, the pronuclear stage embryos were flushed from the donor goat's oviduct at the one and half day after insemination by means of surgical method. In order to obtain higher embryo numbers, every embryo-donating goat had been treated with endocrine so as to achieve the object of superovulation. Such a superovulation treatment comprised of administrating intramuscular of follicular stimulating hormone (FSH) to the embryo-donating goat sequentially for 4 days since eighth day of estrous cycle twice a day with interval of 12 hours and the dosage was decreased daily as 4-, 3-, 2-, and 1-mg, respectively. As the first dosage at the third day, co-administrated with 1000 iu human chorionic gonadotropin (HCG) which resulted in detection of estrous after 54 hours whereupon gave two artificial insemination (AI) with a interval of 12 hours. Then, at about one and half days after conception, the goat one-cell stage embryos were collected with a sterile glass capillary tube via surgical embryo flushing method. The collected embryo was transferred into another petri dish where it was rinsed more than ten times. Thereafter, it was placed under a phase contrast microscope at 400× amplification for gene microinjection manipulation. After a transient in vitro culture, the healthly microinjected embryos were then transferred into recipient oviducts for further conceptus development.
Recombinant hFVIII Transgene Determination
The resulting pups were rapidly screened for the transgene by PCR amplification of tail or ear tissue DNA. PCR was performed using one set of primer, p αLA-124(+): 5′-CTC TCT TGT CAT CCT CTT CC-3′ (SEQ ID: NO.7) and phFVIII-149(−): 5′-GGT TAC GCG TCA AGA TTC TGA-3′ (SEQ ID: NO.8), which defined a 273-bp region spanning the αLA promoter, secretion signal sequence and hFVIII cDNA junctional sequence. For the B domain-deleted hFVIII transgene detection, additional primer pairs was designed, phFVIII-ACJ(+): 5′-AGA CTT TCG GAA CAG AGG CA-3′ (SEQ ID: NO.9) and phFVIII-ACJ(−): 5′-ATC TTT TTC CAG GTC AAC ATC A-3′ (SEQ ID: NO.10), which defined a 751-bp region flanking A-C recombinant junction. The positive PCR screening results for transgenic animals were further confirmed by Southern blot analysis. Ten micrograms of genomic DNA were individurally digested with BamHI, HindIII, PvuII, and XbaI restriction enzymes at 37° C. overnight, electrophoresed on a 0.8% agarose gel, and transferred to a Durose membrane (Stratagene, La Jolla, Calif.). A KpnI fragment of hFVIII-specific cDNA (1.8-kb) was used as a radioactive probe to hybridize the membrane. Blots were subjected to autoradiography for three days at −20° C.
Recombinant hFVIII RNA Expression in Transgenic Animals
The temporal and spatial expression of hFVIII RNA in transgenic animals was analyzed using a reverse transcription-polymerase chain reaction (RT-PCR). Total RNA from different tissues including the heart, liver, lung, muscle, mammary gland, brain, pancrease, and kidney of female transgenic mice during lactation periods (Day 1 to Day 28 post partum) was extracted using the acid guanidinium thiocyanate method (Chomczynski and Sacchi, 1987, Anal. Biochem. 162: 156-159). One microgram of total RNA was treated twice with 10 units of DNase I (Gibco BRL, Gaithersburg, Md.) and phenol-chloroform extracted. RNA pellets were resuspended in 15 μl DEPC-water and then used to synthesize the first-strand cDNA with random primers and SuperScript reverse transcriptase (Gibco BRL, Gaithersburg, Md.) in a total volume of 25 ml. The reaction was carried out at 42° C. for 1 hr.
For further PCR amplification, an aliquot (1/10) of the RT product was adjusted to contain 0.1 μg of each primer and additional buffer was added for a total volume of 50 μl. PCR was performed for 30 cycles (94° C., 1 min; 55° C., 2 min; 72° C., 2 min). The primers used included a pair of hFVIII-specific primers, phFVIII-F2(+):5′-CAT TCT ATT CAT TTC AGT GGA CA-3′ (SEQ ID: NO.11) and phFVIII-R2(−): 5′-GAG ATG TAG AGG CTG GAG AAC T-3′ (SEQ ID: NO.12), and a pair of glyceraldehydes-3-phosphate dehydrogenase (GAPDH), as well as a pair of β-actin. Both β-actin and GAPDH are the universal transcripts in every cell and were used as the internal controls of RT-PCR.
Immunoblot Analysis of Transgenic Milk Proteins
Milk was collected from lactating females as previously described (Simons et al., 1987, Nature 328: 530-532.) and analyzed using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Cheng et al., 1998, Human Gene Therapy 9: 1995-2003). Ten- to twenty-fold dilutions of the collected milk, in 75 mM Tris-HCl buffer at pH 6.8, from different lactation periods were diluted in SDS-PAGE sample buffer with 5% 2-mercaptoethanol and electrophoresed on 7.5% gel. To estimate production levels, HPLC-purified recombinant hFVIII standard was diluted to 10 μg/ml in normal mouse milk and electrophoresed alongside the transgenic milk samples. Proteins were electrotransferred from the gel to a PVDF membrane (NEN Life Science Products, Boston, Mass.). The blots were probed with primary antibodies recognizing hFVIII at 2 to 10 μg/ml and washed with phosphate-buffered saline containing 0.1% Tween-20 (PBS-T). Blots were reacted with horseradish peroxidase (HRP)-conjugated second antibodies at 0.2 mg/ml. Polyclone antibody C6 recognize the heavy chain of hFVIII rang from 80-200 kDa and monoclone antibody D2 recognize the light chain of hFVIII at 75-80 kDa. The blot was then developed with the chemiluminescent ECLTM detection system (Amersham, UK) and exposure to x-ray film. Band intensities were compared by densitometry.
Secretion of hFVIII protein from the transgenic milk was determined quantitatively with the enzyme-linked immunosorbent assay (ELISA) as previously described (Chen et al., 1993, J Virol 67: 2142-2148). Briefly, plates were coated with capture antibodies (ESH-5 and ESH-8, 500 ng each per well; American Diagnostics, Greenwich, Conn.) in carbonate buffer and incubated for 1 hour at 37° C. Plates were washed with 0.05% Tween-20 in phosphate-buffered saline (PBS) and blocked in 50 mM Tris (pH 7.2), 150 mM NaCl, 0.5% gelatin, and 0.05% Tween-20 for 2 hour at 37° C. Purified recombinant FVIII (Hyland, Baxter Healthcare, Calif.), prepared in blocking buffer with 1:50 normal murine milk, served as the standard. Samples and standards were incubated for 1 hour at 37° C. Detection antibody (rabbit anti-human FVIII 1:10,000 dilution) was added and allowed to react with hFVIII at 37° C. for 1 h. After removing the first antibody using four washes with PBS, a second antibody, goat anti-rabbit immunoglobulin antibody conjugated with horseradish peroxidase, was added (1:3,000 dilution) for a further 1 h of incubation. The plates were washed again thoroughly with PBS, and 100 μl of substrate solution (2 mg of 0-phenylenediamine dissolve in 1 ml of 1M phosphate citrate plus 0.02% H2O2) was added for development. After 30 min, 100 μl of 1M H2SO4 was added immediately to stop the reaction and the colors were measured by optical density at 492 nm.
The Feature of α-Lactalbuimn Mammary Expression Cassette
Expression of rFVIII protein in the milk of transgenic animals driven by bovine α-lactalbuimn regulatory sequence seems more efficient than the other transgene constructions, which were controlled by ovine β-lactoglobulin promoter (Niemann et al., 1999, Transgenic Res 8: 237-247.) as well as mouse WAP regulatory sequence (Paleyanda et al., 1997, Nature Biotech 15: 971-975). Expression of the bovine αLA gene has been shown to be the most lactation-specific of all bovine milk protein genes (Goodman and Schanbacher, 1991, Biochem Biophys Res Commun 180:75-84). Use of the αLA 19-aa secretary peptide (SEQ ID: NO: 13) or αS1-casein 15-aa signal peptide (SEQ ID: NO: 14) to lead the rFVIII protein secretion and the bGH polyadenosylation signal to stabilize the steady-state of hFVIII RNA molecules, which may also have contributed to our success. Actually, there are several previous reports demonstrated that mammary specific transgenes driven by αLA promoter have resulted in high levels of gene expression up to 3.7 mg/ml (Hochi et al., 1992, Mol Reprod Dev 33: 160-164; Soulier et al., 1992, Fed Eur Biol Soc Lett 297: 13-20; Maschio et al., 1991, Biochem J 275:459-465) in their milks. This characteristic of the bovine αLA gene makes its regulatory elements potentially useful as a mammary expression system in transgenic animals. In contrast to the caseins and b-lactoglobulin, the production of αLA mRNA increases suddenly at paturition, remains elevated during lactation, and drops sharply at dry-off and during involution. The rFVIII protein profile lead by the αLA promoter and 19-aa secretion signal peptide or αS1-casein 15-aa signal peptide follows a similar trend. The lactation specificity of the regulatory regions used to control mammary expression in transgenic animals may be important when foreign proteins that have biological activities are expressed because these proteins may exhibit their biological function in the animals if they are secreted before tight junctions of mammary epithelial cells are formed.
The Feature of hFVIII Protein Expression
Clotting factor IX (FIX), adenosine deaminase (ADA), and other cDNAs were expressed at high levels from retroviral vectors in rat fibroblasts (Miller, 1992, Nature 357: 455-460; Palmer et al., 1991, Proc Natl Acad Sci USA 88: 1330-13334), whereas the FVIII cDNA was expressed at very low levels from these vectors in primary human fibroblast cells (Hoeben et al., 1990, J Biol Chem 265: 7318-7323; Lynch et al., 1993, Hum Gene Ther 4: 259-272). It has been found that FVIII RNA steady-state levels were reduced 100-fold from a FVIII retroviral vector compared to the same vector expressing other cDNAs, and FVIII vector titers were correspondingly reduced 100-fold compared to other vectors (Lynch et-al., 1993, Hum Gene Ther 4: 259-272). A large part of the inhibitory effect of the FVIII cDNA on expression from a retroviral vector has been localized to a 1.2-kb fragment, derived from the A2 and A3 domains, which decreased the steady-state RNA levels 100- to 200-fold and decreased vector titers 10-fold (Koeberl et al., 1995, Hum Gene Ther 6: 469-479). The mechanism by which the FVIII cDNA inhibitory sequence (INS) decreases RNA and protein expression from expression vectors remain to be established. Recently, experimental data provided ample evidence that the human FVIII cDNA contains elements that repress its own expression at the level of transcription (Hoeben et al., 1995, Blood 85: 2447-2454). Fallaux and coworkers (1996, Mol Cell Biol 16: 4264-4272) identified a 305-bp region derived from exons 9 to 11 that encodes a nuclear-matrix attachment region (MAR), also called the scaffold-attached region (SAR). Yeast MARs elements can play an important role in transcriptional silencing (Newlon and Theis, 1993, Transgenic Res 8: 237-247). The B domain can be deleted without any significant effect on specific procoagulant activity (Pittman et al., 1993, Blood 81:2925-2935; Chao et al., 2000, Blood, 95: 1594-1599). B-domain-deleted hFVIII cDNA (BDD-hFVIII) is 4.3 kb and primary truncate polypeptide (hFVIIIΔB) is 1448 aa (SEQ ID: NO.15) not thought feasible for testing in transgenic animals previously.
The present invention is further depicted in the illustration of examples, but the descriptions made in the examples should not be construed as a limitation on the actual application of the present invention.
Generations of Transgenic Mice, Pigs and Goats Carrying the Full-Length hFVIII Gene in Mammary-Specific Expression Cassette
Out of 79 potential transgenic founder mice, 17 were identified as being transgenic by the PCR screening (
Breeding lines were established from all transgenic founder animals by crossing them with normal ICR mice, Alpine goats (
Generations of Transgenic Mice and Goats Carrying the B-Domain Deleted hFVIII Gene in Mammary-Specific Expression Cassette
B-domain deleted hFVIII transgene construct driven by αLA promoter and αS1-casein secretary peptide sequence was shown in
Out of 65 potential transgenic founder mice, 15 were identified as being transgenic by the two sets of primers, one pair of primer located in 5′-promoter junction, the other pair of primer flanking the B-domain deletion region (
Germ-line transmission ability of transgenic animals carrying hFVIII(ΔB) exogenic DNA in their F1 generation was confirmed by using PCR detection and direct PCR product sequencing. The sequencing result exhibited the transgenic hFVIII(ΔB) fragment was acturally present in their F1 offspring's genome of transgenic goats (
Temporal and Spatial Expression of Exogenous hFVIII RNA in Transgenic Animals Driven by Mammary-Specific Expression Cassette
To characterize the spatial expression of the new developed mammary-specific expression cassette containing hFVIII in this invention, total tissue RNAs were extracted from different organs in the breeding lines established from the transgenic founder mice. Reverse-transcriptional polymerase chain reaction (RT-PCR) analysis was performed using two pair of primer sets, one was phFVIII-F2(+) (SEQ ID: NO.11) and phFVIII-R2(−) (SEQ ID: NO.12) specific to human FVIII cDNA franking 3′-coding region, the other was b-actin primer set as an internal control. As shown in
Temporal expression studies were performed on the F1 transgenic mice in the breeding lines established from founder mice αLAF-15 and -18. Total RNAs were extracted at different physiological states in the transgenic mammary gland using tissue biopsies subjected to RT-PCR detection. The results showed that the recombinant hFVIII mRNA was expressed in all lactating periods (D1, D8, D15 and D22, shown in
To quantify the level of hFVIII mRNA in the mammary glands of transgenic mice, densitometric analysis was performed, comparing the intensity of the 250-bp band in each lane normalized to the level of signal generated upon co-amplification of the mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers. The intensity of the hFVIII transcript increased eight-fold and ten-fold upon Day 8 and Day 15 post-parturition, respectively, while a two-fold decrease was present on Day 22 of the later lactation (
Expression and Secretion of Recombinant hFVIII Proteins into the Transgenic Milks
To determine the production of recombinant hFVIII and to compare the secretion efficiency of rFVIII among the different transgenic mouse lines, milk was collected from five lines of F0, F1 and F2 females during lactation. Proteins from both non-transgenic control and transgenic skim milks were separated in SDS-PAGE and followed by Western blot analysis (
In the transgenic goat (Tg-201), milk was collected from days 3, 11 and 22 during early lactation stage for Western blot assay (
Biological Function Assay for Clotting Activity of Recombinant hFVIII Derived from Transgenic Milks
The coagulant activity of hFVIII was measured by reduction of the activated partial thromboplastin time (APTT) in a one-stage assay (Over, 1984, Scand J. Haematol. 41: 13-24.). Human FVIII dilutions equivalent to a 1:10 to 1:120 dilution of normal plasma were prepared in 50 mM imidazole buffer, pH 7.4, containing 100 mM sodium choride, 0.1% BSA and 0.01% Tween-20. Control and transgenic mouse milks diluted 1:20, 1:30, and 1:40 were incubated in duplicate at 37° C. in hFVIII-deficient plasma (Sigma Diagnostics, St. Louis, Mo.), followed by the automated addition of APTT reagent (Dade Behring, Marburg, Germany). Clot formation was initiated by the addition of 35 mM CaCl2. The clot time, in seconds, was recorded on an ST2 Coagulometer (Stago). Semi-logarithmic analysis was used to plot the clot time versus amount of FVIII. Control mouse milk gave results similar to the reagent blank.
The concentrations of rFVIII ranged from 7.0 to 50.2 μg/ml, over 35- to 200-fold higher than that in normal human plasma (Table 1). The expression levels of rFVIII from three groups on different line carrying low, middle and high copy numbers of transgenes were not significantly different. Recombinant hFVIII production generally tended to increase through the lactation period, but dropped dramatically at the end of lactation. This result was in parallel with the RNA transcript assay (
The functional activity of rFVIII was examined using one-stage clotting assays (Table 1) and this approach demonstrated that rFVIII secreted in the milk of transgenic mice was biologically active. In the clotting assay, the addition of transgenic mouse milk, but not control milk, to FVIII-deficient human plasma resulted in the restoration of normal coagulant activity. Up to 13.41 U/ml of rFVIII procoagulant activity was detected. The activity of rFVIII was detected throughout lactation as shown in Table 1. The same result for rFVIII clotting activity derived from transgenic goat also has been demonstrated and showed in
aOne unit of rFVIII was defined as equivalent to the amount of human FVIII normally present in 1 ml of plasma, approximately 200 ng. Results presented are the average of two independent assays
