David R. Hurwitz

Expression of human serum albumin in the milk of transgenic mice

We have tested the feasibility of producing large quantities of human serum albumin (HSA) in the milk of transgenic livestock by generating transgenic mice as a model system. The sheep β-lactoglobulin (BLG) 5′-regulatory promoter sequences were used to support expression of BLG or HSA in transgenic mice. Transgenic animals generated from the entire BLG gene including 3, 5.5 or 10.8 kb of 5′-sequences demonstrated that 3 kb of 5′-sequences were sufficient to support high levels of expression of BLG, and that the longer 5′-sequences did not improve upon the levels of expression. As such, the 3 kb 5′-sequences were used to drive expression of HSA in BLG-HSA constructs. HSA was not detectably expressed in eight transgenic lines generated from a BLG-HSA construct containing the HSA cDNA. Two transgenic lines of 26 generated, using five different constructs, with an HSA minigene possessing the first intron expressed HSA in their milk. One of these expressed HSA at high levels (2.5 mg ml−1) and has stably transmitted this ability to its progeny. A high percentage of transgenic mouse lines (four of six) generated from a vector containing an HSA minigene possessing introns 1 and 2 expressed HSA in their milk at levels which ranged from 1 to 35 μg ml−1. In a similar trend, levels of expression of HSA by transfected tissue culture cells from BLG-HSA vectors containing an introduced SV40 enhancer were low with the HSA cDNA, increased with the HSA minigene with intron 1 and increased further with the minigene containing introns 1 and 2. This study demonstrates that high levels of HSA can be expressed in the milk of transgenic animals, that introns of the HSA gene play a role in its expression and that transfected cell lines may be used to quickly evaluate the relative expression efficiencies of various vector constructs intended for future transgenic evaluation.

Ectopic expression of β-lactoglobulin/human serum albumin fusion genes in transgenic mice: hormonal regulation andin situ localization

We produced transgenic mice carrying the native sheep β-lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. In contrast, transgenic females carrying the BLG/HSA fusion constructs also expressed the HSA RNA ectopically in skeletal muscle, kidney, brain, spleen, salivary gland and skin. Ectopic expression of HSA RNA was detected only in strains that express the transgene in the mammary gland. There was no obvious correlation between the level of the HSA RNA expressed in the mammary gland and that found ectopically. In three transgenic strains analysed, the expression of HSA RNA in kidney and skeletal muscle increased during pregnancy and lactation, whereas in the brain HSA expression decreased during lactation in one of the strains. HSA protein was synthesized in skeletal muscle and skin of strain #23 and its level was higher in lactating mice compared with virgin mice. Expression of HSA was also analysed in males and was found to be more stringently controlled than in females of the same strains.In situ hybridization analyses localized the expressed transgene in the skin, kidney, brain and salivary glands of various transgenic strains. Distinct strain-specific and cell-type specific HSA expression patterns were observed in the skin. This is in contrast to the exclusive expression of the HSA transgene in epithelial cells surrounding the alveoli of the mammary gland. Taken together, these results suggest that the absence of sufficient mammary-specific regulatory elements in the BLG promoter sequences and/or the juxtaposition of the BLG promoter with the HSA coding sequences leads to novel tissue- and cell-specific expression in ectopic tissues of transgenic mice.

Specific combinations of human serum albumin introns direct high level expression of albumin in transfected COS cells and in the milk of transgenic mice

A new series of expression vectors, each comprised of the β-lactoglobulin (BLG) promoter driving one of a variety of human serum albumin (HSA) minigenes or the entire gene, were evaluated for their ability to direct expression of HSAin vitro in COS tissue culture cells and into the milk of transgenic mice. Vectors directed a hierarchy of expression levelsin vitro, dependent upon the specific complement of HSA introns included. HSA introns acted in a synergistic manner. In addition, minigenes comprised of specific subsets of introns were more efficacious than the entire HSA gene with all of its introns. Transgenic mice expressed as much as 10 mg ml−1 of HSA in their milk. Vectors comprised of specific intron subsets directed levels at 1 mg ml−1 or greater in the milk of 20% of generated transgenics. A statistical correlation between the expression level trendin vitro with the trend of expressionin vivo (% which express) at detectable levels (p=0.0015) and at the level of greater than 0.1 mg ml−1 (p=0.0156) was demonstrated. A weak correlation existed (p=0.0526) atin vivo levels of 1 mg ml−1 or greater. These new vectors are expected to direct the production of high levels of HSA in the milk of a large percentage of generated transgenic dairy animals.

Bone marrow stromal cell-mediated gene therapy for hemophilia A. in vitro expression of human factor VIII with high biological activity requires the inclusion of the proteolytic site at amino acid 1648

To evaluate the potential of the ex vivo bone marrow stromal cell (BMSC) system as a gene therapy for hemophilia A, we studied the in vitro expression of human factor VIII (hFVIII) in canine BMSCs following transfection with plasmid vectors and transduction with retroviral vectors. Vectors were composed of B domain-deleted forms of hFVIII that either retain or delete the proteolytic site at amino acid 1648. On transfection of BMSCs, vectors supported expression and secretion of similar levels of up to 386 mU/10(6) cells/24 hr, even though only 3-9% of the cells expressed hFVIII while 42-48% of transfected cells harbored plasmid vector. Much higher percentages (approximately 70%) of cells expressing hFVIII were achieved when BMSCs were transduced by retroviral vectors, resulting in expression and secretion as high as 1000-4000 mU/10(6) cells/24 hr. Western analysis demonstrated that the B domain-deleted forms possessing the proteolytic site were secreted predominantly as heavy and light chain heterodimers that resemble native forms found in plasma. In contrast, the hFVIII lacking the proteolytic site was expressed mostly as unprocessed, single heavy-light chains. Both hFVIII forms were correctly cleaved and activated by thrombin. The proteolyzed hFVIII form possessed > or = 93% normal biological activity while the unproteolyzed form possessed consistently less than 55% normal biological activity and was therefore considered less suitable for therapeutic application. These results demonstrate that the BMSC system has potential utility in gene therapy for hemophilia A and stress the importance of selecting the appropriate hFVIII structure for prospective clinical use.

Synthesis and secretion of human serum albumin by mammary gland explants of virgin and lactating transgenic mice

Transgenic mice were produced, carrying hybrid genes comprised of the ovine β-lactoglobulin (BLG) milk protein gene promoter and human serum albumin (HSA) coding sequences.In situ hybridization revealed high levels of BLG/HSA hybrid mRNA, confined to the epithelial cells of the lactating mammary gland with a several hundred fold lower concentration in virgin mammary glands. During the first 24 h in culture, exceptionally high levels of HSA were secreted from explants of virgin mice, independent of hormonal control. HSA secretion was reduced considerably during subsequent days in culture and became dependent on the presence of insulin, hydrocortisone and prolactin. This temporal and hormonal pattern of regulation of HSA was different than that found for the secretion of caseins. In contrast to the vast difference in the mRNA content, the amount of HSA secreted from explants derived from lactating mice during the first 24 h in culture was only 2-to 5-fold higher than that found with explants from virgin transgenic mice, suggesting post-transcriptional control of HSA synthesis. The high-level synthesis and secretion of HSA in mammary explants of lactating mice was also dependent on the presence of insulin, hydrocortisone and prolactin. This study confirms previous suggestion that mammary explants from virgin transgenics may serve as a powerful tool for screening the potential of transgenic animals to secrete foreign proteins in their milk.

Co-integration of β-lactoglobulin/human serum albumin hybrid genes with the entire β-lactoglobulin gene or the matrix attachment region element: Repression of human serum albumin and β-lactoglobulin expression in the mammary gland and dual regulation of the transgenes

The effect of co‐integration of the entire β‐lactoglobulin (BLG) gene or matrix attachment region (MAR) sequences on the expression of various BLG/human serum albumin (HSA) gene constructs was tested in transgenic mice. These former sequences were chosen because of their reported ability to insulate transgenes from the neighboring host genomic DNA sequences and/or to provide a more permissive transcriptional environment. When introduced alone, a cDNA‐based BLG/HSA construct was expressed in 60% of transgenic strains and HSA was secreted at levels up to 0.3 mg/ml into the milk. Upon co‐integration with either the entire BLG gene or MAR element, HSA RNA and protein expression were completely abrogated. While the co‐integrated BLG gene suppressed the proportion of expresser strains carrying cDNA as well as genomic BLG/HSA constructs, the MAR element only exerted its negative effect on the cDNA‐based BLG/HSA construct. In transgenics expressing both HSA and BLG, the tissue specificity and developmental patterns of BLG expression were altered and resembled the less stringent pattern of the BLG/HSA expression. These results demonstrate that rescue of transgene expression through co‐integration with BLG or MAR sequences do not apply universally.

HSA production by mammary explants of virgin transgenic mice: A reliable tool for predicting levels of secretion into milk

SUMMARY Lactating females of eight independent strains of transgenic mice carrying a human serum albumin (HSA) gene or minigene under the control of the Blactoglobulin (BLG) promoter secrete strain-specific amounts of HSA into their milk, ranging from 0-4.5 mg per ml. The HSA produced in the milk of all transgenic strains appeared to be identical. The ability of mammary explants from virgin females of these strains to de novo synthesize and secrete HSA immediately upon explantation, or on day 5 of culture in the presence of insulin and prolactin, was evaluated based upon total de novo protein synthesis as well as equal tissue weight. Levels of de novo HSA synthesis and secretion by explants were compared with levels of secretion of HSA in the milk of lactating females of these same strains. The correlation of relative densitometric values of HSA production in vivo versus in vitro ranged from r=0.70 to r=0.92. In all cases, correlation improved when explants were cultured for 5 days in the presence of hormones compared to explants evaluated immediately upon explantation.

Heterogeneous Expression and Synthesis of Human Serum Albumin in the Mammary Gland of Transgenic Mice

Limited information on the production and secretion of the individual milk proteins at the cellular level in the mammary gland is available. It is generally assumed that all active epithelial cells within the lactating mammary gland of mice share a comparable ability to produce and secrete all the components of milk. This is based on ultrastructural studies and observations on cell membrane turnover related to fat globule membrane loss and vesicular protein exocytosis.