Henry Daniell

Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants

Abstract The use of plants for medicinal purposes dates back thousands of years but genetic engineering of plants to produce desired biopharmaceuticals is much more recent. As the demand for biopharmaceuticals is expected to increase, it would be wise to ensure that they will be available in significantly larger amounts, on a cost-effective basis. Currently, the cost of biopharmaceuticals limits their availability. Plant-derived biopharmaceuticals are cheap to produce and store, easy to scale up for mass production, and safer than those derived from animals. Here, we discuss recent developments in this field and possible environmental concerns.

Analysis of 81 genes from 64 plastid genomes resolves relationships in angiosperms and identifies genome-scale evolutionary patterns

Angiosperms are the largest and most successful clade of land plants with >250,000 species distributed in nearly every terrestrial habitat. Many phylogenetic studies have been based on DNA sequences of one to several genes, but, despite decades of intensive efforts, relationships among early diverging lineages and several of the major clades remain either incompletely resolved or weakly supported. We performed phylogenetic analyses of 81 plastid genes in 64 sequenced genomes, including 13 new genomes, to estimate relationships among the major angiosperm clades, and the resulting trees are used to examine the evolution of gene and intron content. Phylogenetic trees from multiple methods, including model-based approaches, provide strong support for the position of Amborella as the earliest diverging lineage of flowering plants, followed by Nymphaeales and Austrobaileyales. The plastid genome trees also provide strong support for a sister relationship between eudicots and monocots, and this group is sister to a clade that includes Chloranthales and magnoliids. Resolution of relationships among the major clades of angiosperms provides the necessary framework for addressing numerous evolutionary questions regarding the rapid diversification of angiosperms. Gene and intron content are highly conserved among the early diverging angiosperms and basal eudicots, but 62 independent gene and intron losses are limited to the more derived monocot and eudicot clades. Moreover, a lineage-specific correlation was detected between rates of nucleotide substitutions, indels, and genomic rearrangements.

Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

In nuclear transgenic plants, expression of multiple genes requires introduction of individual genes and time-consuming subsequent backcrosses to reconstitute multi-subunit proteins or pathways, a problem that is compounded by variable expression levels. In order to accomplish expression of multiple genes in a single transformation event, we have introduced several genes into the chromoplast genome. We confirmed stable integration of the cry2Aa2 operon by PCR and Southern blot analyses in T0 and T1 transgenic plants. Foreign protein accumulated at 45.3% of the total soluble protein in mature leaves and remained stable even in old bleached leaves (46.1%), thereby increasing the efficacy and safety of transgenic plants throughout the growing season. This represents the highest level of foreign gene expression reported in transgenic plants to date. Insects that are normally difficult to control (10-day old cotton bollworm, beet armyworm) were killed 100% after consuming transgenic leaves. Electron micrographs showed the presence of the insecticidal protein folded into cuboidal crystals. Formation of crystals of foreign proteins (due to hyperexpression and folding by the putative chaperonin, ORF 2) provides a simple method of purification by centrifugation and enhances stability by protection from cellular proteases. Demonstration of expression of an operon in transgenic plants paves the way to engineering new pathways in plants in a single transformation event.

Molecular strategies for gene containment in transgenic crops

The potential of genetically modified (GM) crops to transfer foreign genes through pollen to related plant species has been cited as an environmental concern. Until more is known concerning the environmental impact of novel genes on indigenous crops and weeds, practical and regulatory considerations will likely require the adoption of gene-containment approaches for future generations of GM crops. Most molecular approaches with potential for controlling gene flow among crops and weeds have thus far focused on maternal inheritance, male sterility, and seed sterility. Several other containment strategies may also prove useful in restricting gene flow, including apomixis (vegetative propagation and asexual seed formation), cleistogamy (self-fertilization without opening of the flower), genome incompatibility, chemical induction/deletion of transgenes, fruit-specific excision of transgenes, and transgenic mitigation (transgenes that compromise fitness in the hybrid). As yet, however, no strategy has proved broadly applicable to all crop species, and a combination of approaches may prove most effective for engineering the next generation of GM crops.

PROGRESSIVE OSTEOPOROSIS DURING ANDROGEN DEPRIVATION THERAPY FOR PROSTATE CANCER

PURPOSE Hypogonadism is a prominent risk factor for osteoporosis in older men. However, bone loss during androgen ablation therapy for prostate cancer has rarely been quantitated. MATERIALS AND METHODS Femoral neck bone mineral density was determined in 26 men before orchiectomy or chemical castration as initial hormone therapy for prostate cancer and at 6-month intervals thereafter for 6 to 42 months. Measurements were made in 16 other men at 12 to 24 months beginning 3 to 8 years after the onset of castration. Baseline and post-castration bone loss was related to several host and tumor characteristics, and compared to similar measurements in 12 control subjects. RESULTS Average age corrected baseline femoral neck bone mineral density was higher in controls than in treated men and remained essentially unchanged for 2 years. Following orchiectomy average bone mineral density decreased 2.4% and 7.6%, respectively, during years 1 and 2 (2-year loss 2.5% to 17.0%), with similar losses documented in men undergoing chemical castration. Average bone mineral density decreased 1.4% to 2.6% per year 3 to 8 years after uninterrupted androgen deprivation. Age corrected baseline bone mineral density was greater in men who were obese, younger than 75 years or participated in regular exercise but the influence of each characteristic could not be isolated. Post-castration bone loss was greater in men who were obese, younger than 75 years without regular exercise. CONCLUSIONS Chemical or surgical castration in men with prostate cancer is usually followed by greatly accelerated bone loss which may be superimposed on a bone mass already depleted before hormonal therapy. Baseline bone mass and subsequent bone loss may be influenced by host obesity, age and exercise habits.

Osteoporosis After Orchiectomy for Prostate Cancer

PURPOSE The possibility of increased osteoporosis and osteoporotic fractures following therapeutic orchiectomy in men with prostate cancer was investigated. MATERIALS AND METHODS A total of 235 men with nonstage A prostate cancer diagnosed between 1983 and 1990 was analyzed for therapeutic orchiectomy, other osteoporotic risk factors and subsequent hospital treatment for osteoporotic fractures. The 17 castrated men alive in 1995 were interviewed, and femoral neck bone mineral density was compared to that of 23 controls of similar age. RESULTS Risk factors for osteoporosis, including smoking, slender habitus and atrophic testes, were common among men treated with orchiectomy. Of the men in the study cohort 10 had osteoporotic fractures: 8 of 59 treated with and 2 of 176 without orchiectomy (13.6 versus 1.1%, p < 0.001). First fracture cumulative incidence rates 7 years after castration or diagnosis were 28 and 1%, respectively (p < 0.001). Osteoporotic fractures were much more common than pathological fractures or those due to major trauma (1 each). Bone mineral density averaged 0.91, 0.84, 0.79 and 0.66 gm./cm.2 in 9 controls without prostate cancer, 14 men with prostate cancer before orchiectomy, 9 men at 9 to 60 and 8 men at 60 to 115 months after orchiectomy, respectively. Of the 16 men surviving for longer than 60 months after orchiectomy 6 had osteoporotic fractures, as did 5 of 6 and 5 of 7 with a bone mineral density of less than 0.70 gm./cm.2 and less than 75% of normal for age, respectively. CONCLUSIONS Orchiectomy for prostate cancer is frequently followed by severe osteoporosis, some of which had developed before castration. Appropriate therapy should be identified that does not diminish the antitumorigenic effectiveness of androgen ablation.

Expression of the Native Cholera Toxin B Subunit Gene and Assembly as Functional Oligomers in Transgenic Tobacco Chloroplasts

Abstract The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1% of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expresssion levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast- synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast-synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of many proteins is essential for their function, this investigation demonstrates the potential for other foreign multimeric proteins to be properly expressed and assembled in transgenic chloroplasts.

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

Chloroplast genomes defied the laws of Mendelian inheritance at the dawn of plant genetics, and continue to defy the mainstream approach to biotechnology, leading the field in an environmentally friendly direction. Recent success in engineering the chloroplast genome for resistance to herbicides, insects, disease and drought, and for production of biopharmaceuticals, has opened the door to a new era in biotechnology. The successful engineering of tomato chromoplasts for high-level transgene expression in fruits, coupled to hyper-expression of vaccine antigens, and the use of plant-derived antibiotic-free selectable markers, augur well for oral delivery of edible vaccines and biopharmaceuticals that are currently beyond the reach of those who need them most.

Plastid-Expressed Betaine Aldehyde Dehydrogenase Gene in Carrot Cultured Cells, Roots, and Leaves Confers Enhanced Salt Tolerance

Salinity is one of the major factors that limits geographical distribution of plants and adversely affects crop productivity and quality. We report here high-level expression of betaine aldehyde dehydrogenase (BADH) in cultured cells, roots, and leaves of carrot (Daucus carota) via plastid genetic engineering. Homoplasmic transgenic plants exhibiting high levels of salt tolerance were regenerated from bombarded cell cultures via somatic embryogenesis. Transformation efficiency of carrot somatic embryos was very high, with one transgenic event per approximately seven bombarded plates under optimal conditions. In vitro transgenic carrot cells transformed with the badh transgene were visually green in color when compared to untransformed carrot cells, and this offered a visual selection for transgenic lines. BADH enzyme activity was enhanced 8-fold in transgenic carrot cell cultures, grew 7-fold more, and accumulated 50- to 54-fold more betaine (93–101 μmol g−1 dry weight of β-Ala betaine and Gly betaine) than untransformed cells grown in liquid medium containing 100 mm NaCl. Transgenic carrot plants expressing BADH grew in the presence of high concentrations of NaCl (up to 400 mm), the highest level of salt tolerance reported so far among genetically modified crop plants. BADH expression was 74.8% in non-green edible parts (carrots) containing chromoplasts, and 53% in proplastids of cultured cells when compared to chloroplasts (100%) in leaves. Demonstration of plastid transformation via somatic embryogenesis utilizing non-green tissues as recipients of foreign DNA for the first time overcomes two of the major obstacles in extending this technology to important crop plants.

Plant-made vaccine antigens and biopharmaceuticals

Plant cells are ideal bioreactors for the production and oral delivery of vaccines and biopharmaceuticals, eliminating the need for expensive fermentation, purification, cold storage, transportation and sterile delivery. Plant-made vaccines have been developed for two decades but none has advanced beyond Phase I. However, two plant-made biopharmaceuticals are now advancing through Phase II and Phase III human clinical trials. In this review, we evaluate the advantages and disadvantages of different plant expression systems (stable nuclear and chloroplast or transient viral) and their current limitations or challenges. We provide suggestions for advancing this valuable concept for clinical applications and conclude that greater research emphasis is needed on large-scale production, purification, functional characterization, oral delivery and preclinical evaluation.