In This Issue:
Baculovirus Field Test OK'd By EPA
Update From APHIS On Biotech Activities
Maize Is Transformed By Agrobacterium
Cassava Biotechnology: A Reality Check?
DNA-Treated Sperm As A Gene Transfer Vector - Revisited
Recombinant Proteins From Milk Of Transgenic Animals - Part 2:
Targeting Genes To The Mammary Gland And Regulating Production
Biological Control Of Mosquitoes With Genetically Engineered Bacteria
An Electronic Journal BIOSAFETY
Mid-Year Cooling Of Hot '96 Public Capital Markets
American Cyanamid will conduct field trials in 12 states to test the efficacy of the genetically engineered biocontrol agent against tobacco budworm and the cabbage looper on cotton, tobacco and leafy vegetables such as cabbage, broccoli and lettuce. Less than 100 grams of active ingredient will be used to treat a total of 7.4 acres.
Similar field tests were conducted in 1995 in which the EPA requested soil sampling data to evaluate survival and persistence of the baculovirus. These data were not available when the 1996 release was being considered, however the agency felt that they were not needed to evaluate the potential for the new trials to cause unreasonable adverse effects on the environment.
In the 1996 field tests, soil samples are to be taken at specified points during the course of the release: prior to the initial application of the recombinant virus; following application; just prior to spraying with wildtype baculovirus; and after allowing sufficient time for dispersal of the wildtype virus in the soil. The soil samples are to be used in a bioassay with a highly sensitive susceptible insect to detect infectious polyhedra; PCR is to be used to detect the recombinant gene construct. At the end of the trial, applications of lime are to be used to inactivate residual virus in the soil.
Alan Wood, Boyce Thompson Institute for Plant Research, raised several issues concerning the proposed release in comments to the EPA. He noted that the AaIT virus has an increased rate of infection compared with the unmodified virus, an unexpected and unexplained phenotype that could be due to uncharacterized genetic changes in the recombinant strain. Wood also questioned the feasibility of using lime to raise soil pH to inactivate the virus and added that the amount of lime needed may itself cause environmental effects.
Pat Traynor
Information Systems for Biotechnology
traynor@nbiap.biochem.vt.edu
>Field Testing of Agricultural Crop Varieties Continues and
Expands
Field testing of new agricultural crop varieties continues its
rapid pace of expansion approximately doubling each year. Since
1987, APHIS has approved or acknowledged 2,380 field trials at
9,467 field sites. The notification option, which simplifies
regulatory requirements for certain field trials involving
particular types of modified crops, has been in effect since 1993
and now accounts for approximately 86% of all current field
testing. Since the beginning of 1994, 1,723 field trials (at 7,922
test sites) were approved or acknowledged, of which 1,487 were
conducted under notification and 236 under permit. Derivatives of
45 different plant species have been field tested to date, with a
wide variety of modifications. Corn continues as the major crop
being field tested under our regulations. In the past 2 years we
have seen the first field trials involving Arabidopsis thaliana,
barley, broccoli, carrot, chicory, cranberry, creeping bentgrass,
eggplant, gladiolus, grape, pea, pepper, raspberry, strawberry,
sugarcane, sweetgum, watermelon, and wheat.
In an effort to expand public access and awareness of the progress in the development of work with transgenic plants, APHIS has made available on the Internet information on both field testing and commercialization of new varieties. This information, which is updated daily and provides direct public access to information formerly available only upon written request, is proving very useful both to companies and to individual researchers who wish to track the progress of agricultural biotechnology.
New Varieties Determined to be No Longer Regulated Articles
In the past several months, we have seen the acceleration of
requests for determinations by APHIS that particular field-tested
organisms have no potential for plant pest risk and should no
longer be regulated. These requests, from developers of new
products produced through biotechnology, facilitate the entry of
the products into the marketplace. Thirteen new products in four
crop plants were the subject of such determinations in the past
year:
Three types of products that have been approved merit particular mention here. Virus-resistant squash, the first of which was approved in 1994, was the first crop considered for which there are populations of interbreeding wild relatives in the major areas of squash cultivation in the United States, and with a trait that has the potential in theory to alter the fitness of the crop plant or its wild relatives. The analysis demonstrated that there was no potential for increased weediness of either the crop or its relatives as a result of cultivation in agriculture of the new squash variety. Insect-resistant crops, of which a potato developed by Monsanto Agricultural Company was the first approved in March 1995, may have the potential to offer an alternative to the use of chemical insecticides. The insect-resistant corn developed by Ciba Seeds was the first new corn variety that has been determined not to pose a potential plant pest risk. The modification in the corn line may offer the potential for control of European corn borer, which has not heretofore been effectively managed. Corn is the leading crop in terms of economic importance in the United States.
First Field Trial of a Transgenic Arthropod Approved
In February 1996, APHIS granted a permit for the first limited
field trial release of a transgenic arthropod, Metaseiulus
occidentalis (Acari: Phytoseiidae), in Alachua County, Florida.
This organism, a transgenic predatory mite, may be potentially
useful for the control of spider mite in strawberries and other
crops. The particular transgenic organism was modified only by the
addition of marker DNA sequences, which are not expressed and do
not alter any of its phenotypic characteristics. Upon receipt of
the application, APHIS made it available to the public
electronically and established an expert team for its review. An
Environmental Assessment and Finding of No Significant Impact was
prepared in support of the agency decision to authorize the field
trial, and these documents have also been made available
electronically.
Regulatory and Policy Developments in Progress
We continually reexamine our regulations and strive to keep abreast
of the latest scientific developments to ensure that we are
responsive to the demands of the rapidly expanding technology and
the public. In the interest of such flexibility and
responsiveness, new regulations were proposed in the Federal
Register on August 22, 1995, which, if finalized, would enable
APHIS to extend an existing determination of nonregulated status to
additional organisms that resemble the organism for which the
determination was initially made and extend the notification system
to enable field testing under performance standards of most crop
plant species.
The proposed rule also announces APHIS' intent to develop guidelines on various topics to provide information relevant to our regulations to developers of new varieties and to other interested persons. In addition, the proposed rule would lessen certain paperwork requirements for the research community and for State agricultural officials. The comment period on the proposed rule closed on October 23, 1995.
In April 1995, APHIS convened a conference to discuss safety issues related to the use of transgenic virus-resistant plants in agriculture. APHIS is also discussing these topics in parallel in the international arena through the Organization for Economic Cooperation and Development (OECD).
Efforts on International Harmonization of Regulatory
Approaches
This is the first year in which several new agricultural
biotechnology products developed in the United States are entering
the marketplace nationally and internationally, both as produce and
as bulk commodities. A key effort we have undertaken is to ensure
that these products can enter existing export markets without the
imposition of undue regulatory burdens. We are working on several
fronts to bring about international harmonization of regulatory
approaches for the products of agricultural biotechnology.
(1) We have continued our ongoing bilateral environmental consultations with the Commission of the European Union, and have established a Permanent Technical Working Group on Biotechnology and the Environment. There are, in addition, other informal meetings with representatives of the Commission of the European Union and the key trading partners to discuss developments on policies relating to commercialization of, and trade in, new agricultural commodities.
(2) Under the joint auspices of the OECD Environment and Agriculture Directorates, the United States has taken the lead in a project on the Commercialization of Agricultural Products Derived Through Modern Biotechnology. The aims of this project are to assist Member countries in their oversight of these organisms, specifically in their efforts to ensure safety, to make oversight policies more transparent, and to facilitate trade. The project was initiated with a questionnaire, completed in 1994, which surveyed the regulatory approaches and data considered by participating countries in the evaluation of these products. The project has continued in several directions, which together are aimed at the goals of harmonization and mutual acceptance of data and assessments. One area in which much progress is being made is in the development of scientific consensus documents on specific topics relevant to the environmental biosafety of transgenic plants, such as the biology of particular crop plants and issues associated with the introduction of particular traits into plants (e.g., the use of coat protein genes from plant viruses to confer virus resistance on host plants).
(3) Regular meetings continue to be held with Canada and Mexico to address safety issues relevant to biotechnology-derived commodities that are under regulatory review or already approved in one of our three nations, and to facilitate trade in these products.
(4) APHIS continues to be an active participant in discussions by the Conference of Parties (COP) to the Convention on Biological Diversity. The COP, of which the United States is not a member, has decided that it will negotiate a binding protocol that may affect international transfer of certain biotechnology products. APHIS is working along with other Federal agencies to ensure that appropriate policies are agreed upon to guarantee the safe international use and development of new products derived through biotechnology.
Michael Schechtman, Ph.D.
USDA, APHIS, BBEP, BCTA
mschechtman@aphis.usda.gov
In earlier reports from other labs, some progress in transformation of wheat, rice and maize has been reported, however the frequency was low and in some cases the events were not fully characterized. The new report presents detailed molecular and genetic evidence of stable integration, expression and Mendelian inheritance of transgenes introduced into immature maize embryos. The technique outperformed the biolistic "gene gun" approach, resulting in transformation frequencies of 5 to 30%.
Negative effects associated with other protocols -- abnormal morphology, reduced fertility, and impairment of agronomically important traits -- were not seen. Almost all of the transformants were morphologically normal and 70% were fully fertile. By Southern blot and sequence analysis, most transgenic plants contained a single copy of the T-DNA with no notable rearrangements. Border sequences were similar to those in dicotyledons and rice, suggesting a similar molecular mechanism for T-DNA transfer and insertion.
As seen with several other crops, transformation efficiency was influenced by multiple factors including plant genotype and embryo stage, culture medium, bacterial concentration, vector, and selectable marker gene. The authors suggest that the main difficulty in transformation was not the delivery of DNA into plant cells, but the recovery of cells in which T-DNA had integrated into the chromosomes.
Pat Traynor
Information Systems for Biotechnology
traynor@nbiap.biochem.vt.edu
At issue are the nature of the crop as cultivated in tropical and subtropical regions, and the infrastructure needed to deliver new products to cassava farmers. Cassava has a "nebulous and dynamic gene pool," a reference to its high heterozygosity and allopolyploid nature which, combined with low natural fertility and a lack of resistance genes, seriously hamper conventional breeding efforts. A hardy plant, it will grow in marginal soils unsuited for other crops; poor yields under these conditions are further reduced as much as 70-80% by weeds, pests and diseases.
Cassava is grown by numerous small-scale farmers who vegetatively propagate their crop, thus there are thousands of different land-races, each adapted to local conditions. Assuming genes for disease and pest resistance, nutritional quality, or stress tolerance can be successfully introduced, how many (and which) local cultivars will be transformed? Perhaps a more significant issue is the lack of an established communication network linking cassava growers and breeders. How, exactly, will genetically engineered varieties be delivered into the hands of individual farmers?
These concerns are echoed by Indra K. Vasil in an accompanying analysis which notes that the technology to improve cassava, like almost all agbiotech R&D, is a product of developed countries, not in African or South American countries where cassava is a staple crop for millions of people. He calls on donor agencies which support international agricultural development to work towards correcting the imbalance by ensuring transfer of the technology to countries where it will have practical benefits.
Part of the answer may be found in organizations such as the Cassava Biotechnology Network (CBN), a coordination project funded by the Netherlands Directorate General for International Cooperation, Special Programme on Biotechnology and Development Cooperation. CBN's objectives are to integrate the needs of small-scale cassava farmers, processors, and consumers into biotechnology research planning; stimulate research on high priority topics; and foster the exchange of information, techniques, and research materials. CBN will hold its Third Internationsl Scientific Meeting in Kampala, Uganda this August (contact Dr. Ann Marie Thro, Coordinator, Cali, Colombia; fax +57 2 445-0273; a.thro@cgnet.com).
Pat Traynor
Information Systems for Biotechnology
traynor@nbiap.biochem.vt.edu
In subsequent years it was reported that DNA could bind to and be incorporated into spermatozoa from a diverse array of animals and insects such as mouse, pig, goat, sheep, cattle, chicken, carp, sea urchin, honeybee, and blowfly. Liposome or electroporation methods were often employed to enhance DNA entry into sperm cells. The goal was to use these sperm as vectors in standard artificial insemination or in vitro fertilization procedures to generate transgenic animals. The successful production of transgenic fish (salmon and zebrafish) and poultry (chicken) using DNA-treated sperm was reported in 1991-1993. One unexpected result from these studies was the persistence of the plasmid transgene as an extrachromosomal element. In the case of the zebrafish, however, germline transmission was still shown to occur.
In 1995, researchers from Austria reported in Animal Biotechnology the production of transgenic cattle following artificial insemination with DNA-treated sperm. One in 86 calves or fetuses examined were transgenic by Southern blot analysis. In the first 1996 issue of Animal Biotechnology, the Italian group which first published the sperm-mediated gene transfer method, reported the generation of transgenic pigs using DNA-treated sperm. Of the 82 pigs examined, Southern blot data showed five were transgenic. Interestingly, different DNA plasmids were found to be incorporated into swine blastocysts with widely varying efficiencies, suggesting that the transformation process is DNA sequence-dependent. One major drawback to the sperm-mediated gene transfer method is the observation that rearrangement of plasmid sequences is a common occurrence. This could result in the inactivation of transgene expression.
Sperm-mediated gene transfer is starting to show some promise in livestock species. However, a number of issues, such as understanding the molecular basis of DNA binding to sperm and its subsequent nuclear internalization, still need to be investigated and resolved before this method becomes as routinely utilized as the more established method of microinjecting DNA directly into the zygote pronucleus.
Dr. Eric A. Wong
Department of Animal and Poultry Sciences
Virginia Tech
ewong@vt.edu
RECOMBINANT PROTEINS FROM MILK OF TRANSGENIC ANIMALS - Part
2
TARGETING GENES TO THE MAMMARY GLAND AND REGULATING PRODUCTION
Promoters -- the elements upstream from most genes which regulate
gene function -- from mice, rats, rabbits, sheep, goats, and cattle
have been examined in vivo for their ability to support useful
levels of heterologous protein expression in the mammary gland of
transgenic animals. Results of work to date suggest that most of
these elements are capable of promoting expression, sometimes at
low levels, but that yet-unidentified sequences in some yield much
higher, more desirable levels of expression.
It is also becoming apparent that hormone-inducible transcriptional regulatory elements occur upstream from milk protein genes, and transfected cells display the effect of these, as well as constitutive, elements. In the transgenic animal, chromosomal structure in milk protein genes may also be involved in the tight regulation which is often seen. A major roadblock caused by these regulatory strictures -- the need to test modifications in transgenic animals, at great expense of time and money -- may be avoided by methods currently in development.
Genetic regulatory elements which have been studied in detail include those from the mouse whey acidic protein (WAP) gene. WAP regulatory elements can be used to direct expression of foreign genes to the mammary gland in mice, but also in other species, such as sheep, goats, and pigs. In one report of WAP-directed expression of a transgene in the sheep mammary gland, functional WAP was secreted into milk at 100 - 500 mg per liter. Unfortunately, ectopic expression was also observed, with minimal amounts of WAP found in salivary gland, spleen, liver, lung, heart muscle, kidney, and bone marrow of some transgenic sheep.
Another regulatory system used in transgene expression studies is that from the sheep beta-lactoglobulin gene. Milk yield in transgenic mice bearing this gene and regulatory system was at normal levels, but nearly one-third of the total milk protein (which was equivalent in transgenic and nontransgenic mice) consisted of beta-lactoglobulin, suggesting that the recombinant protein was synthesized in place of, rather than in addition to, normal milk proteins. Explants of transgenic mammary tissue displayed significantly higher total protein synthesis, implying that an in vivo limiting factor was removed in the organ culture system. These results point out a possible determinant of the overall success of this approach: high-level expression by the mammary gland is necessary, but may be compromised by lower-than- desirable production capacity of the mammary gland.
Lines of transgenic mice have also been prepared by introducing constructs consisting of regulatory elements from the rabbit WAP (rWAP) gene linked to the human growth hormone (HGH) structural gene. Milk was produced by less than 50% of the lines, and HGH was found in the bloostream of most or all of these. It may be that ectopic production of this hormone resulted in sterility. However, in those producing milk, the concentration of biologically-active, recombinant HGH in milk ranged from 4 to 22 mg per ml.
Transgenic rabbits may be useful in situations where demand does not exceed 1 kg of recombinant protein per year. Production of recombinant HGH, under the regulation of mouse WAP elements, has been noted in milk and blood of transgenic rabbits, and at least some of the primary transgenic animals transmitted the foreign gene to offspring.
Some Problems Remain
In spite of the promise of recombinant protein production in milk,
several problems must be solved before this process becomes
routine. The vectors carrying the genes coding for the proteins of
interest are still of somewhat unpredictable efficiency, and,
therefore, the expression level of genes associated with milk
protein gene control regions is often unpredictable a priori.
Recombinant proteins secreted in milk are not always processed
satisfactorily; although results of work completed to the present
(about 20 proteins produced on an experimental scale) suggests that
problems of this sort are not insurmountable, the importance of
this cannot yet be discounted. Some have suggested that migration
of recombinant proteins from the mammary alveolar compartment to
the blood can have negative health effects on lactating animals.
Furthermore, it is not inconceivable that pathogenic agents could
pass through processing with the product of interest.
A possible alternative to genetic and physical manipulation of embryos is organ-targeted use of replication-defective retroviruses carrying a gene or genes for protein(s) of interest. Such viruses, bearing the structural gene for HGH, have been introduced into the mammary gland of goats during hormone-induced mammogenesis. Secretion of HGH into the milk followed with the onset of lactation.
Products in Development
New attempts to produce recombinant proteins in mice and larger
animals are being reported continually. Some may be of interest as
examples of the potential of this technology. Mouse WAP or sheep
beta-lactoglobulin regulatory elements have been used to target
expression of recombinant human extracellular superoxide dismutase
to the mammary gland of transgenic mice. Recombinant protein was
fully compatible with native protein in activity and structure.
Scale-up to production in transgenic farm animals could provide
enough recombinant superoxide dismutase for therapeutic use.
Similar work has been done with transgenic mice expressing human
serum albumin under the control of the sheep beta-lactoglobulin
regulatory elements or the goat beta-casein gene, which encodes the
most abundant protein of goat milk.
Human factor IX has been produced in transgenic mice and sheep, and the full activity of the recombinant protein suggests that appropriate post-translational modification was carried out by the mammary gland. Human alpha 1-antitrypsin, produced in the milk of transgenic sheep, is now being prepared on an industrial scale. Human factor VIII and erythropoietin for direct therapeutic use, and human lysozyme and K-casein for beneficial alteration of milk are also in preparation. Recombinant antithrombin, expressed in goat milk at concentrations of 4 g per l, is likely to enter human Phase I clinical trials in the near future. Transgenic goats are now producing a long-acting form of tissue plasminogen activator, and transgenic mice have been constructed to express glutamate decarboxylase for use in type I diabetes. Doubly-transgenic mice produce both furin (intracellular endoprotease required for post-translational processing of proteins) and human protein C, which requires extensive and specific proteolytic processing. Lactoferrin and fibrinogen are also expressed by transgenic animals, and at least one company is preparing gene-deleted cows to produce nutraceuticals" such as lactose-free milk.
As noted in earlier articles, the first transgenic product to be used in people is (or, by now, has been) organs for xenotransplantation. Of particular interest has been the use of livers from pigs engineered to express proteins that block complement-mediated hyperacute rejection. Transgenic pig livers for in vivo perfusion in patients too ill to undergo transplant may be in use as this is printed.
J. Glenn Songer
University of Arizona
gsonger@ccit.arizona.edu
Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis produce potent protein toxins that have been used effectively to control mosquito larval populations. However, these bacteria have not met with the same commercial success as chemical pesticides because the bacterial spores are UV-sensitive, resulting in their rapid inactivation near the surface of water where the larvae feed. Furthermore, the spores rapidly settle from the zone in which the larvae feed, restricting the duration of effective control. These disadvantages limit the use of Bacillus sp. as effective biological control agents. To overcome these problems, researchers have genetically engineered other bacteria to express Bacillus mosquitocidal toxin proteins, but success utilizing this strategy has been limited mainly due to low expression of the introduced toxin genes.
In the March issue of Nature Biotechnology, researchers in Singapore report the production of genetically engineered Asticcacaulis excentricus expressing the binary toxin genes of Bacillus sphaericus. A.excentricus is an aerobic, Gram-negative, motile bacterium that normally reproduces near the water surface. Laboratory studies have demonstrated that the recombinant A. excentricus expresses mosquito larvicidal activity that is close to that of the natural high-toxicity strains of B.sphaericus. The real advantage with A. excentricus is its ability to persist for weeks in the larval feeding zone.
How well this organism can adapt to the number of diverse habitats in which mosquitoes breed while maintaining high level toxin production remains to be determined in field trials. If this biological control method proves to be efficacious, then a program that rotates chemical pesticides with biological control could successfully regulate mosquito population growth while minimizing the probability of selecting for pesticide- or toxin-resistant mosquitoes.
Eric A. Wong
Department of Animal and Poultry Sciences
Virginia Tech
ewong@vt.edu
As has traditionally been the case, biopharmaceutical companies represent the majority of firms within the biotech industry that have benefited from receptive public markets. In light of the impact that drug-focused firms have on overall biotech sector stocks, key factors that contributed to a banner 1995 year include positive clinical trial announcements, numerous FDA product approvals, and an overall positive public market, which across industries funded over 500 offerings worth a record breaking $25 billion (2). In part due to carry over from the success in 1995, 1996 is shaping up to be the best year in history for raising public capital in the biotech industry. In the first 5 months of 1996, biotechnology firms have attracted $2.54 billion in public financing, putting the industry on pace to far surpass even the phenomenal public financing year of 1991 (3). Table 1 shows examples of companies that have pursued public financing in 1996.
In spite of the extraordinary amounts of money that have flowed into biotechnology in 1996, the market appears to be taking a downturn at the mid-year point, illustrating how quickly the window to public capital can close. One securities analyst noted in late June that biotech companies were beginning to be required to have a unique story to tell as well as an attractive stock price in order to get money in a new "antagonistic market." The analyst did not feel that it was a bear market and expected biotech stocks to come back in the second part of the year (4).
Whether or not the public market's mid-year mood swing will be the start of a more dramatic shift in investor sentiment away from biotechnology is yet to be seen. While there are those that believe that this is only a brief detour on an otherwise straight climb to a record breaking year, others are concerned that this may be the beginning of a major market correction for biotechnology stocks (1). Just as positive clinical data helped open the markets in 1995, trial results in the second half of 1996 will again likely determine the availability of public capital for the remainder of the year and beyond.
How have agricultural biotechnology firms done in this financing environment? Only two of 66 companies that have filed with the SEC for public offerings are classified by our Institute as focusing primarily in areas related to agricultural biotechnology (5). These companies are Ergo Science and GalaGen. Agricultural biotech companies appear to be focusing more on partnering as a means of securing capital for product research and development rather than tapping public markets that have been a key financial resource for their biopharmaceutical brethren.
References:
William O. Bullock
Institute for Biotechnology Information, LLC
Research Triangle Park, NC
http://www.biotechinfo.com
The material in this News Report is compiled by NBIAP's Information Systems for Biotechnology, a joint project of USDA/CSREES and the Virginia Polytechnic Institute and State University. It does not necessarily reflect the views of the U.S. Department of Agriculture or of Virginia Tech. The News Report may be freely photocopied or otherwise distributed without charge. P.L. Traynor, Editor.
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