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TITLE:  GM Pharmaceuticals from Common Green Alga
SOURCE: The Institute of Science in Society, UK
        http://www.i-sis.org.uk
DATE:   22 Jul 2005

------------------- archive: http://www.genet-info.org/ -------------------


GM Pharmaceuticals from Common Green Alga

Amid widespread protests against using crop plants to produce genetically
modified pharmaceuticals, companies are turning to the single-cell green
alga Chlamydomonas touted as a "safe" alternative; but not when it is to
be grown in largescale outdoor bioreactors Prof. Joe Cummins and Dr. Mae-
Wan Ho

A fully referenced version of this article is posted on ISIS members'
website http://www.i-sis.org.uk/full/PICGA.php. Details here http://
www.i-sis.org.uk/membership.php

Largescale production of GM algae producing human proteins

The Hawaii Department of Agriculture received an application from Mera
Pharmaceuticals, originally filed 1 November 2004, for a permit to begin
large-scale production of a genetically modified (GM) alga, Chlamydomonas
reinhardtii, producing a human immunoglobulin-A protein against a variant
of the herpes simplex virus. On 17 April 2005, Mera Pharmaceuticals
revised their application to include seven additional GM strains
expressing a range of human antibodies, interleukins and nerve growth
factors (see Box) [1]. The case for the first GM strain was heard on 25
May 2005, and the Plant Quarantine Honolulu Office did not grant the permit.

The application for the seven other GM strains had been scheduled for 7
October 2005, but was brought forward to 29 June. On that occasion, the
state Board of Agriculture gave its approval [2]. Mera Pharmaceuticals
will begin the process of importing the microalgae from The Scripps
Institute, La Jolla, California, even as opponents are seeking to block
the permit, which was approved on a six-to- two vote after nearly three
hours of testimony and an hour of discussion.

Single cell green algae are serving as green cell factories for producing
pharmaceuticals, and have been touted as a safe alternative to producing
them in crop plants, because they can be contained in the laboratory [3,
4]. Unfortunately, Mera's proposed large-scale cultivation is not
contained. Anything from 500 to 26 000 litres of culture are housed in
transparent "Outdoor Photobioreactors", which are cooled with cold
seawater with added chlorine [1]. The exposed facilities are prone to
weather, storm and other damages, resulting in immediate massive
contamination of the marine environment. In addition, the use of
chlorinated seawater for cooling will be expected to impact on marine
life. Neither of these concerns appeared to have been addressed. Henry
Curtis, executive director of Life of the Island, told the Board that the
nonprofit organization will file for a contested case hearing.


GM Chlamydomonas strains to be imported into large-scale culture facility
in Hawaii
1. Strain Hsv8, producing a full-length human immunoglobulin-A against a
variant of the herpes simplex virus
2. Strain aFceR 1r-1, producing a protein targeting the Fc portion of the
IgE molecule, thereby limiting the interaction between circulating IgE
molecules and receptors on mast cells, which is turn limits the release
of histamines and reduces inflammation.
3. Strain aTNFr-1, producing IgG1 anti-tumor necrosis factor antibody.
4. Strain a TNr-1, producing IgG1 anti-microbial antibody.
5. Strain aCRr-1, producing IgG1d anti-cell proliferation antibody.
6. Strain aBSSsr-1, producing anti-cancer cell specific antibody.
7. Strain aIL 10r-1, producing various interleukins (including
interleukin 10, interleukin 13. interleukin 5 and interleukin 3)
8. Strain aARTr-1, producing neurotrophic factors to stimulate the growth
of new nerve tissue.


The GM algae will be imported into Kona on the island of Hawaii to be
grown in the outdoor bioreactor system at Keahole Point at the state's
aquaculture park Nelha. As Nancy Redfeather, Director of Hawaii Genetic
Engineering Action Network (Hawaii GEAN ) points out, this type of "field
trial" of a biopharmaceutical algae has never been attempted before in
the United States, and all government agencies, the Food and Drug
Administration (FDA), the US Department of Agriculture (USDA) and
Environment Protection Agency (EPA) had waived oversight of the trial.
The native Hawaii algal systems have not been well documented; nor has
the ecology of Chlamydomonas itself. Apart from some last minute attempt
to conduct basic environmental experiments of the survival of GM algae,
there are no peer-review studies, or studies of any kind to back up Mera
Pharmaceutical's claim of "no harm to the environment or human health."

Written and oral testimony by the public was overwhelmingly opposed to
the project. The board also ignored testimony and reports by a number of
local algae experts from the University of Hawaii, Manoa, the State
Biologist and Maui Country District Health Officer. Other scientists
providing testimony include R. Malcolm Brown Jr., the Johnson and Johnson
Centennial Chair in Plant Cell Biology at the University of Texas at
Austin; Marti Crouch, Doug Sherman from Friends of the Earth, and Joe
Cummins and Mae-Wan Ho from ISIS.

Malcolm Brown's message to the board was, "Hawaii is still the supreme
ecosystem on earth to understand the dynamics of evolution and natural
selection. Let's not forever lose this opportunity because a few
commercial operations thoughtlessly tried to construct mass scaleup of
genetically modified organisms in Hawaii."

Nancy Redfeather said, "It was indeed a sad day for the native algae of
Hawaii island.


The Chlamydomonas reinhardtii transformation

Chlamydomonas reinhardtii is a preferred organism for molecular pharming
by chloroplast transformation because its nuclear and chloroplast genomes
have both been sequenced, and it has a long history of laboratory
culture. In addition, it has a single chloroplast, which makes it easy to
produce a uniformly transformed culture. One technical drawback with the
alga is codon bias related to the high GC content of the algal DNA, To
achieve significant production, the code of human and many other genes
must be altered to fit the bias of the algal cell, For that reason the
human pharmaceutical products are produced from synthetic approximations
of the human gene [5]. The synthetic human DNA in the alga, and all the
more so, the expressed transgene, should not be deemed equivalent to the
human gene and gene product until they have been tested for untoward
effects on humans and other organisms in the environment. It is already
known that the proteins are not subject to post- translational
modifications [3] as they would be in human cells, and hence likely to be
treated as 'foreign' by the human immune system.

Mayfield and Franklin described construction of transgenic Chlamydomonas
reinhardtii whose chloroplasts had been modified to express human
antibodies [6]. The human genes were extensively adjusted for codon bias.
Either the rbcl (ribulose-1,5-bisphosphate carboxylase of the
chloroplast) promoter or the atpA (alpha-subunit of the chloroplast ATP
synthase of the chloroplast) promoter were used to drive the antibody
gene, with the rbcL transcription terminator following the human gene. A
16S ribosomal subunit with resistance to the antibiotic accompanied the
human gene transformation. Using this system, IgA antibody directed
against herpes simplex virus were produced [6], as well as single chain
antibodies against the herpes virus [3]. The numerous codon alterations
to optimize production of recombinant protein in the alga have been
described [7]. Code optimization need not change the amino acid sequence
of the protein produced from the recombinant gene, but Mera's petition
contained no proof that the protein produced from the synthetic gene was
identical to the original human gene, nor the fact that the transgenes
were synthetic approximations to the human genes.

Although not prominently stated, the GM strains probably all contained in
addition a kanamycin resistance marker gene [1], which is necessary to
select for the transformed cells.


Risks from the GM alga

The claim that the risk from contact with the recombinant products was
negligible even in the worst case is groundless, because no experiments
were reported to support that conclusion.

As pointed out already ("Molecular pharming by chloroplast
transformation", this series), producing pharmaceuticals in chloroplasts
entails specific risks due to the large quantities of transgenic proteins
produced and the hazards of horizontal gene transfer to bacteria due to
homologies between the chloroplast and bacterial genomes.

Large quantities of transgenic proteins are produced from multiple copies
of transgenes present, in the case of Chlamydomonas, about 50 to 100 per
cell. Strains 2-6 (see Box) produce antibodies that bind to
immunologically active proteins that could lead to anaphylaxis (severe
life- threatening allergic reaction) following repeated exposure. Strain
7 produce interleukins, potent regulators of immune functions active in
minute quantities. Pulmonary exposure to interleukin 13, for example,
causes inflammation, mucus hypersecretion, physiologic abnormalities
associated with asthma [8], while interleukin 10 is a powerful immune
suppressant [9]. Strain 8 produces unspecified neurotrophic factors to
stimulate growth of nerve tissue, again, potent molecules active at very
low concentrations, whose effects, especially at high concentrations are
completely unknown.

An additional hazard from the gene products is that they are in all cases
not the same as the human protein, because of the changes in making the
synthetic gene copies, and because there is no post-translational
processing. They may hence be treated as immunologically 'foreign' by the
human immune system, resulting in dangerous complications.

Horizontal transfer of the GM Chlamydomonas transgenes are likely to
occur in all environments, particularly in the soil, where Chlamydomonas
is commonly found, but also in the marine environment and in the
gastrointestinal tract of all animals. Horizontal transfer of transgenes
can occur both from the accidental release of genetically modified
Chlamydomonas reinhardtii itself, or from the intentional release of
transgenic DNA in the large amounts of transgenic wastes that are likely
to be discharged from the large scale culture facilities into the environment.

As already mentioned, horizontal transfer and recombination of transgenes
could create new bacteria and viruses that cause diseases and spread
antibiotic resistance marker genes to the pathogens




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