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ISIS Report 22/07/05

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. Details here

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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