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[SANET-MG] algal recombinant pharmaceuticals in Hawaii



June 18, 2005
Prof. Joe Cummins
“Algal recombinant pharmaceuticals in Hawaii”
The Hawaii Department of Agriculture ,on 25 May 2005, heard a proposal from Mera Pharmaceuticals for a permit to begin large scale production of a genetically modified alga, Chlamydomonas reinhardtii, modified to express a full length immunoglobulin-A moleculae against a variant of the herpes simplex virus .the permit was denied by thee Plant Quarantine Honolulu Office. However another application from Mera Pharmaceutical has been scheduled for October 7,2005, it calls for an additional 7 strains to be imported and produced in the Mera facility in Hawaii. The strains included the following:
 1.  Strain aFceR 1r-1:Molecule targeting the Fc
portion of the IgE molecule limiting the interaction
between circulating IgE molecules and receptors on
mast cells, which is turn limits the release of
histamines and reduces inflammation.
2.  Strain aTNFr-1: IgG1 anti-tumor necrosis factor
antibody.
3.  Strain a TNr-1: IgG1 anti-microbial antibody.
4.  Strain aCRr-1:  IgG1d anti-cell proliferation
antibody.
5.  Strain aBSSsr-1:  Anti-Cancer cell specific
antibody.
6. Strain aIL 10r-1: Various interleukins (various including interleukin 10, interleukin 13. interleukin 5 and interleukin 3)
7.  Strain aARTr-1:  Neurotrophic factors to stimulate
frowth of new nerve tissue.
The petition from Mera Pharmaceutical described the Chlamydomas strains in broad detail with little or no molecular detail describing the actual genetic makeup of the strains. The petition provided some broad and groundless assurances such as in the “worst case scenario” in a complete failure of there reactor growth facility the risk from a health perspective would be negligible because the strains , as well as their expressed proteins are completely harmless unless injected. Since the petition did not provide adequate information about the genetic and molecular modifications of the alga the following discussion will deal with some considerations about the genetic modification Chalamydomonas and actual concerns about the products produced in the modified algae. Transgenic microalgae serve as green cell factories for producing pharmaceuticals. Either chromosomal or chloroplast transgenes can be produced with ease and a number of transgenic algae have been prepared. Chlamydomonas reinhardii is a preferred organism for such experiments because its genome has been sequenced and it has a long history of laboratory culture One technical drawback with the alga is codon bias related to the high GC content in 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(1). The synthetic human DNA in the alga should not be deemed equivalent until it has been tested for untoward effects on humans and the environmental biota. Mayfield and Franklin described construction of transgenic Chlamydomonas reinhardii whose chloroplasts had been modified for expression of human antibodies. The human genes were synthetic approximation extensively adjusted for codon bias. Either the rbcl promoter (ribulose-1,5-bisphosphate carboxylase of the chloroplast) or the atpA promoter (alpha -subunit of the chloroplast ATP synthase of the chloroplast) were used to drive the antibody gene. The rbcL transcription terminator followed the human gene. A 16s (small) ribosomal subunit with resistance to the antibiotic accompanied the human gene transformation(2). IgA antibody directed against herpes simplex virus were produced in Chlamydomonas reinhardii (3). Single chain antibodies against the herpes virus were produced in Chlamydomonas reinhardii (4). In order to achieve adequate production of the human gene products the DNA code of the human gene the genetic code must be optimized for the codon bias of Chlamymononas. The code letter alterations are rather extensive so that the synthetic human DNA inserted into Chlamydomonas bears little resemblance to the original human gene. The numerous codon alterations to optimized production of recombinant protein in the alga have been described (5). Of course, code optimization need not lead to alteration in the amino acid sequence of the protein produced from the recombinant gene but the protein produced from the synthetic gene must be proved to be identical to the protein produced from the original human gene. In the petition for production both codon optimization by producing synthetic gene and the proof that the protein produced from the synthetic gene was identical to the original human gene was not mentioned. The petition failed to provide minimal information about the genetic makeup of the strains to be introduced into Hawaii. On top of that the fact that synthetic approximations of human genes was not mentioned. The claim that the risk of contacting the recombinant products was negligible even in the worst case seems to have been groundless because there were no experiments reported that support that conclusion. For example, strain aFceR produces 1r-1 molecule targeting the IgE to reduce inflammation. The petition did not provide evidence showing that the protein was inactivated on ingestion nor did it discuss problematic side effects. Strain TNFr-1 produces anti-tumour necrosis factor antibody, as above there was no evidence that the protein was inactivated on ingestion nor was there any discussion of the a potential immune response leading to anaphylaxis following repeated exposure. Strain TNr-1 produces IgG1d antimicrobial antibody, that antibody seems unlikely to be inactivated by ingestion and repeated exposure is likely to result in anaphylaxis. Strain aCRr-1 produces anti-cancer proliferation antibody, there was no evidence presented on the destruction of the antibody on ingestion nor of the problem of anaphylaxis following repeated exposure. Strain aIL 10r-1produces various interleukins (various including interleukin 10, interleukin 13. interleukin 5 and interleukin 3). Interleukins are very potent regulators of the immune system and they are active in minute quantities. The petition failed to provide evidence that the interleukins were destroyed on ingestion but pulmonary exposure to interleukin 13 , for example, causes inflammation, mucus hypersecretion , physiologic abnormalities associated with asthma (6). Interleukin 10 is a powerful immune suppressant (7).The many adverse effects of the interleukins should have been discussed in the application but were not. Strain aARTr-1 produced unspecified neurotrophic factors to stimulate growth of nerve tissue. Since the factors were ill defined it is not possible to evaluate any effects of the nerve factors or their stability. In conclusion, the Mera Pharmaceuticals application for a permit to produce recombinant pharmaceuticals in Hawaii employing recombinant Chlamydomonas algae was defiecient in the main areas related to the safety of the recombinant microbes. The molecular genetic description of the genetic modification were simply ignored, there was no acknowledgement that the strains were modified with synthetic approximations of the human genes and no effort was made to insure that the products of the synthetic genes were identical to the human proteins that they approximated. Even if the products of the synthetic genes can be shown to be identical to the products that they approximate the claim that such products are inactive on ingestion should have been documented with experiments. It staggers the mind to think that an application lacking fundamental safety information might be approved anywhere.
References
1. Leon-Banares R, Gonzalez-Ballester D, Galvan A. and Fernandez E. Transgenic microalgae as green cell-factories. Trends Biotechnol. 2004 Jan;22(1):45-52 2. Mayfield,S. and Franklin,S. Expression of human antibodies in eukaryotic micro-algae Vaccine 2005 ,23,1828-32 3. Franklin S and Mayfield S . Recent developments in the production of human therapeutic proteins in eukaryotic algae. Expert Opin Biol Ther. 2005 Feb;5(2):225-35 4. Mayfield SP, Franklin SE and Lerner RA. Expression and assembly of a fully active antibody in algae. Proc Natl Acad Sci U S A. 2003 Jan 21;100(2):438-42 5. Franklin S, Ngo B, Efuet E and Mayfield S. Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast. Plant J. 2002 Jun;30(6):733-44 6. Zhu Z, Homer RJ, Wang Z, Chen Q, Geba GP, Wang J, Zhang Y and Elias J Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J Clin Invest. 1999 Mar;103(6):779-88 7. Kamradt T, Goggel R and Erb K Induction, exacerbation and inhibition of allergic and autoimmune diseases by infection. Trends Immunol. 2005 May;26(5):260-7

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