Genetics Society of Malaysia

Transgenic Research In Molecular Pharming

H. Y. Yeang

Abstract

Molecular 'pharming' can be a cost-eficient wa.y to prodttce pepticle-based phar-maceuticals. Genes codingfor proteins of pharmaceutical importance are insertedintct farm animals to produce the foreign proteins in the milk of the transgenicaniruals. Similarly, crop plants are genetically transformed (or act as hosts oftransgenic viruses) to setne as sources of commercially valuable recombinant pro-teins. Protein recovery from the milk oJ'transgenic animals is continual and there-

fore sustained, although building up large numbers of productive transformantscould be problematic. On the other hand, while it is cheap to maintain transgenicplants and easy to mukiply them, continual harvesting of the target protein is notnormally feasible. The transgenic rubber tree combines the favourable attributesof transgenic animals and transgenic plants in terms of low muintenance cost, ease

of propagation and contirutal harryest.

Animals have been used for the production ofvarious biomedical products in the service ofmankind. More recently, DNA engineering has

enabled foreign genes to be inserted into ani-mals with the aim of expressing foreign pro-teins (commonly therapeutic proteins) in themilk of the animals. Milk proteins are secreted

in large quantities by the mammary gland, andthis hence becomes the natural bioreactor thatenables sustained yield of the target protein. Inthe laboratory, small animals such as mice and

rabbits are used in experiments, but for com-mercial production, larger animals, usually farmanimals such as sheep, goats and cows, are

Biotechnology and Strategic Research UnitRubber Research Institute of Malaysia

Email: hyyeang@lgm.gov.my

ts2

favoured (Wall et al., 1997)" Many of the targetproteins are human proteins such as hormones,

enzymes, blood coagulating factors and immu-nological agents. Protein-based pharmaceuti-cals already inproduction, at least in the labora-tory, include anti-thrombin. tissue plasminogenactivator, anti-tr,?sin, human serum albumin,human alpha-glucosidase, human lactoferrin and

various monoclonal antibodies.

The transformation ofanimals for'pharming'begins with the preparation of a gene conshuctin which a specific milk protein promoter is li-gated to the gene coding for the protein of in-terest. Gene transfer is then effected by micro-injecting copies of the cloned gene into the pro-embryo of a recently fertilised egg with the aidof special optics and a micromanipulator sys-

tem. Generally, I to 5% of the manipulatedembryos of farm animals produce transgenics,although the figure could be higher in labora-

Thircl National Congress on Genetics, lB-19 November 1998

tory mice. The level of protein expression inanimals for the same gene consffuct varies notonly betu,een species, but aiso befween indi-viduais. depencling on the opp.lrtune insertionof the gene. 'lhe recent success rn animal clon-rng is obviously advantageous for the niuitipli-cation of selected individual transgemcs (Wilrnut

et al.. 1!i97). although anrmai clonlng i:: s:ill far*rrm being a ro*Iine prc.,cedure today.

I-ik* transi:egic animatr-.,, plants cal L,l 14*-

iretrcaii y rran s f!.)rrnrcl to sx.press prof eil*'r:;l <i:c

nhar*raceuticuJl Flants are cheaper tr: marn-iain than animais and their rnultip[cation thoughinbred seeds is srmple and etficient. Many plants

are also arnenable to vegetative propagation tbrclonal multiplicatron. Growr in the lieid, theyrequire little more than sunlight, water and ba-src horticulturai input to thrive. As ptrarma-e:eutical manufacturing factories, plants are so-

iar-powered and ecologicaliy friendly. In plantfransformation' gene transfer is commonly ef-fected by the bombardment o1'the appropriatepiani tissue (e.g. callus tissue, embryos, etc.iwith DNA of the desired protein using a par-ticie gun, or through mediation by.4grobacteriunr harbounng the gene of interest.

There is active ongoing research on the pro-duction of recombinant vaccines that are ex-pressed in the edible parts of plants, e.g. in ba-

nanas and potato flrbers (Mason and Amtzen,1995). 'fhese 'edible'vaccines could serve as

inexpensive and easy-to-handle vaccines thatwould be especially useful in deveioping coun-tries. The hepatitis B virus (HBV) protein thathas been successfully expressed in tobacco has

been shown to induce the intended immune re-

sponse in mice.

In a variation ofthe strategy to produce for-eign proteins in a crop plant through genetictransfonnation, the plant itself is not trfr,ns-

formed. Instead. the crop plant ac,ts as a hostfor a plant virus vector (e.g. tobacco mosaicvirus, potato virus X) that harbours the foreigngene. The recombinatrt protein that is

synthesised by the virus is attached to the virusprotein coat and harvested with the plant orplant part (Cruz et al., 1996).

The many advantages of transgenic plants

for 'pharming' notwithstanding, their one stg-nificant weakness is the difficulty in recoveringthe recomhrnant protein. Unlike transgenic ani-mal* where protein production is targeted in $*rnilk i:arvt:ltrng of the recombinant protern Mr-;oives riestr',rrtion of tl-re plant or a portrl,n li'1"i. r\5 a. i :::::ii, rlrilietrl re$rJv{1i,1. :s ii}l' ft a-:i,:t ..

lrnual pro,;ess. T'he next harve,,,:i mtgh'; ,lrave i,,r

""varf imiil tjre next repianting seasflx, Evm r.v'hr'it:

i,he li;n'esterlplant is nr-lt complrtel_v- iii:.rfro.i {:c

ie:.9,. if oi'li:! sc!fi)e *.erial parts am renr'r'ecli, r,i

ro,rau!, sliii r*qriile a consrdera'bir perrl..d ri1'r icovcr.v for r,:-.qrcwth before a subs"'quent hal.vest is r:ossihie

'I aking into consideration the strengths nfthe rransglenic animal (continual protein prodtrc-tion in the miik) and the transgenic plant (lorvcost of maintenance, simple clonal propagation )lbr recombinant protein production, it wouldobviously be beneficial to have a productionsystem that combines both advantages. The

transgenic rubber tree meets this requirement.Among plants, the rubber tree is unique in thatit produces voluminous sap (latex) upon taB-

ping, which is a non-destructive method of la"tex exfraction. Latex serum contains about 1..5".i

native proteins, and so the mechanisms for pr*-tein production are present. The free can bt:

tapped every altemate day throughout the yr:ar

without pause. Hence, the transgenic Heve:a.

system allorvs for continual production of tlretarget protein (Arokiaraj et al, 1998).

Successful molecular'pharming' is stili ;;r

its infancy. Whether the recombinant pharnra'ceutical comes from a transgenic animal or r,

transgenic plant, technical and logistic proirlem,:

abound in the scaling up ofproductron frora thr.

laboratory to the field. Even with successfulgene expression, several other crucia! fictorswould determine its ultimate coorrnercial viabil-ity. As the protein is a three-dimensionai moi-ecule with complex folding characteristics. rt i.t

not known if such properties can be faithf'ullvreplicated. Post-translational modification, ifit occurs in the recombinant protein, may alsobe flawed. Having taken heed of all these fac-tors, it might ultimately be the cost-effective-

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Genetics Society of Malaysia

ness of the transgenic plant or animal that willdetermine its success as a conrmercial produc-tion unit. Existing technology already enables

recombinant proteins to be produced by micro-organisms (bacteria, yeasts and fungi) inbioreactors. The 'pharm' would need to matchor better the bioreactor system before it can be

considered a viable alternative. Whetherkansgenic plants or animals are used, factorssuch as the global market size and price elastic-ity of demand for the product would be impor-tant considerations.

References

Arokiaraj, P., Yeang, H.Y., Cheong, K.F.,Hamzah. S., Jones, H., Coomber, S. and

Charlwood, B.V. (1998). CaMV 35S pro-moter directs b-glucuronidase expression inthe latrciferous system of transgenic Heveabrasiliensis (rubber tree). Plant Cell Re-ports, l7:621-625.

Cruz, S.S., Chapman, S., Roberts, A.G., Rob-efis, I.M., Prior, D.A. and Oparka, K.J.( 1996). Assembly and movement of a plant

virus carrying a green fluorescent protein

overcoat. Proceedings of the NationalAcademy of Science, USA, 93:6286-6290.

Mason, H.S. and Arntzen, C.J. (1995).Transgenic plants as vaccine productionsystems. Trends in Biotechnology, l3 :388-

392.

Wall, R.J., Kerr, D.E. and Bondioli, K.R.(1997). Transgenic dairy cattle: geneticengineering on a large scale. Joumal ofDairy Scienc e, 80 :2213 -2224.

Wilmut,I., Schnieke, A.8., McWhir, J., Kind,A.J. and Campbell, K.H. (1997). Viable off-spring derived from fetal and adult mam-malian cells, Nature, 385:8 l0-813.

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