Expression and Manufacture of Pharmaceutical Proteins in Genetically Engineered Horticultural Plants - Transgenic Horticultural Crops: Challenges and Opportunities - page 87

expression.cassette,.and.the.target.protein-coding.sequences.will.be.expressed.as.a.
by-product.of.the.viral.genome.replication.cycle.under.the.control.of.subviral.pro-
moters. 19,20 . In. a. variation,.recombinant.viral.genomes.can.be.integrated.into.plant.
chromosomes. via. transformation.. The. subsequent. transcription. of. viral. RNA. will.
initiate.vial.replication.and,.in.turn,.drive.the.expression.of.the.target.pharmaceutical.
protein. 21 .Overall,.the.robust.transcription.and.ampliied.translation.of.the.plant.viral.
components.allow.the.transient.expression.systems.to.yield.high-level.accumulation.
of.recombinant.proteins.
Viral. “deconstruction”. is. a. recent. application. of. using. viral. genomes. to. cre-
ate.transient.transgene.expression. 22,23 .This.newly.deconstructed.viral.vector.sys-
tem.is.based.on. in planta .assembly.of.replication-competent.tobacco.mosaic.virus.
(TMV).and.potato.virus.X.(PVX).genomes.from.separate.provector.modules. 23,24 .
Agrobacterium tumefaciens . is. used. to. deliver. DNA. modules. along. with. a. sepa-
rate. construct. that. produces. a. recombination. integrase.. Upon. its. expression,. the.
integrase.assembles.separate.DNA.modules.into.a.replication-competent.TMV.or.
PVX.genome.under.the.control.of.a.plant.promoter..This.assembled.DNA.construct.
is. then. transcribed. and. spliced. to. generate. a. functional. infective. replicon.. This.
“Maginfection”.system. combines. the. advantages.of. three. biological. systems. 22,24 .
First,.the.laborious.in.vitro.process.of.generating.RNA-based.vectors.is.eliminated.
due.to.the.use.of. Agrobacterium .as.viral.vector.delivery.tool..Second,.the.deletion.
of.viral.coat.protein.genes.in.this.system.allows.the.speed.and.high.protein.yield.
of.a.viral.system.without.the.threat.of.creating.functional.infectious.particles..In.
addition,. “Maginfection”. integrates. the. posttranslational. processing. capacity. of.
eukaryotic. plant. cells.for. producing. complex. proteins..Peaks. of. PMP. accumula-
tion.are.usually.detected.between.7.and.14.days.after.vector.delivery. 24 .This.system.
thus.gains.the.lexibility.of.nuclear.gene.expression.with.the.speed.and.expression.
ampliication.of.viral.vectors.
All.three.plant.expression.strategies.are.likely.to.have.their.place.in.the.com-
mercial. realm. of. producing. pharmaceutical. proteins.. Expression. systems. using.
stably. transformed. plants. represent. an. inexpensive. and. permanent. genetic. prop-
agation. source. for. pharmaceutical. protein. production. 25 . However,. this. develop-
ment.of.technology.has.been.hindered.by.several.scientiic.and.regulatory.issues..
First,.it.requires.a.relatively.long.time.frame.to.create.and.select.the.initial.stable.
transgenic.plants..In.addition,.the.expression.level.of.the.transgene.product.can.be.
luctuated.wildly.or.unstable.not.only.between.individual.plants.but.also.between.
different.generations.of.the.same.plant.line..These.uncertainties.are.often.caused.
by.the.randomness.of.transgene.insertion.into.the.plant.nuclear.genome.(the.“posi-
tion. effect”). and. by. the. phenomenon. of. posttranscriptional. or. siRNA-dependent.
gene.silencing..The.latter.may.be.triggered.by.the.presence.of.a.particular.mRNA.
in.high.concentration,.leading.to.both.immediate.and.potential.permanent.produc-
tion.instability. 26-31 .The.potential.risk.of.unwanted.transgene.outlow.from.ields.
with. genetically. modiied. (GM). plants. to. neighboring.ields. with. non-GM. crops.
or.their.wild.relatives.has.also.raised.regulatory.and.public.acceptance.issues.for.
this.technology..These.scientiic.and.regulatory.hurtles.have.been.addressed.by.a.
growing.number.of.academic.and.industrial.laboratories..For.example,.in.response.
to.regular.concerns.for.potential.transgene.escape.through.outcrossing,.transgenic.