Week 11 Flashcards
What is an overview of current plant derived pharmaceuticals?
Morphine and its derivatives are mainly extracted from poppies by the ton.
Vinblastine and vincristine (anti-cancer drugs) are isolated from the leaves of the Madagascar periwinkle plant.
Artemisinin (anti-malarial) is extracted from the plant Artemisia annua, sweet wormwood
What is an overview of the molecules looked for by the pharmaceutical industry?
METABOLITES (SMALL MOLECULES) AND THE PHARMACEUTICAL INDUSTRY IS LOOKING FOR ALTERNATIVE METHODS OF PRODUCTION.
What exmaples of protein-based pharmaeutical molecules could plants be used to make?
Vaccines, particularly virus-like particles (VLPs).
Antibodies for immunotherapy (cancer treatments, passive
immunisation)
Enzymes for enzyme replacement therapies (e.g. Gaucher’s disease).
What are the advantages of using plants to manufactoring pharmaceuticla proteins?
Produce large biomass
Require minimal inputs
Keep themselves sterile
Have no problem of contamination with animal pathogens
Possibility of edible vaccines?
How can you use plants for protein expression?
Possibility arose from developments in 1980s regarding Agrobacterium-mediated transformation
Followed by the development of transient methods based on the use of plant virus-based vectors and agroinfiltration in the 1990s
What are the categories for plant host for protein farming?
Whole food plant
Whole non-food plant
Unusual plants
What is an overview of whole food plant for protein farming?
e.g. potato, soya, maize
Low risk from plant contaminants
What is an overview of whole non-food plant for protein farming?
e.g. tobacco, Arabidopsis
Low risk of entering food chain
What is an overview of unusual plants for protein farming?
(Duckweed, moss, algae)
Might allow adaptation of traditional microorganism culture techniques
What is an overview of containment when working with plants?
Physical e.g. in containment greenhouses, tissue culture vessels. Problems of expense.
Biological e.g. chloroplast transformation to prevent pollen transfer, use of disabled viral vectors
What is an overview of the different types of stable transformation?
Nuclear transformation – Standard method: genetically modify genome in cell nucleus
Plastid transformation – Modify chloroplast genome, allows better containment, high yield – but a prokaryotic environment
What are the advantages of stable transformation?
Introduced sequence is heritable and true-breeding
(homozygous) lines of plants can be created
Site of expression can be controlled by use of tissue-specific promoters, with no limit on complexity
Well-established technology for creation of plants with different phenotypes such as herbicide resistance
What is an example of a promoter used to maximsie expression levels?
35S promoter from Cauliflower Mosaic Virus. Can be constitutive, tissuespecific
What is an example of a 5’ UTR used to maximsie expression levels?
Cowpea Mosaic Virus RNA-2 5’ UTR with or without translantionenhancing mutation
What is an example of a coding sequence and optional used to maximsie expression levels?
Coding sequence for a protein, fusion protein, synthetic protein
Optional: tag or signal peptide (Nor C- terminal)
What is an example of a 3’ UTR used to maximsie expression levels?
Nopaline synthase (Nos) gene terminator from Agrobacterium tumefaciens TDNA – evolved for plant cell use
What is an early attemot at an edible vaccine?
EDIBLE VACCINE AGAINST NOROVIRUS
What is an overview of the Norovirus?
Small RNA virus which causes intestinal infection
Results in diarrhoea and vomiting particularly in winter and when people are in close contact
58kDa coat protein will self-assemble into particles in heterologous system
Particles will raise antibodies when administered to mice
What is an overview of the pharmancy/lab experince with norovirus?
Cannot grow virus in culture
No vaccine or treatment available
How did they make a norovirus in plants?
Make cassette containing CaMV 35S promoter, translational enhancer, sequence of NV coat protein (CP) and nos terminator
Transform tobacco and potato
Examine expression and aggregation state of CP in regenerated plants
What is an overview the results of a norovirus vaccine in plants?
Expression detected in plants
Expressed protein forms NV particles
What is an overview the mice and human trials of a norovirus vaccine in plants?
Plant-derived particles stimulate mucosal immune response when orally administered to mice
When peeled raw potatoes were fed to human volunteers increased levels of NV antibodies were found
What are the problems with edible norovirus vaccines?
How do you test whether it works?? (As it cant be cultured)
Do you want to eat raw potatoes????
Problems in controlling the dose.
How effective is antibody production in plants?
As early as 1989, it was shown that functional IgG molecules can be synthesised in plants.
IgGs consist of 2 heavy (H) and 2 light (L) chains linked by disulphide bonds
What is an potential advantage of antibody production in plants?
Such molecules have large number of uses in both diagnostics and treatments.
Normally produced in mammalian cells but very expensive
What is an overview of EU sixth framework producing antibodies in plants?
Ran from 2004 to 2012 and involved 33 partners including academic institutions, SMEs and large industry.
Target was the IgG molecule 2G12 which has broad anti=HIV neutralising activity in vitro.
Potential use as a topical microbicide to prevent HIV transmission
What is an overview of the making of antibodies in plants to help reduce transmission of plants?
Transformed with plasmid containing LC and HC genes, linked to selectable and visible markers
High expressers used to generate production lines
Assayed for 2G12 (HIV antibody)
What is the outcome of producing antibodies in plants to help reduce transmission of plants?
Antibodies were substantially similar in physical proterties to mammalian CHO derieved excpet glycan structuture
Indentical in terms of functional properties in all test and efficacious in HIV neutralisation assays
Can be scaled up am pass GMP compliancy
What is an overview of 2G12 production and purification?
GMP compliant downstream processsing achieved
Preclincal toxicology tests approved
Clinical trial plant approved
Phase 1 clinical trials
What is an overview of phase 1 trial plant 2G12 roadmap?
Started in 2008
November 2008, clinical trial application to MHRA
March 2009 initiate clinical trial
Concluded in 2011
What did the 2G12 plant production show?
Plants can produce materials to clinical trial standards
Development of methods to make and extract antibodies
Difficult to devise methods demostrating efficacy
And ability to scale up production
Why is 2G12 plant production not needed?
HIV is now far more treatable by anti-retroviral drugs
Why is cell culure prefered?
More analogous to conventional cell culture
High degree of control of whole process
What is an example of research into plant cell culture vaccine production?
Dow agrosciences aginst Newcastle disease virus (mostly bird virus)
Plasmid made Hemagglutinin-neuraminidase (HN) protein (attachment protein), inserted and isolated in cell cultures
What is an overview of Newcastle disease virus plant vaccine?
Production was effective in disease treatment
LIscence in FDA in 2006
Very costly so wasnt put on market
What is an overview of Protalix?
Company investigated enzyme replacement therapy production
Focused on Gauchers disease which glucocerebrosdide accomulates in cells and organs
What is a disadvantage of Gauchers disease enzyme replacement therapy?
Intravenous recombinant glucocerebrosidase treats symtptoms like skeletal abnormalities and decrease liver size
Costs $200,000 a year and is for life
What is an overview of Protalix glucocerebrosidase production?
Used carrot suspension cultures to produce GCB enzyme
Obtained FDA approbal for human use in 2012 the first approval for human use plant made proteins
Currently used as the orphan drug for treatment
What is the main outcomes from Protalix glucocerebrosidase production?
Plant made material has shown superior pharmokinetics to mammalian cell-made material
Though only given chance due to problems with mammalia cell made material
What is an example of moss based treatments?
Germany produced moss that made antibodies, replacement enzymes and fusion toxins
What is an example of ageing based treatments?
Human growth factor used as anti-wrinkle agents produced in barley
Made in Iceland
What are the limitations of stable transformation approach?
Transformation can be difficult and time consuming especially in crop species
Large variabillity in properties of resultant transgenic plants making evaluation of contructs difficult
Often low level of gene expression meaning only useful for long term consistent production not rapid response to emerging disease
What are alternative role transgenic plants can do?
Modify plants which the proteins are to be expressed
Functions such as glycolysation can be silenced ie to stop fucose and xylose production
Additional functions can be added such as production of sialic acid
What is an overview of transient expression?
Foreign seqeunce to be introduced into developed plants
No heritbale genome change
Expression occurs after a few days
A batch process in response to demand
What are the methods for transient expression?
Replicating virus-based vector
Agrobacterium-mediated expression
Both approached use agro-infiltration
What are the scales of transient expression?
Bench scale (µg of yield)
Pilot scale (mg of yield)
Industrial scale (kg of yield)
What is an overview of a replicating system?
Transcripts produced are amplified within the cells and can spread to neighbouring cells
Very high levels of expression are achived quickily
What are the problems with a replicating system?
Replication is slowed down when the size of the replicon is increased
Practical limitations of the size of proteins made
Difficult to express multiple polypeptides in the same cell due to issues of virus exclusion and competition
What is an overview of the replicating Magnicon sytem?
Developed by Icon genetics
Based on decontructed version of TMV
Agroinculation to deliver 5’ abd 3’ molecuel and Phi recombinase creating functional virus in plants
How does the Magnicon system work?
Virus replicated and spreads cell to cell but but no virons (coat protein deleted)
Gives very high levels of expression
Widely used by academic and commercial labs
What is an overview of zMAPP?
A combination of 3 antibodies to neutralise ebola virus
Produced in N.benthamiana using Magnicon TMV system
Given experimentally to a few ebola sufferes, including Willaim Pooley in 2014
Though small trial meant difficult to assess efficacy
What is an overview of transient expression with non-replicating system?
Translation of mRNA synthesised of T-DNA without amplification or spread of the RNA
Agroinfriltration procedure
mRNA translatable and stable to compact gene silencing
No limit on gene size and co-expressed number
Control the expression levels of different constructs
What is a CPMV-HT non-replicating system overview?
Measured with a GFP protein and UV light
CPMV-HT 5’ overexpression increased by 15 times
What is an overview of pEAQ vector series?
Allow for simple one-step cloning into small binary vector
Modular design allows the expression of multiple proteins
What are the advanatges of using virus like particles?
Similar structure to original virus but not infectious
Reptitive
Toll-Like Receptor ligands
Used in nanotechnology
Useful source of vaccines
What is an overview of virus like particles production method by transient expression?
Plasmid based capsid gene
Amplification of plasmid in E.coli
Transformation of Agrobacterium
Formed by recombination
Confirm by western blot anti-NNV antibodies
What are non-eveloped virus like proteins made by CPMV-HT?
Bovine paplioma virus
Hepatitis B core particles
Blue tongue virus
Poliovirus
What is an overview of polio disease?
Destroys motor neurons
Results in poliomyelitis
Bulbar polio the nerve cells of the brain stem get attacked
RNA virus spread by oral-faecal route
What is the structure of polio?
MAde up of three structural proteins VP1, VP2 and VP3
What is an overview of polio vaccines?
Salk vaccines (IPV) - Consists of inactivated poliovirus, introduced in the 1950s and admistered by Jab
Sabin vaccines (OPV) - Live attenuated virus administered orally, introduced in the 1960s
Highly effective at eliminating most global cases
What are the problems of current polio vaccines?
OPV can cuase vaccine-associated paralytic poliomyelitis and allows for circulting vaccine derived poliovirus
IPV provides poor cut immunity meaning silent spread. Production levels need to be in large quantities
What are the problems with using virus like particles?
In absence of RNA, VLPs are relatively unstable
Rapidly undergo native to heated cells changing antigenticy making them immunologically useless
How do you make VLPs?
P1 - contains all the structural regions
P3 - Protease 3c and 3d to help make (to high expression causes host shut off)
All seperate plasmids
What was the result of the first plant made polio VLPs?
Entirely the active heated or C form at 1.58 optical density
Type 1 IPV - 1.38 optical density at 1/20 dilution in D form compared to 0.2 optical density at 1/20 dilution
How do you thermostablise polio VLPs?
Serial dilution picking those that best resist increasing temperatures
Use these as base for plasmid, and innoculate
This thermostabilsed
What were the results of the thermopolio VLPs?
D polio - 3 optical value of D virus, also 2.6 optical value of C, though doesnt matter at 1/20
Type 3 IPV - 2 optical value and 0.6 Optical value at diluiton 1/20
What is the efficacy of viral like particle immune response?
Neutralising antibody values that mirror those of current IPV vaccines
What is a potential problem with enveloped virus?
More complicated than non-enveloped as they contain host components (membranes)
Enveloped proteins are usually glycosylated
Maturation often uses host-encoded proteases eg furin
Correct assembly involves interactions between different compartments in a coordination fashion
Why are enveloped virus vaccines important?
Many important viruses are envoloped: dengue, zika, influenza and coronavirus
How can this technology be applied to covid?
Has to handled under phase 3 containment which is expensive
So VLP can be made to study mechanisms such as attachment and be more viable to more people
What is needed to make covid VLPs?
S, M and E enveloped proteins,
Glycosylation apparatus
What is useful to know about covid VLPs?
S, M and E are produced from seperate subgenomic RNAs in the virus, makign design of expression cassettes easier
Some of the major antigenic sites are structural rather than linear
What is an overview of the important envelop protein in covid?
Only S is needed for VLPs Membranes
Makes formed coronavirus structure
These react to the immune system
What is an overview of glycosylation in covid?
All bar one are glycosylated
Can be overcome by making in plants which lack fucos and xylose transferase enzymes
How can you commertialise VLPS?
Either sell licensing to other companies interested in it or make your own facility
What is an overview of commertlising covid VLPs?
Sold to Medicago (now part of Mitsubishi Tanabe) reported the results of a phase 3 efficacy study
In 2021, Medicago announced sucessful phase 3 efficacy and safety
What was the result of medicago plant covid trials?
Based on modified version of the S protein though issue about WHO approval due to involvement of tobacco company
Medicargo was shut down in 2023 by parent company
What is needed to build your own facility?
Growth of plants under controlled contained conditions
Mechanism for the efficient agroinfiltration
Facility to extraction of products
Efficient way of disposing waste
What is an overview of Leaf expression systems?
On Norwich Research Park
Opened on 23rd Jan 2017
What are the differences between plant and animal pharmeceutical expression?
Difference in glycosylation paterns - not concrete whther it matters
Difference in degree of cross-linking between expression systems
Can plant based production compete?
Even if plants are not used as final production platform it can contribute to the development of new pharamaceuticals and help other emerging tech like mRNA
