Cell Factories - Molecular Pharming

Question 1

Molecular pharming summary

Answer 1

New technology, mostly untried but capable of very large scale production using agricultural technologies
-> Includes production of pharma in transgenic livestock (e.g secreted in milk) and in transgenic plants

Question 2

Potential issue with molecular pharming?

Answer 2

Segregation from food chain: PT processing is not necessarily correct

Question 3

Transgenic livestock in molecular pharming..

Answer 3

Using livestock as bioreactors for production of pharma useful proteins in milk

Question 4

Advantage of transgenic livestock in molecular pharming..

Answer 4

Large scale production using normal agricultural practice for collecting milk
- from which secreted protein would be collected

Question 5

Transgenesis in molecular pharming

Answer 5

Based on delivery of DNA to in vitro-fertilized eggs or other embryonic manipulations. Done by…

• microinjection into the zygote pronucleus
• Or by liposome-transfection of embryo-derived fibroblasts

The resulting transgenic embryos are implanted into a surrogate mother and the offspring checked for transgene presence and expression. Successful integrants can then be used for subsequent breeding to develop a transgenic herd.

For particularly valuable individuals, there is also the potential to clone animals (using the techniques developed to produce Dolly the sheep).

Question 6

Liposome-transfection of embryo-derived fibroblasts in transgenesis of molecular pharming

Answer 6

• cultured until transgene insertion is confirmed
• then microinjected into the blastocyst of an early embryo (essentially embryonic stem cell manipulation).
• latter approach also allows “gene targeting” to specific loci:
  although inefficient:
• ensures a stable genetic locus,
• and can allow regulated transgene expression, if a transgene is targeted to a gene encoding a milk protein (e.g.casein)

Question 7

ATryn

Answer 7

By GTC Biotherapeutics (now rEVO Biologics) = only milk-produced protein licensed for therapeutic use.

• > Recombinant form of human antitrhrombin: blood clotting inhibitor
• > 1/5000 have antitrhrombin deficiency

Recombinant form =
- produced in goats milk t/f covered in dif glycosides (specific to goats milk) so not recommended for people w/ goats milk allergy

Question 8

Plant - produced pharmaceuticals in molecular pharming

Answer 8

The potential for ^ production scaled than fermentation-based technologies

• w/o need for ultra-clean plant and machinery
• OR controlled growth conditions

Estimated that 250 acres of greenhouse space = sufficient to grow enough transgenic potato to meet S.asia annual demand for hep B vaccine

Question 9

Why are most crop-based pharma compound productions based around expression in cereal seeds?

Answer 9

1) Cereal production is supported by agri technologies for harvest
2) Cereal seeds have ^ protein content associated w/ low water content = easy storage and no cold chain needed to maintain stability of products
- important in dev world
3) seeds also provide protection against fungal metabolites

Question 10

How to achieve ^ transgene expression in plant produced pharmas?

Answer 10

Through the use of highly active endosperm-specific promoters
AND
Regulation of gene expression using regulatable aleurone-specific promoters

Question 11

Barley for transgenic production

Answer 11

Maltagene = developed barley as a production system for the manufacture of recombinant forms of
- Lactoferrin,
- Lysozyme (antimicrobial proteins found in milk, tears and other secretions)
- Human Serum Albumin (used in blood-substitutes for tranfusion during surgery:
such substitutes are important in an age when donated blood may be infected with HIV, Hepatitis C etc)
- Hepatitis B Surface Antigen.

Question 12

Advantages of barley for transgenic production

Answer 12

• Strongly self pollinating, t/f “pharma” crops are unlikely to cross-pollinate food crops
• Very good phenotypic marker available (seed colour) enabling segregation of pharma- and food-crops
• Malting characteristics of barley are very well understood, enabling regulated expression of transgenes during the malting process.
• Process is carried out on an industrial scale in all breweries, so it utilises existing technology.

Question 13

Maize for pharming

Answer 13

Widely grown in the USA, and genetically very well characterised.

“ProdiGene”
currently produces two maize-derived recombinant products:
- “Trypzean” – recombinant trypsin
-“Aprolizean” – the serine proteinase inhibitor aprotinin. Used as a proteinase inhibitor in the downstream processing and purification of recombinant proteins, and also medically in the treatment of cardiac surgery patients.

Both have widespread use in mammalian cell culture media.

Question 14

Rice in pharming

Answer 14

Ventria
Markets rice-produced transferrin under the trade name:
- “Lacromin”
Transferrin is used as a mammalian cell culture medium component = replaces bovine serum albumin-based products that carry a potential risk of contamination with prions (BSE/CJD) .

Currently in commercial use in the culture of cells for monoclonal antibody production
- is sold in kilogram quantities, and it outperforms the “traditional” bovine products.

Question 15

Oilseeds in pharming

Answer 15

Non cereal systems are based on production in oilseeds - in particular, oilseed rape.
Company Stratosome:
has developed a technology for targeting transgenic protein production to the oil bodies.

Question 16

How does oilseeds work in pharming?

Answer 16

In oilseeds:

• oil is stored in oil bodies that are contained within a protein coat, or shell
• The protein subunits of this coat are amphiphilic polypeptides called oleosins, which comprise hydrophobic alpha-helices that sit in the oil, and hydrophilic chains that form the interface with the aqueous cytosol.

Recombinant proteins are fused with the cytosolic domain of the oleosin, so that rapid purification is possible.
- When an aqueous homogenate is made of plant tissue rich in oils, the oil bodies form a layer on the surface after centrifugation.
This enables rapid and straightforward purification of the oleosin-fusion proteins.

Question 17

Problem w/ plant-derived phamas?

Answer 17

Proteins become glycosylated after translation.

In mammals, glycosylation is often required for optimal activitry, but it has to be the right glycosylation.
- Plant glycoproteins are often decorated with fucose and xylose, which can be highly antigenic moieties.

It is therefore necessary to “humanise” the recombinant protein.

Question 18

Greenovation…

Answer 18

Uses moss in which gene targeting has been used to knock out the glycosyltranferase genes that regulate the addition of plant-specific sugars to glycoproteins.

• This results in the production of glycoproteins that are much more similar in profile to the native human proteins, and which have been found to be:
  (i) non-immunogenic and
  (ii) superior activity compared with the original human antibody.

Question 19

Mossess in pharming

Answer 19

Mosses were among the first land plants, so are intrinsically interesting from an evolutionary standpoint
– but they are also being exploited for the biotechnological production of pharmaceutical proteins (“Bryotechnology”).

P. patens

• is the best characterised moss
• it has a sequenced genome (the first non-flowering land plant to be sequenced) and can be easily transformed.
• a particular utility is that transforming DNA integrates at homologous loci, allowing very efficient “gene targeting” – like yeast.

Additionally, moss tissues are one-cell thick, have eukaryotic secretion systems and export proteins to the medium.