MOLECULAR PHARMING

By: Vikas CJ

History

Introduction

Conclusion

Applications of transgenic animals in pharmaceuticals

Methods of creating transgenic animals

Contents

Reference

The term ‘pharming’ comes from a combination of the words ‘farming’ and ‘pharmaceuticals’

Molecular pharming is the process of using transgenic animals to produce human drugs and therapeutic proteins.

Using genetically modified plants or animals to produce pharmaceuticals, also called as Gene Pharming; part of Molecular Farming

Introduction

Transgenic animals are animals which have been genetically transformed by splicing and inserting foreign, animal or human genes into their chromosomes. The inserted gene, when successful, enables an animal to make a certain pharmaceutical protein in its milk, urine, blood, sperm, or eggs, or to grow rejection-resistant organs for transplant.

• The term transgenic was first used by J.W. Gordon and F.H. Ruddle in 1981.

• The first chimeric mice were produced in 1974 by Brinster.

History

BrinsterRuddle

In 1986 Gossler et al introduced the embryonic stem (ES) cell-mediated gene transfer.

In 1976 Jaenisch introduced the retrovirus-mediated transgenesis.

In 1981 Gordon and Ruddle performed the DNA microinjection method to creat transgenic mouse.

• DNA microinjection.

• Embryonic stem cell-mediated gene transfer.

• Retrovirus-mediated gene transfer.

Methods To Creat Transgenic Animals

• This method involves the direct microinjection of a chosen gene construct (a single gene or a combination of genes) from another member of the same species or from a different species, into the pronucleus of a fertilized ovum.

• The manipulated fertilized ovum is transferred into the oviduct of a recipient female, or foster mother

• This method is one of the first methods that proved to be effective in mammals.

1. DNA microinjection.Gordon and Ruddle used DNA microinjection technique for the 1st time in mice

• This method involves prior insertion of the desired DNA sequence by homologous recombination into an in vitro culture of embryonic stem (ES) cells.

• These cells are then incorporated into an embryo at the blastocyst stage of development.

• This technique is of particular importance for the study of the genetic control of developmental processes.

2. Embryonic stem cell-mediated gene transfer.

• To increase the probability of expression, transgene transfer is mediated by means of a carrier or vector, generally a virus or a plasmid

• A retrovirus is any virus in the family Retroviridae that has RNA as its nucleic acid and uses the enzyme reverse transcriptase to copy its genome into the DNA of the host cell's chromosomes.

• Transmission of the transgene is possible only if the retrovirus integrates into some of the germ cells.

3. Retrovirus-mediated gene transfer.

• In medical research, transgenic animals are used to identify the functions of specific factors in complex homeostatic systems through over- or under-expression of a modified gene (the inserted transgene)

• In toxicology: as responsive test animals (detection of toxicants)

• In mammalian developmental genetics;

A representative list of purposes for which transgenic animals have been used indicates the wide ranging application of this biotechnology

• In molecular biology, the analysis of the regulation of gene expression makes use of the evaluation of a specific genetic change at the level of the whole animal;

• In the pharmaceutical industry, targeted production of pharmaceutical proteins, drug production and product efficacy testing;

• In biotechnology: as producers of specific proteins;

• Developing animals specially created for use in xenografting.

Transgenic Animals And Their Applications

The animal whose genetic material is taken from another organism is called as transgenic animal. The artificial genes are inserted in the germline cells of the organisms so they pass from one generation to the other. First transgenic animal was mouse which is the most important animal for doing experiments in the laboratory. But after that many other animals were also genetically modified through genetic engineering techniques for example rabbit, pig, sheep,fish and cattle.

Uses of transgenic cows There are many potential uses of transgenic cows in biomedicine and agriculture.

Biomedicine• Making therapeutic protiens Transgenic cows can be used as ‘biofactories’ to produce

human therapeutic proteins (proteins that are used to treat diseases)• In June 2006, the first therapeutic protein made in a transgenic animal was approved for

use in Europe and the USA. ATryn, a human antithrombin protein, is made in transgenic cows. The protein prevents blood clots in patients who don’t make their own version of this protein.

• At AgResearch, they’ve generated cows that produce human myelin basic protein. Treatment with human myelin basic protein may help reduce the symptoms of multiple sclerosis.

• Transgenic cows are made to produce proteins lactoferrin and interferons in their milk.• Prion free cows resistant to mad cow disease.

Transgenic cows

• Transgenic cow used to make human myloma basic producing gene for the tretment of multiple sclerosis.

Trangenic sheep

Transgenic sheep are made to produce CFTR

Until recently, the transgenes introduced into sheep inserted randomly in the genome and often worked poorly. However, in July 2000, success at inserting a transgene into a specific gene locus was reported. The gene was the human gene for alpha1-antitrypsin, and two of the animals expressed large quantities of the human protein in their milk.

α1-antitrypsin is produced in milk. Used to cure emphsysema, a lung

disease in man.

Cystic fibrosis

Transgenic pig The production of human haemoglobin in the blood of transgenic pigs for isolation and treatment of trauma patients is one of the interesting applications being studied. The production of Protein C, in-activator of certain human coagulation factors Va and VIIIa in the milk of pigs has been studied. It has been found that the mammary epithelial cells of the pigs are capable of making the coagulation factors VIII and IX biologically active due to post transcriptional modifications.

The production of human hepatocytes in transgenic pigs to help in the transplantation of the regenerated human hepatocytes to patients of liver failure from the transgenic pigs shows great promise.

Transgenic pig made to produce tissue plasminogen activator thrombosis

Transgenic chicken

Transgenic Chickens grow faster than sheep and goats and large numbers can be grown in close quarters;Synthesize several grams of protein in the "white" of their eggs.

Preliminary results from transgenesis method indicate that it may be possible for chickens to produce as much as 0.1 g of human protein in each egg that they lay.Transgenic chicken are made to produce monoclonal antibodies for the vaccines production

Transgenic mice

Alzheimer’s mouse– In the brain of Alzheimer’s patients, dead

nerve cells are entangled in a protein called amyloid.

– Mouse made by introducing amyloid precursor gene into fertilized egg of mice.

Oncomouse– Mouse model to study cancer– Made by inserting activated oncogenes.

Smart mouse– Biological model engineered to overexpress NR2B receptor in the synaptic

pathway.– This makes the mice learn faster like juveniles throughout their lives.

Transgenic Fish

•The transgenic technology is becoming increasingly popular as a powerful experimental tool in developmental biology of model fish, notably the zebrafish (Danio rerio) and medaka (Oryzias latipes).

•Now the GFP transgenic fish system is actively used to investigate gene expression patterns, tissue/organ development, tissue-specific promoters/enhancers, cell lineage and migration, upstream regulatory genes,mutagenesis screening and characterization

A plasmid containing human coagulating factor VII complementory DNA regulated by a cytomegalovirus promoter was microinjected into fertilized eggs of zebra fish, african catfish, and tilapia. The active form of hfVII was detected in the fish embryos by various assays. This positive expression of human therapeutic protein in fish embryos demonstrates the possibility of exploitation of transgenic fish as bioreactors.

Superfish– Increased growth and size– Growth hormone gene inserted into fertilized egg.– Transgenic salmon grows about 10 – 11 times faster than

normal fish.

Pharming products currently in development

Animal Drug/protein UseSheep CFTR treatment of cystic fibrosisSheep tissue plasminogen activator treatment of thrombosisSheep factor VIII, IX treatment of hemophiliasheep fibrinogen treatment of wound healingpig, tissue plasminogen activator treatment of thrombosisPig factor VIII, IX treatment of hemophiliaGoat human protein C treatment of thrombosisgoat antithrombin 3 treatment of thrombosisGoat glutamic acid decarboxylase treatment of type 1 diabetesGoat Pro542 treatment of HIVcow alpha-lactalbumin anti-infectionCow factor VIII treatment of hemophiliaCow fibrinogen wound healingcow collagen I,collagen II tissue repaircow lactoferrin treatment of GI tract infection,and infectious arthritisCow human serum albumin maintains blood volumechicken, cow, goat monoclonal antibodies other vaccine production

Conclusion The ultimate aim of biopharma development is to improve the quality

of life and to extend longevity. The quest for new drugs is never ending, as is the need to understand disease causes beyond the symptoms. The world market is growing for human pharmaceutical products. Producing transgenic animals is still relatively expensive, however, costs are trending down and transgenic animals have certain advantages over traditional laboratory methods for producing human proteins and drug. The future for biopharmaceuticals production is indeed extremely bright and offers an good opportunity.

Reference•Prathibha Nallari, V Venugopal Rao, Medical Biotechnology.pg no151-162.•Satyanarayana U, Biotechnology, Uppala author-publisher interlinks, page no-480-490•Channarayappa, Molecular Biotechnology – Principles and Practices (2006), 1st edition, University Press Pvt Ltd, Hyderabad.•http://people.ucalgary.ca/~browder/transgenic.html •Kumaresan V, Biotechnology, Saras publications, page no-233-243

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