Role Of Genetic Engineering In Improvement Of

Pharmaceutical Production of Microorganisms

Dr.Nawfal Hussein AldujailiDepartment of biology ,College of Science

University of Kufa, 24 March , 2011

Conference Marcht 24, 2011

Strain Improvement - After an organism producing a valuable product is

identified, it becomes necessary to increase the product yield from fermentation to minimise production costs. Product yields can be increased by

(i) developing a suitable medium for fermentation,

(ii) refining the fermentation process and

(iii) improving the productivity of the strain.

Strain Improvement The techniques and approaches used to genetically

modify strains, to increase the production of the desired product are called strain improvement or strain development.

Strain improvement is based on the following three approaches:

(i) mutant selection, (ii) recombination, and(iii) recombinant DNA technology.

Strain improvement Virtually all biotherapeutic agents in clinical use are

biotech pharmaceuticals.

A biotech pharmaceutical is simply any medically useful drug whose manufacture

involves microorganisms or substances that living organisms produce (e.g., enzymes).

Most biotech pharmaceuticals are recombinant—that is,

produced by genetic engineering. Insulin was among the earliest recombinant drugs.

Recombinant DNA Technology

The ability to combine the DNA of one organism with the DNA of another organism.

Recombinant DNA technology was first used in the 1970’s with bacteria.

1. Remove bacterial DNA (plasmid).

2. Cut the Bacterial DNA with “restriction enzymes”.

3. Cut the DNA from another organism with “restriction enzymes”.

4. Combine the cut pieces of DNA together with another enzyme and insert them into bacteria.

5. Reproduce the recombinant bacteria.

6. The foreign genes will be expressed in the bacteria.

Bacterial Transformation• The ability of bacteria to

take in DNA from their surrounding environment

• Bacteria must be made competent to take up DNA

Microorganisms as Tools

Yeast are Important Too!Single celled eukaryote Kingdom: FungiOver 1.5 million speciesSource of antibiotics, blood cholesterol lowering

drugsAble to do post translational modificationsGrow anaerobic or aerobicExamples: Pichia pastoris (grows to a higher

density than most laboratory strains), has a no. of strong promoters, can be used in batch processes

Cloning and Expression Techniques• Fusion Proteins

Microorganisms as Tools

Yeast Two-Hybrid System• Used to study protein interactions

Microorganisms as Tools

Recombinant Microorganisms

The revolutionary exploitation of microbial genetic discoveries in the 1970s, 1980s and 1990s depended heavily upon the solid structure of industrial microbiology, described above.

The major microbial hosts for production of recombinant proteins are E. coli, B. subtilis, S. cerevisiae, Pichia pastoris, Hansenula polymorpha and Aspergillus niger.

The use of recombinant microorganisms provided the techniques and experience necessary for the successful application of higher organisms, such as mammalian and insect cell culture, and transgenic animals and plants as hosts for the production of glycosylated recombinant proteins.

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The Production of Commercial Products by Recombinants Microorganisms

Molecular biotechnology can be used to enhance the production of many commercially important compounds e.g. Vitamins Amino acids Antibiotics

We will be investigating the use of recombinant organisms to improve or enhance the production of : Restriction enzymes Ascorbic acid Microbial synthesis of the dye indigo Production of xanthan gum

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Therapeutic Agents Before the advent of molecular biotechnology

most human proteins were available in only small (limited) quantities.

Today hundreds of genes for human proteins have been cloned, sequenced, expressed in the host cells and are being tested as therapeutic agents (drugs) in humans.

Types of biomolecules produced through recombinant DNA technology

Recombinant Hormones Insulin (and its analogs), growth hormone, follicle stimulating hormone,

salmon calcitonin.

Blood products Albumin, thrombolytics, fibrinolytics, and clotting factors ( Factor VII, Factor

IX, tissue plasminogen activator, recombinant hirudin )

Cytokines and growth factors Interferons, interleukins and colony stimulating factors (Interferon, α, β and γ,

erythropoietin, interlukin-2, GM-CSF, GCSF )

Monoclonal antibodies and related products Mouse, chimeric or humanized; whole molecule or fragment; single chain or

bispecific; and conjugated (rituximab, trastuzmab, infliximab, bevacizumab)

Recombinant Vaccines Recombinant protein or peptides, DNA plasmid and anti-idiotype

(HBsAg vaccine, HPV vaccine)

Recombinant Enzymes Dornase– α (Pulomozyme), Acid glucosidase (Myozyme), α –L-

iduronidase (Aldurazyme) and Urate Oxidase

Miscellaneous products Bone morphogenic protein, conjugate antibody, pegylated

recombinant proteins, antagonist

The market for recombinant therapeutics has considerably improved with generation of new molecules and new expression systems. Even though the overall world market has been around $30-40 billion it is expected to reach around $75 billion by end of this decade.

In fact the major money producer has been few biomolecules such as insulin, erythropoietin, interferon and hormones. These few molecules take a major share of biopharmaceutical market (Table 2). Among these biomolecules, erythropoietin is the most valuable product followed by insulin, growth factors and interleukin. It is projected that erythropoietin market will be around $10 billion by next five years.

Human therapeutics from recombinant DNA technology

One of the greatest benefit of the recombinant DNA technology has been the production of human therapeutics such as hormones, growth factors and antibodies which are not only scarcely available but also are very costly for human use. Ever since the recombinant insulin was produced by Eli Lilly in 1982, considerable efforts has been made world wide to clone and express many therapeutically important proteins, which are otherwise difficult to produce either by extraction from the natural sources or by chemical synthesis.

Therapeutic proteins are preferred over conventional drugs because of their higher specificity and absence of side effects. Therapeutic proteins are less toxic than chemical drugs and are neither carcinogenic nor teratogenic. Further, once the biologically active form of a protein is identified for medical application, its further development into a medicinal product involves fewer risks than chemical drugs. Notable diseases for which recombinant therapeutics have been produced include diabetes, hemophilia, hepatitis, myocardial infarction and various cancers.

Recombinant therapeutics include proteins that help the body to fight infection or to carry out specific functions such as blood factors, hormones, growth factors, interferons and interleukins. Starting with simple protein such as insulin and then growth hormone, recombinant biopharmaceuticals has increased considerably in recent years.

Till today, around 165 biopharmaceuticals (recombinant proteins, antibodies, and nucleic acid based drugs) have been approved. Table 1 lists the type of biomolecules that have been produced by the recombinant DNA technology. This includes hormones, growth factors, blood products, monoclonal antibody, enzymes and many others.

Production using recombinant DNA technology has made these molecules available for the treatment of human diseases at a relatively lower cost. Availability of large amount of pure molecules has helped in development of its different modified form to have improved pharmacokinetic parameters.

Pegylated proteins and controlled release formulation of biomolecules have become reality with improved characteristics. This has not only helped in cheap availability of the biomolecules for health care but also has led to development of new molecules having improved performances. The best example being different varieties of insulin analogs (long acting , slow release, acid stable etc). Others are being pegylated proteins such as peg-interferon and peg-antibodies and growth factors.

The most notable applications of the recombinant technology having direct impact on humanity have been:

1. Large scale production of therapeutic protein such as insulin, hormones, vaccine and interleukins using recombinant microorganisms.

2. Production of humanized monoclonal antibodies for therapeutic application

3. Production of insect resistant cotton plant by incorporation of insecticidal toxin of Bacillus thuringiensis (Bt cotton plant).

4. Production of golden rice (rice having vitamin A) by incorporating three genes required for its synthesis in rice plant.

5. Bioremediation by the use of recombinant organisms and 6. Use of genetic engineering techniques in forensic medicine.

Hormones Recombinant human insulin became the first

manufactured, or commercial, recombinant pharmaceutical in 1982, when the FDA approved human insulin for the treatment of cases of diabetes that require the hormone.

Before the development of recombinant human insulin, animals (notably pigs and cattle) were the only nonhuman sources of insulin. Animal insulin, however, differs slightly but significantly from human insulin and can elicit troublesome immune responses.

The therapeutic effects of recombinant human insulin in humans are identical to those of porcine insulin, and it acts as quickly as porcine insulin, but its immune-system side

effects are relatively infrequent. Further, it can satisfy medical needs more readily and more affordably. Other recombinant hormones include those described below. Regular insulin ordinarily must be injected 30 to 45 minutes before meals to control blood glucose levels. Lispro (Humalog)—a recombinant insulinlike substance—is faster-acting than regular insulin. Because injection of lispro is appropriate within 15 minutes before meals, using it instead of regular insulin may be more convenient for some patients. ( ) Lispro.

Erythropoietin (EPO), a hormone produced by the kidneys, stimulates the bone marrow to produce red blood cells. The FDA has approved recombinant EPO—epoetin alfa—for the treatment of anemia due to chronic renal failure. Epoetin alfa.

Human growth hormone (hGH) is used to counter growth failure in children that is due to a lack of hGH production by the body. Before the introduction of recombinant hGH the hormone was derived from human cadavers. Cadaver-derived hGH was susceptible to contamination with slow viruses that attack nerve tissue. Such infective agents caused fatal illnesses in some patients. Recombinant hGH has greatly improved the long-term treatment of children whose bodies do not produce enough hGH.

Recombinant human growth hormone.

1. Clotting Factors Inadequate bodily synthesis of any of the many clotting factors can

prevent effective clotting. The FDA has approved two clotting-related recombinant drugs: abciximab for the prevention of blood clotting as

an adjunct to angioplasty, and recombinant antihemophiliac factor (rAHF) for the treatment of hemophilia A. Hemophilia A is a lifelong hereditary disorder characterized by slow clotting and consequent difficulty in controlling blood loss, even from minor injuries. About 20,000 persons in the United States alone have this condition, which is due to a deficiency of antihemophiliac factor (AHF, or

factor VIII). Before the introduction of rAHF, treatment of hemophilia A required protein concentrates from human plasma. Such concentrates could contain contaminants (e.g., HIV), and the lifetime

treatment of a single patient required thousands of blood contributions. Persons with hemophilia B lack factor IX. They require either factor IX concentrates from pooled human blood or factor IX from cell cultures (some of which are genetically engineered).

Using Microbes Against Other Microbes• Antibiotics• Act in a few key ways

• Prevent replication• Kill directly• Damage cell wall or prevent its synthesis

Using Microbes for a Variety of Applications

Improving Antibiotic Production 12,000 antibiotics have been identified

Most from Gram-positive soil bacterium Streptomyces Some from fungi and other Gram-positive or Gram-

negative bacteria

100,000 tons of antibiotics produced per year 200-300 new discoveries per year

Cost to bring new one to market very high 1-2% prove useful

Fleming’s fungal production 2 U/ml, now 70,000 U/ml Biotechnology…

Cloning Antibiotic Genes

Mutate antibiotic producing strain to antibiotic negative

Introduce plasmids from genomic library of wt strain

Use clones to screen large insert library

Pathway may require up to 20-30 steps…

Production of Penicillins and Cephalosporins

Most Common Microbially Synthesized Antibiotics

Antibiotics Produced by Strptomyces Strains and Those Transformed by Plasmids Pij2303 and Pij2315

Vaccines In every modern vaccine the main or sole active ingredient consists of killed

microorganisms, nonvirulent microorganisms, microbial products (e.g., toxins), or microbial components that have been purified. All these active ingredients are antigens: substances that can stimulate the immune system to produce specific antibodies. Such stimulation leaves the immune system prepared to destroy bacteria and viruses whose antigens correspond to the antibodies it has learned to produce. Although conventionally produced vaccines are generally harmless, some of them may, rarely, contain infectious contaminants.

Vaccines whose active ingredients are recombinant antigens do not carry this slight risk. More than 350 million persons worldwide are infected with the virus that causes hepatitis B, a major cause of chronic inflammation of the liver, cirrhosis of the liver, and liver cancer. ( ) Hepatitis B kills a million people each year worldwide. About 1.25 million Americans harbor the hepatitis B virus (HBV);

30 percent of them will eventually develop a serious liver disease. About 300,000 children and adults in the U.S. become infected with HBV each year, and 5,000 Americans die annually from liver disease

Some of the foreign genes that have been expressed in recombinant vaccinia viruses.

Vaccines

First was a vaccine against smallpox (cowpox provides immunity)• DPT-diphtheria, pertussis, and tetanus• MMR –measles, mumps, and rubella• OPV- oral polio vaccine (Sabin)

A Primer on Antibodies• Antigen- foreign substances that stimulate an immune

response• Types of leukocytes or white blood cells

• B-lymphocytes: antibody-mediated immunity• T-lymphocytes: cellular immunity• Macrophages: “cell eating” (phagocytosis)

Vaccines

Heavy chain

Light chain

IgA – first line of defense

IgG and IgM – activates macrophages

Vaccines

Antigens stimulate antibody production in the immune system

Vaccines

Mechanism of Antibody Action

How are vaccines made?• They can be part of a pathogen (e.g. a toxin) or

whole organism that is dead or alive but attenuated (doesn’t cause disease)

• Subunit (toxin) or another part of the pathogen• Attenuated (doesn’t cause disease)• Inactivated (killed)

What about flu vaccines (why do we have to get a shot every year?)

Vaccines

Recombinant VaccinesRecombinant Vaccines

Vaccines – provide immunity to infectious microorganisms

Attenuated Vaccine Inactivated Vaccine Subunit Vaccine

Recombinant VaccinesRecombinant Vaccines

Recombinant Vaccines• A vaccine produced from a cloned gene

Video: Constructing Vaccines

Recombinant VaccinesRecombinant Vaccines

DNA vaccines• Direct injection of

plasmid DNA containing genes encoding specific antigenic proteins

Bacterial and Viral Targets for Vaccines

HIV

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Principles of Vaccination A vaccine renders the recipient resistant to

infection. During vaccination a vaccine is injected or given

orally. The host produces antibodies for a particular

pathogen. Upon further exposure the pathogen is

inactivated by the antibodies and disease state prevented.

Generally to produce a vaccine the pathogen is grown in culture and inactivated or nonvirulent forms are used for vaccination.

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Principles of Vaccination There are many disadvantages and they

include: Not all organisms can be cultured. The procedure is expensive and sometimes

unsafe. New pathogens keep occurring. For some pathogens e.g. HIV vaccination is

not appropriate. why?

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New Generation of Vaccines

Recombinant DNA technology is being used to produce a new generation of vaccines.

Virulence genes are deleted and organism is still able to stimulate an immune response.

Live nonpathogenic strains can carry antigenic determinants from pathogenic strains.

If the agent cannot be maintained in culture, genes of proteins for antigenic determinants can be cloned and expressed in an alternative host e.g. E. coli.

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New Generation of Vaccines There are three types of vaccines we will be

discussing: Subunit (protein) vaccines Attenuated vaccines Vector vaccines Subunit Vaccines Antibodies usually bind to surface proteins of the

pathogen or proteins generated after the disruption of the pathogen.

Binding of antibodies to these proteins will stimulate an immune response.

Therefore proteins can be use to stimulate an immune response.

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Principles of Vaccination

It has been showed that the capsid or envelope proteins are enough to illicit an immune response. E.g:

Herpes simplex virus envelop glycoprotein O. Foot and mouth disease virus capsid protein (VP1) Extracellular proteins produced by Mycobacterium

tuberculosis.

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A Subunit Vaccine for M. tuberculosis (e 2nd)

Tuberculosis is caused by Mycobacterium tuberculosis.

The bacterium form lesions in the tissues and organs causing cell death. Often the lung is affected.

About 2 billion people are infected and there are 3 million deaths/year.

Currently tuberculosis is controlled by a vaccine called BCG (Bacillus Calmette-Guerin) which is a strain of M. bovis.

M. bovis often responds to diagnostic test for M. tuberculosis.

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A Subunit Vaccine for M. tuberculosis

Six extracellular proteins of M. tuberculosis were purified.

Separately and in combinations these proteins were used to immunized guinea pigs.

These animals were then challenged with M. tuberculosis.

After 9-10 weeks examination showed that some combinations of the purified proteins provided the same level of protection as the BCG vaccine.

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Attenuated Vaccines Attenuated vaccines often consists of a

pathogenic strains in which the virulent genes are deleted or modified.

The Development of a Live Cholera Vaccine. Live vaccines are more effective than a killed or

subunit (protein) vaccines. With this in mind a live vaccine for cholera was

developed. Cholera is characterized by fever, dehydration

abdominal pain and diarrhea.

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A Live Cholera Vaccine The causal agent of cholera is Vibrio cholerae

and is transmitted through contaminated water. V. cholerae produces a enterotoxin with an A

subunit and 5 B subunits. Presently the cholera vaccine consist of a phenol-

killed V. cholerae and it only last 3-6 months. A live vaccine would be more effective. In the sequence of the A peptide a tetracycline

resistance gene is inserted.

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A Live Cholera Vaccine

A plasmid with A peptide was digested with 2 restriction enzymes Cla1 and Xba1.

This removes 550 bases of A peptide. A Xba1 linker was added and T4 ligase used to

ligate the DNA. This plasmid was mixed with V. cholerae with tetracycline resistant gene.

By conjugation the plasmid was transferred to the strain with the tetR gene inserted into it’s chromosomal DNA.

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Production of a Live Cholera Vaccine

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A Live Cholera Vaccine

By recombination the A peptide with the tetR gene was replaced by the deleted A peptide.

The final result is V. cholerae with a 550 bp of the A peptide deleted.

If this can be used as a vaccine is being tested.

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Production of a Live Cholera Vaccine

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Vector Vaccine

A vector vaccine is a vaccine which is introduced by a vector e.g. vaccinia virus.

The vaccinia virus as a live vaccine led to the globally eradication of the smallpox virus.

The genome of the vaccinia virus has been completely sequenced.

The virus replicates in the cytoplasm rather than in the nucleus.

The vaccinia virus is generally nonpathogenic.

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Vector Vaccine

These characteristics makes the vaccinia virus a good candidate for a virus vector to carry gene for antigenic determinants form other pathogens.

The procedure involves: The DNA sequence for the specific antigen is

inserted into a plasmid beside the vaccinia virus promoter in the middle of a non-essential gene e.g. thymidine kinase.

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Vector Vaccine The plasmid is used to transform thymdine

kinase negative cells which were previously infected with the vaccinia virus.

Recombination between the plasmid and vaccinia virus chromosomal DNA results in transfer of antigen gene from the recombinant plasmid to the vaccinia virus.

Thus virus can now be used as a vaccine for the specific antigen.

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Insertion of antigen gene into vaccinia virus genome

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Vector Vaccine

A number of antigen genes have been inserted into the vaccinia virus genome e.g.

Rabies virus G proteinHepatitis B surface antigen Influenza virus NP and HA proteins. A recombinant vaccinia virus vaccine for rabies is

able to elicit neutralizing antibodies in foxes which is a major carrier of the disease.

Monoclonal Antibodies Other Biotech Drugs Listed below are a few of the hundreds of other biotech drugs that are either

in clinical use or undergoing scientific investigation. Biotech vaccines undergoing investigation include vaccines for acellular

pertussis (whooping cough), AIDS, herpes simplex, Lyme disease, and melanoma.

Two new recombinant interferons are undergoing investigation: consensus interferon, for treating hepatitis C; and recombinant beta interferon 1a, for multiple sclerosis.

Recombinant PTK (protein tyrosine kinase) inhibitors may have therapeutic utility against diseases marked by cell proliferation, such as cancer, atherosclerosis, and psoriasis. Protein tyrosine kinases contribute to cell division and are the targets of these biotech drugs.

Recombinant human interleukin-3 is undergoing clinical investigation as an adjunct to traditional cancer chemotherapy.

Two recombinant growth factors (cytokines that regulate cell division) are undergoing major clinical trials: recombinant human insulin-like growth factor (rhIGF-1) and recombinant human platelet-derived

growth factor-BB (PDGF). PDGF can contribute to wound healing.

In December 1997 the FDA approved clinical testing of a recombinant version of the cytokine myeloid progenitor inhibitory factor-1 (MPIF-1). MPIF-1 can keep certain normal cells, including many immunologically important cells, from dividing and can thus protect them from anticancer drugs that target rapidly multiplying cells. When such anticancer drugs affect normal cells that divide rapidly, hair loss, nausea, and immunosuppression can result.

Injecting the recombinant protein fibroblast growth factor (FGF-1) into the human myocardium increases the blood supply to the heart by inducing blood-vessel formation. ( ) Such treatment, called a "biologic bypass" or "biobypass," does not require surgery. FGF-1 is injectable nonsurgically into the myocardium by cardiac catheterization. A biobypass may benefit persons with coronary artery disease whose arteries are not reparable surgically. (A gene-therapy form of biobypass, VEGF gene therapy, is described below.)

In January 1998 advisors to the FDA recommended that the agency approve Apligraf, a recombinant skin replacer, for the treatment of leg ulcers due to poor circulation; and DermaGraft, another such product, for the treatment of diabetic ulcers. About 800,000 diabetic foot ulcers occur in the U.S. annually, and they lead to most of the lower-leg amputations that approximately 60,000 diabetics

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Production of Monoclonal Antibodies

Monoclonal antibodies results from a clone of a B lymphocyte producing a single antibody which will bind to a specific epitope of an antigen.

What is a polyclonal antibody?

Monoclonal antibodies are produced: Fusion of a myeloma (B cell which has become

cancerous) with a spleen cell that is immunized with a specific antigen.

The resulting hybridomas are tested for the production of a monoclonal antibodies.

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Production of Monoclonal Antibodies

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Production of Human Monoclonal Antibodies by E. coli

Hybridoma cells grow relatively slow and require expensive media.

To circumvent this problem human monoclonal antibodies are grown in E. coli.

The produce involves: mRNA is isolated from the B cell. cDNA is synthesized from the mRNA by the

enzyme reverse transciptase. Both heavy and light chains are amplified

separately from the cDNA using PCR. The amplified products are cut with restriction

enzymes and cloned into Lambda vector.

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Production of Human Monoclonal Antibodies by E. coli

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Production of Human Monoclonal Antibodies by E. coli

During cloning different light and heavy chains are cloned.

The DNA of one heavy and one light chain are cloned into the same vector.

Many different combinations of H and L chains are cloned together in the same vector.

Lambda is not useful for producing large amounts of proteins.

The L and H chains are excised from Lambda and cloned into an E. coli plasmid and the recombinant plasmid transformed into E. coli.

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Production of Human Monoclonal Antibodies by E. coli

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Production of Human Monoclonal Antibodies by E. coli

E. coli will produce large amounts of monoclonal antibodies which are harvested.

These monoclonal antibodies can be used for: Diagnostic purposes e.g detection of HIV by

ELISA. Therapeutically for the treatment of infection.

Biosynthesis of Amino Acids Amino acids uses in food industry Flavor enhances Antioxidants Nutritional supplements Amino acid uses in agriculture Feed additives Amino acid uses in medicine Infusion solutions Amino Acid uses in industry Starting materials for polymer and cosmetic

production

Commercial Applications of Amino Acids

Cysteine Biosynthesis by E. coli

Serine acetyltransferase is feedback inhibited

Site-directed mutagenesis Transform into E. coli

strain that does not degrade cysteine

Even better feedback insensitive enzyme genes isolated as cDNAs from Arabidopsis and transformed into strain

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Enzymes as Therapeutic Agents/ DNase1

• Cystic fibrosis (CF) is one of the most fatal heredity diseases among European and their descendants with ~30,000 cases in the US and 23,000 in Canada.

• Furthermore among European descendants it occurs in 1 in 2,500 live birth and 1 in 25 are carriers.

• It is caused by more than 500 different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

• Individuals with CF are highly susceptible to bacterial infection and antibiotic treatment often results in resistant strains.

Thanks for your

attention

A practical example: Manufacturing human insulin

• Insulin a hormone that regulates the level of sugar in the blood

• • People with defective genes for insulin have diabetes and must take insulin shots

• • Before recombinant DNA, insulin was obtained from animal tissue:

• Goal: • to insert human insulin gene into a

bacteria so that the bacteria can produce it for our use.

• Relatively inexpensive (already done)

Steps for making insulin

• Plasmids are obtained from bacteria cells, cleaved with a restriction endonuclease

• • Human chromosomes (DNA) are collected and cleaved with the same restriction enzyme

• – many fragments of chromosomal DNA are formed, but only some of the fragments contain the insulin gene.

• • Chromosomal and plasmid DNA fragments are mixed together with enzyme called DNA ligase

• recombinant plasmids are then put back into bacteria, yeast or some other rapidly dividing type of cell

• • We screen our library to identify which bacterial colonies contain recombinant plasmids with the insulin gene --

• We now have a collection of recombinant DNA plasmids

• – some contain the insulin gene, while others do not

• – this collection of plasmids is called a DNA library.

Therapeutic proteins• Recombinant insulin in bacteria

Using Microbes for a Variety of Applications

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Nucleic Acids as Therapeutic agents (e 3rd)

• Many human disorders e.g. cancer and inflammatory conditions (virus, parasites) are often caused by overproduction of a normal protein.

• Theoretically a small ss nucleic acid can hybridize to a specific gene or mRNA and diminish transcription or translation.

• An oligonucleotide (oligo) that binds to a gene and blocks transcription is an antigene.

• An oligo that binds to mRNA and blocks translation is called an antisense oligo.

• Ribozyme (catalytic RNA) and interfering RNA ( RNAi) can target specific mRNA for degradation.

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Antisense RNA

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Antisense Oligonucleotide

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Antisense RNA• Episomally based expression vectors with cDNA for

insulin-like growth factor 1 (ILGF-1) receptors were constructed in the antisense version.

• ILGF-1 is prevalent in malignant glioma a common form of brain cancer and prostate carcinoma.

• Culture of glioma cells when transfected with the antisense version of ILGF-1 in ZnSO4 lost its tumurous properties.

• A similar treatment of mice which were injected with prostate carcinoma cells caused small or no tumor to develop.

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Antisense Oligonucleotides

• Antisense deoxynucleotides can also be used as therapeutic agents.

• However when injected into the body is deoxynucleotides are susceptible to degradation.

• To prevent this modified deoxynucleotides are used including phosphorothioate, phosphoramidate and polyamide.

• Free oligos are usually introduced into to the body encapsulated in a liposome.

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Modified Deoxynucleotides

Phosphodiester linkage Phosphorothioate linkage

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Modified Deoxynucleotides

Phosphoramidite linkage Polyamide linkage

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Liposome

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Therapeutic Nucleic Acids• Several preclinical trials have been conducted

with antisense oligos.• Narrowing of the coronary and carotid arties can

lead to heart attacks and strokes.• This condition is alleviated by angioplasty.• This involved inserting a balloon into the

blocked artery and inflating it.• This often results in injury of the artery and

subsequent healing can result in blockage in 40% of patients within 6 months.

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Therapeutic Nucleic Acids• When antisense oligo that target the mRNA for a

protein essential for the cell cycle was applied to rat carotid arteries after angioplasty the reoccurring blockage was reduced by 90%.

• Furthermore smooth muscle cell proliferation is implicated in other disease such as:Ω AtherosclerosisΩ HypertensionΩ Diabetics mellitusΩ Failing of coronary bypass graft

• Similar antisense therapeutics could be used to help alleviate these conditions.

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Antisense Oligos and Psoriasis• Antisense oligos have also been tested in the treatment

of psoriasis.• Psoriasis is uncontrollable epidermal growth.• ILGF-1 receptors are implicated in the pathogenesis of

psoriasis.• 15 nt antisense oligo were transferred into keratinocytes

using liposome and the amount of ILGF-1 protein was decreased by 45-65%.

• When mouse with human psoriasis lesions were injected with anitsense oligi complementary to ILGF-1 receptor mRNA there was significant reduction (58-69%) in epidermal thickness.

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Interfering RNA• The addition of dsRNA to an animal cell causes the

degradation of the mRNA from which it is derived.• This process is called gene silencing or RNA inference

(RNAi).• Gene silencing has been shown to be a natural

mechanism which plant and animals use to protect against viruses.

• The dsRNA that is introduced is cleaved by dicer-like dsRNAse into ssRNA of 21-23 nt.

• These short oligos complex with RISC ( RNA inference inducing silencing complex) which degrade the mRNA complimentary to the oligos.

• This process can be used to target specific mRNA.

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RNA1 as Therapeutic Agents

• A viral vector was used to deliver a small fragment of RNA to brain cells of mice with SCA1 (human neurodegenerative disease spinocerebellar ataxia 1).

• This suppress the SCA1 gene and the mice has normal coordination and movement.

• Scientists are optimistic about using RNAi to treat other neurological diseases such as Alzheimer’s and Hunting’s disease.

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HIV Therapeutic Agents (e 2nd)

• Acquired immune deficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HIV).

• The target of HIV are the T helper cells (TH).• TH cells play a pivotal role in the immune system by

the release of cytokines which stimulate other immune cells.

• The gp120 glycoprotein of HIV binds to CD4 receptors of TH cells.

• The TH cells become infected with the virus and are destroyed, slowly shutting down the immune system.

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Interaction of HIV with CD4

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HIV Therapeutic Agents• HIV antiviral strategies may include:• Production of antibodies to CD4 (will block CD4

receptors on TH cells and prevent infection by HIV).• Production excess CD4 protein (react with gp120

protein therefore HIV cannot infect TH cells).• Both strategies do not destroy HIV but only block

infection.• To stop HIV infection we need to develop strategies

which will destroy HIV.

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HIV Therapeutic Agents

• One strategy which will protect TH cells and destroy HIV include the production of a fusion protein.

• The fusion protein will have 2 parts CD4 protein attached to the Fc portion of an immunoglobulin (CD4 immunoadhesion).

• The CD4 portion will attach to the gp120 protein of HIV or virus infected cells.

• The immunoglobulin portion will initiate a cytotoxic response to destroy the virus or virus infected cell.

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CD4 Immunoadehesion

Fusion Protein

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HIV Therapeutic Agents• Another strategy involves making a second fusion protein.• The CD4 sequence is ligated to the sequence of Pseudomonas

exotoxin A to form a fusion protein.• HIV infected cells have gp120 proteins on their surfaces.• The CD4 portion of the fusion protein will attach to the infected

cells.• The fusion protein will enter the cells and initiate the killing of

the infected cell.• Pseudomonas exotoxin A inactivates the protein synthesis by

affecting elongation factor EF-2. This prevents further protein synthesis and eventually causes death of the infected cell.

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CD4-Pseudomonas Exotoxin Fusion Protein