Advanced Biotechnological tools

for Human health care

Presentated By:-Dr. Shiv Om Pratap Dixit

Head of Department Department of Biotechnology

SRGI, JhansiEmail: drshivompratap@gmail.com

Review Article

Contents:

Introduction

Principle of Gene Expression/Manufacturing of

desired products

Applications of Biotechnology for Human Health Care

Introduction Biotechnology means, ‘the utilization of Biological and

Engineering principles to enhance researches and productivity by using Microorganisms and their derivatives or modifying products for Human welfare.

Advancements of Biotechnological tools have been widely employing for the laboratory researches, industrial

productions, medicines, pharmaceuticals, healthcare, agriculture, animal husbandry, environment and food

industry etc. Biotech companies are making pharmaceutical at

molecular level by ‘Recombinant DNA technology’, which entails genetic manipulation of cells through Genetic engineering principles.

The ‘Hybridoma Technology’ is a technique, used for the production of Monoclonal antibody as a ‘Magic bullet’ in the treatment of several diseases.

Biotech companies have been designing and producing safer vaccines by various manipulated Organisms (GMOs), which are transformed through Genetic Engineering by minimizing the risks of infections.

These products of many Pharmaceutical companies are widely using for the prevention, diagnosis or treatment of many types of diseases have been made through Biotechnological aspects.

Introduction Continue…….

Introduction Continue…….

Pharmaceutical companies are widely utilizing ‘Biotechnological

tools and techniques’ of for the manufacturing of drugs, pharmacogenomics-agents, genetic testing and disease diagnosis etc.

Study of Genetic variation underlying differential responses to drugs.

The more important benefit of pharmaceutical biotechnology leads in the form of better ‘Vaccinal’ immunization’.

Biotechnological Pathway to Regulate the Gene Expression:

The Central Dogma Molecular bases of Life

replication

Recombinant Technology: A basic tools for the production of Industrial Products through Biotechnological Aspects

The Insertion of a desired gene in to a ‘Vector Molecules’ followed by Ligation and Transfection in to host cell for the Gene Expression to obtained desired product is known ‘RDT’.For exp. Production of Metabolites, Pharmaceutical product, Insulin, HCG, Proteins, Transgenic animals & plants, recombinant or Vaccine s etc.

Applications of Biotechnology for Human Health Care1.Molecular marker based disease

diagnosis

These are most efficient tools of disease diagnosis on molecular level, to identify the presence or absence of a particular nucleotide sequences of a Pathogen through PCR amplifications with immense reliability.

Recent advances of molecular tools have opened up a new horizons for the assessment of genetic variability

as well as disease diagnosis at the DNA level. These Molecular markers are; Microsatellites or STRs,

RFLPs, VNTRs consist a short repeat of nucleotide sequences of genome from coding or non coding regions.

PCR: A Novel Technique of Determination Pathogenic Nucleotides Sequences

Molecular markers based disease diagnosis Continuous ……

The efficiency of Molecular markers has increased in multifold through advanced marker, Single Nucleotide Polymorphism (SNPs) for the identification of a particular disease upon the basis of presence or absence of a particular single nucleotide of a pathogen.

The polymorphism of SNPs can provide efficient confirmation of Cancer, Genetic disease, Breast

cancer, Diabetes, Hemophilia etc. immediately through

PCR amplifications. The ‘DNA chip’ technology, modern method to screen SNPs hybridization in the current laboratory diagnosis methods at molecular label for the potent confirmation of a Pathogen in the host.

Molecular markers based disease diagnosis Continue…..

Molecular marker system requires prior Genomic

knowledge of a pathogen and a specific RNA primer designed by using Gene Tool, Gene Map through the use of NCBI, US, Chinese data base resources available till date.

2. Gene Therapy

The advancements of Molecular Biology and Biochemistry have enhanced the genetic basis of a disease inheritance.

It is a replacement of a defective gene with efficient gene to overcome the genetic disorders.

The new gene insert, that encodes specific proteins overcome several deficiencies, responsible for multiple disorders after Gene Expression.

Gene therapy primarily involves selection and improvement of desired gene followed by insertion for efficient gene expression of desired proteins/metabolites.

The human Gene therapy trials for deficiencies of TNFs, ADA, SCID, Ils, multi drug resistance, growth hormones, Phenylalanine hydroxylase, hemophilia (IX) etc. have been completed on animals/humans

Ashi DeSilva : Enzyme deficiency No Immune System

3. Disease Diagnosis The presence of a pathogen like; bacteria, virus and fungi causing disease, can be detected by identifying and confirming foreign genes inside host.

The nucleic acid DNA/RNA is a genetic material of almost pathogens having characteristic information to contribute in the identification of a specific pathogen.

These are the most reliable and potent Biotechnological tools rather than traditional and indirect symptoms based diagnostic methods.

For the laboratory diagnosis of a pathogenic, DNA sequences information is essential.

Nucleic acid Hybridization and DNA probing are widely used for the detection of infectious; Tuberculosis, Malaria, Aids,

Human Papilloma virus and genetic diseases like: Cystic fibrosis,

SCID, Alzheimer’s disease etc.

4. Medical forensics

It is also known as ‘DNA fingerprinting’ or DNA profiling, in the practice of modern medical forensics.

A minute amount of DNA from blood stains, secretary body fluids, hairs, skin etc.

This method involves DNA sample, Restriction Fragments

Length polymorphism (RFLPs), electrophoresis,

blotting and Hybridization techniques for the detection of a culprits.

DNA profiling has wide range of applications, related to Medical forensics like; identification of criminals, settlements of paternity disputes , use of immigration tests cases and disputes etc.

5. Pharmaceutical ProductsCertain proteins could have pharmacological properties

which can be utilized as a pharmaceutical agent to cure many human diseases; infectious viral, bacterial

diseases and common diseases such as Diabetes, Cancer etc.

However, availability of such therapeutic agents was very limited due to their cost and old conventional methods of processing and poor yields as used earlier.

These were also associated with several complications like; dose, mode of administration, and harmful side effects to the humans.

The common pharmaceutical products which are produced through Biotechnological aspects on commercial scales are as follows-

biotechbuddy.googlepages.com

Target Cell

Therapeutic Protein

Transgene

Nucleus

Production Strategies of Therapeutic Proteins

Pharmaceutical Products Continue…….

a) Recombinants Proteins: The advent tools of Biotechnology; Recombinant DNA Technology (RDT), Genetic Engineering, Gene Expression study have opened a new vista to pharmaceutical industry for Human health care.

This RDT was started in late seventies (1970) to produce specific proteins which open a new horizon for Pharmaceutical researches.

Certain Pharmaceutical proteins can be used as therapeutic agents and are listed in the Table No.1, which are approved by US Food and Drug Administration agency.

S. N Recombinant Protein Disease

1. Hemoglobin, Erythroproietin Anamia2. Interleukins-I receptor Asthama3. Tissue plasminogen

activator (tPA), UrokinaseBlood Clots

4. Epidermal Growth Factors Burns5. Interferons, Tumor Necrosis

Factor, Interleukins,Macrophages activating Factors

Cancer

6. Insulin, Insulin Like Growth factor

Diabetes

7. Choronic Gonadotropic Hormones

Female Infertility

8. Superdioxide dimutase Freeradical Damage

9. Growth Hormone, Somatotropin-C

Growth disorder

10. Heart Attacks Prourokinase11. Hepatitis –B Vaccines Hepatitis12. ILs, B-Cells, growth factors Immune

disorders13. Erythropoietin Kidney

disorders14. Interferon Multi sclerosis 15. Calcitonin Osteomalacia16. Endorphins andenkephalins Pain17. Adrenocorticotropic

hormonesRheumatic disease

18. Urogestron Ulcer19. Interferons Viral infections20. DNAse I Cysticfibrosis

Table No. 1. List of Pharmaceutical Proteins

Activase (tissue plasminogen activator) heart attack Humulin (Insulin) diabetesEngerix-B (Hepatitis B Vaccine) hepatitis B infectionEpogen (erythropoietin) anemiaHerceptin Breast CancerReopro cardiac ischemiaBenefix hemophiliaRituxan non-Hodgkin’s lymphoma

Medicinal Applications -Approved Drugs Products

b) Second Generation Therapeutic Proteins

A class of Proteins which are produced by Protein engineering through ‘Site directed mutagenesis’ generally known as ‘Muteins’.

These proteins have changes in their amino acids sequences through Protein Engineering to modify a protein with regards to its stability, solubility, specificity, substrate affinity, pharmacokinetics etc.

These Proteins found most efficient and reliable for the purpose of human health care e.g. Insulinlespro, Alteplase etc.

c) Insulin

Amongst the earliest uses of biotechnology in pharmaceutical manufacturing, is ‘Recombinant Technology’ to produce human insulin, Escherichia coli

by Genentech in 1978. Insulin Protein having a artificial ‘genes’ for each of two

protein chains (A& B ) that comprise the insulin molecule on assembling .

The artificial gene was inserted into plasmids having multiple genes and resistances genes .

Production of Insulin on Commercial level

Insulin molecules

d) Growth hormone

In 1979, Genentech produced human growth hormone (HCG) by inserting DNA encodes ,into a plasmid.

E. coli bacteria was used for the Gene expression

through ‘Recombinant DNA technology’.The gene fragment having total 551 base pairs was

inserted into the plasmid was prepared by reverse transcription of the mRNA found in Pituitary glands by to produce human growth hormone HGH on the industrial scale.

e). Vaccines

These are inactive/attenuated, purified lived or dead pathogenic metabolites, toxins, toxiccoids, proteins, polysaccharides which can generates Immune responses for prolonged immunity as Vaccinal immunization..

These are of mainly three types-• Dead Bacteria or inactivated viruses • Live non virulent or attenuated strains• Viral fragments or Bacterial molecules as sub unit

vaccines These vaccines also known as conventional vaccines are

widely accepted for many diseases; Small pox, Chicken pox, Cholera, Typhoid, Tuberculosis, Poliomyetis, etc.

f ) Recombinant Vaccines

Vaccination is a preventive mode of immunization through modern generation of vaccines with the addition of manipulated genes to produced specific proteins or metabolite as vaccines.

There are several modern strategies to establish a novel recombinant vaccines. These are of two types-

1) Sub-Unit Recombinant Vaccines: These are the components of pathogenic organisms including proteins, peptides and DNA particles.2) Attenuated Recombinant Vaccines: These are Genetically Modified Organisms (GMOs) like bacteria and virus which are modified in such a way that they could only produce immune responses but not diseases.

S. No. Disease Types Pathogenic Organisms1. Acute respiratory Disease Viral Influenza A&B

2. AIDS Viral HIV3. Liver damage Viral Virecella4. Genital Ulcer Viral Herpes Virus5. Cholera Bacterial Vibrio Cholere6. Diarrhea Bacterial E. coli7. Tetanus Bacterial Clostridium Tetany8. Leprosy Bacterial Mycobacterium Leprae9. Pneumonia Bacterial S. Pneumonia

10. Tuberculosis Bacterial Mycobacterium tuberculosis

11. Filariasis Parasites Wuchereria banfrofti12. Malaria Parasites Plasmodium species13. Sleeping Sickness parasites Trypanosoma species

Table No.2. Recombinant Vaccines which developed or developing to human health care.

Recombinant Vaccines continue……..

g) DNA Vaccines

This is a novel approach of a potent immunization through direct administration of genetic material DNA/RNA in the host to evoke immune responses.

These DNA vaccines can be administered through; nasal spray, intramuscular injection, gene gun transfer to reproduce immune responses for efficient immunization.

There are some disadvantages of DNA vaccines; Cancer, alteration in gene expression and problem in the post translational modifications may take place.

Micro- projectile: Partical Gun Instrument

Gene Expression of desire DNA Fragment

Strategies to Design a DNA Vaccine

h) Monoclonal Antibodies (MAbs)

These are ‘Immunoglobulins’ found epitope specific, developed by Georg Kohler and C. Milstein in 1975 through ‘Hybridoma Technology’.

These MAbs are the results of a fusion between B-lymphocytes and Myeloma cells followed by HAT selection, De-novo synthesis, Salvage pathways.

These MAbs having wide acceptability for both diagnostic; Cancer, bacterial infections AIDS as well as therapeutic agents, catalytic proteins, autoantibody fingerprinting.

These MAbs also can be utilized as a ‘Magic Bullet’ to treat potent antigens like cancer by producing antigen specific MAbs.

Monoclonal Antibodies (MAbs): Principle for the Production of Mabs

through ‘Hybridoma technology’

Production of Mabs through ‘Hybridoma technology’

i ) Assisted Reproductive Technology

The manipulations in the reproduction in animals and human are collectively considered under Assisted Reproductive Technology (ART) for increasing the rate of reproduction of a desired species with the advances of Biotechnology.

All mammals having the capability to produce millions of sperms to fertilize limited number of female ovum having complexity due to many reasons, known as ‘Infertility’.

The ART having multiple prospective of artificial insemination, embryo transfer, In-vitro fertilization, embryo cloning in-contrasting traditional breeding strategies.

There are certain reasons of male and female infertility; Oligospermia, Azoospermia, tubal and unfunctional ovaries, non functional uterus, and idiopathic infertility, respectively which can be overcome through ‘In-vitro Fertilization’ (IVF) applications for the human health care.

Reproductive Cloning

For Exp. Test Tube Babies for the overcome of Male & Female infertility

Assisted Reproductive Technology Continue ……………….

7. Transgenic Animals

These are the advancements of Biotechnology to improve our ‘live stock’ which are important resources of our daily need, also known as Genetically Modified Organisms (GMOs) to obtained desired traits of economic importance for human welfare.

Transgenic animals mostly suited as ‘Bioreactors’ due to its capabilities to produce metabolites, hormones, enzymes, pharmaceuticals and the study of researches, gene expression analysis.

The term ‘Transgenesis’ is; insert the gene of interest, inside the host for the expression of desired proteins/enzymes/hormones to determine the desired traits of importance.

These transgenic animals having better productive traits, pharmaceutical agents, metabolites having great significance.

The following steps are used for the production of transgenic animals- manipulation of desired gene, selection of an efficient viral vectors, recombinant technology, gene expression analysis and confirmation of ‘Transgene’.

For exp transgenic ‘mice’ is widely used for the laboratory purposes.

Transgenic Animals Continue …….

Transgenic livestock: a Novel Approaches for the ‘Molecular Farming’ By using as a ‘Bioreactor’

8. Molecular Farming: Resent Advances for the Human Health Care

The modern approaches to synthesize specific metabolites, growth regulators, growth promoters in-vivo by using transgenic animals as a ‘Bioreactor’ directly to the human consumptions for the better nutritional health care.

The desired gene found responsible for the production of desired metabolites introduced inside the female at the embryonic stage and they secreted in their milk after their expression.

The transgenic cow and goat are currently being used for the production of interferon, tumor necrosis factor (TNFs) for the multiple purposes for better immunity.

9. Animal Cell Culture: A boon for the Pharmaceutical Researches

This is the most important area of today’s research having great potential of researches as well as commercial production for the health care and human welfare through cell and tissue culture techniques.

Generally, more valuable products are commercially produced through cell culture like; vaccines, monoclonal antibodies, pharmaceuticals agents, drugs, researches of cancer, genetic manipulation, study of gene expression and drug testing etc.

Animal cell culture is a specific in-vitro culture conducted under the hygienic conditions by using nutrients, growth regulators and promoters for better output.

In the modern time, several valuable pharmacological agents having great medicinal importance have been produced, using animal cell culture or genetically engineered cells.

1.Requirements of Animal Cell Culture:a) Substrate for cell growth like; plastics , glasses, Palladium followed by substrate treatments by using purified fibrinectin or collagens, gelatin or poly D-lysine, UV light for sterilization purposes.

b) Culture media: Natural and synthetic media (Serum media) c) Sterilization : Glass wares, equipments and culture media.

2. Equipments for Animal Cell Culture: Laminar air flow, Co2 incubator, Centrifuge and

inverted microscope.3. Establishment of Cell Culture:

The cell culture is maintained as a Primary culture and Secondary culture like Finite cell lines; IMR 90 from human lungs and Hela from the human epithelial cells.

Animal Cell Culture: A boon for the Pharmaceutical Researches

Continue……..

Animal Cell Culture: A boon for the Pharmaceutical Researches

Continue……..

Role of Animal Cell Culture in ‘Testing of Drugs’:

Animal cell culture has a significant role in testing of pharmacological agents without any social ethics regarding the animals. Earlier many animals had to suffer or even got dead due to strong chemical nature and side effects of these active pharmacological drugs. With the help of animal cell culture, the drugs efficacy can be done safely on animal cells without harming the animal lives such as; Tissue Plasminogen Activator (tPA), commercial insulin (Humulin), erythropoietin and blood clotting factors on commercial scale.

10. Pregnancy Kits

These are the instant methods to detect pregnancies by using Biotechnological tools.

These kits having a ‘Substrate’ with oxidative enzymes to oxidized ‘Human Chorionic Growth hormone’ (HCG) found in the urine of females to initiate Placental development for embryo attachment with uterine wall.

Now if the urine sample has HCG than it will be oxidized by Substrate enzyme to produce a specific color, as an indication of positive sign of pregnancy.

It can be detected through Pregnancy kits, by taking 1-3 drops of urine in the given space in the Pregnancy Kits as a sample for testing.

11. Transplantation or Grafting

Earlier, autografting, xenografting and allografting methods were employed, during the surgery for transplantation.

These methods have more complications and minimal rate of success due to rejections through blood groups and antigenic determinant sites, genesis etc.

Animal cell culture is playing an important role in transplantation by producing artificial skin from the same genesis under laboratory conditions by using biotechnological aspects.

The cells/tissues are taken from the patient and grow artificially and transplanted in the host with larger successive rate.

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