Brief introduction to Biotechnology with reference to Pharmaceutical Sciences

1) Definitions of Biotechnology: Some of the definitions of biotechnology are mentioned below:

  1. As per the Spinks Report (1980) biotechnology is defined as ‘the application of biological organisms, systems or processes to the manufacturing and service industries1.
  2. As per the United States, Congress’s Office of Technology Assessment biotechnology is ‘any technique that used living organisms to make or modify a product, to improve plants or animals or to develop microorganisms for specific uses1.
  3. Biotechnology means any scientific application that uses biological systems, living organisms, or derivatives thereof, to produce or alter products or processes for particular use’25.

2) History of Biotechnology (in relation to Pharma industry)

The history of biotechnology can be divided into 3 phases:

  1. Phase I (1980 to 1989): The major transformation of biotech started in the 1980s. During this period, recombinant proteins were used to treat major life-threatening diseases such as diabetes, anemia, etc. Also, during this period the hepatitis antigen vaccine was developed using recombinant DNA technology. During this period for the approval of a biotech product, the government followed the regulatory guidelines followed for a small drug molecule’s approval. However, the FDA officially fixed a separate guideline, application, and review process for the approval of biological/ biotech products. The biotech products developed during the 1980s were developed for endocrine and blood disorders. Insulin was the first product obtained by recombinant DNA technology which was approved by FDA7.
  2. Phase II (1990 to 1999): During this period the pathophysiology of various diseases was clearly understood and the therapeutic potential of monoclonal antibodies was revealed. Due to advancements in molecular engineering, monoclonal antibodies (chimeric, humanized, and fully-humanized) were produced. Manipulation of the protein structure to obtain a protein which has therapeutically improved properties was also possible with molecular engineering. For example, Genentech’s thrombolytic alteplase used in the treatment of myocardial infarction was created by modifying the amino acid sequence and glycosylation of Tenecteplase. Due to this Tenecteplase could be administered by bolus instead of infusion. To accelerate the approval of biological products the regulatory authorities established the Prescription Drug User Fee Act (PDUFA) of 1992. In 1997 the Food and Drug Administration Modernization Act was brought in to enforce. Due to this act and PDUFA 1992, the approval process for biotech products was accelerated7,8.
  3. Phase III (2000 onwards): From 2000 onwards the evolution of the biotech industry started. Pegylation of protein molecules, improving pharmacokinetics and dosing schema were developed. For example, Pegylated compounds included interferon (peg-interferon alpha) for hepatitis C. Fusion protein technology became a major product development strategy. Full proteins (both recombinant and synthetic) were replaced by peptide molecules for diseases like diabetes and hereditary angioedema. Monoclonal antibody conjugates were produced due to which antibody served as a drug and delivery vehicle. Several orphan drugs were developed to treat genetic disorders and fatal diseases. During this period, the regulatory authorities focused on monitoring the adverse drug reactions, efficacy data, and the FDA established a new risk management plan. In the 2000s the major pharma companies either partnered or acquired the pharma companies7.

3) Scope and development of biotechnology with relation to pharma:

Most of the conventional medicines used for the treatment of various diseases were synthetic small size molecules. However, from the 1980s onwards the biotech startups-initiated research on biological molecules to check their therapeutic efficacy against diseases. Biotechnology is a multidisciplinary field of science and technology and its scope is extended to various sectors of science and technology. Biotechnology helps the pharma industry to develop new, safe, efficacious products. Since the 2000s application of biotechnology in the pharma industry is constantly increasing7.

4) Applications of Biotechnology in the pharma industry:

Some of the common applications of biotechnology in the pharma industry are discussed below:

  1. DNA fingerprinting: The technique of DNA fingerprinting was discovered by Great Britain geneticist Alec J. Jeffrey in the year 1984. DNA fingerprinting is a technique used for authentication of various plant parts and herbal drug standardization. In DNA fingerprinting, bar-code like DNA patterns are generated after amplification of chromosomal DNA of the plant material. The sequence of DNA patterns produced is identified by DNA markers which are very specific for each plant species. Thus, DNA fingerprinting is an effective analytical tool used for herbal drug standardization, authentication of plant parts. In addition to herbal drug standardization DNA fingerprinting is also used to detect diseases cystic fibrosis, Huntington’s disease, hemophilia, etc. Detection of such diseases in the early stages increases the chances of patients been cured completely2.

The various techniques used for DNA fingerprinting are listed below:

  1. Hybridization based:
  2. Variable Number Tandem Repeat (VNTR)
  3. Probe hybridization with Micro and Minisatellite
  4. Random Genomic Clone
  5. cDNA Clone
  6. Restriction Fragment Length Polymorphism (RFLP)
  7. PCR based:
  8. Inter Simple Sequence Repeat (ISSR)
  9. Random Amplification Polymorphic DNA (RAPD)/Arbitrary Primed PCR
  10. Amplified Fragment Length Polymorphism (AFLP)
  11. DNA Amplification Fingerprinting (DAF)
  12. Sequence-based
  13. Simple Sequence Repeats (SSR)
  14. Sequence Characterized Amplified Region (SCAR)
  15. Cleaved Amplified Polymorphic Sequence (CAPS)
  16. Single Nucleotide Polymorphism (SNP)2.
  17. Stem Cell in research and stem cell therapy:

Stem cells are undifferentiated cells that undergo division to give rise to a daughter cell which is similar to itself. These daughter cells further undergo division to form specific cells such as adipocytes, neuronal cells, macrophages, etc.

Diseases such as cancer and birth defects mainly arise because of abnormal cell division. Stem cells help in identifying the signals and mechanism of abnormal cell division and thus helps in proposing the new strategies to combat such diseases. Stem cells are used by researchers to screen for new potential drug molecules. For example, the cancer cell line is used to screen novel anti-cancer agents, macrophage cell line infected with Mycobacterium tuberculosis is used to screen potential antimycobacterial agents25.

  • Gene therapy: Gene therapy is one of the promising therapeutic techniques used in the treatment of cancer and other genetic disorders. Gene therapy is a technique to correct the abnormal genes. In gene therapy, a functional/ therapeutic is been transferred inside the target cells3. Vectors such as adenoviruses, simplex virus, liposomes, polyethylene glycol (PEG), gene gun are used for transferring the new gene inside the target cell. The new gene will ameliorate the abnormal metabolic event or the new gene will make a new functional protien3,5.
  • Transgenic plants: Transgenic plants is a class of genetically modified plants. The recombinant DNA also is known as rDNA technology which is used to produce medicinal plants with required traits and characteristics. Some of the significant traits possessed by transgenic plants are listed below:
  • Higher production of secondary metabolites
  • Production of plants resistant to pests, viruses, bacteria
  • Drought resistant crop
  • Production of hybrid seeds.
  • Production of plants with an increased rate of photosynthesis
  • Stress-resistant medicinal plants.

Example: Bacillus thuringiensis cotton also abbreviated as BT cotton is a genetically modified plant which is resistant to pest and insects. Bt cotton was produced by genetically modifying the cotton genome to express a microbial protein from Bacillus thuringiensis bacteria19.

  • Diagnosis of diseases: With the help of various techniques in biotechnology, infectious and inherited diseases are detected. Some of the most commonly used detection techniques for the diagnosis of diseases are mentioned below:
  • Agarose Gel Electrophoresis and Polyacrylamide gel electrophoresis
  • Blotting techniques:
  • Nucleic acid blotting
  • Southern blot analysis
  • Protein blotting
  • Northern blot analysis
  • Dot blot technique
  • Autoradiography
  • PCR-based techniques:
  • Inter Simple Sequence Repeat (ISSR)
  • Random Amplification Polymorphic DNA (RAPD)/Arbitrary Primed PCR
  • Amplified Fragment Length Polymorphism (AFLP)
  • Hybridization techniques
  • Variable Number Tandem Repeat (VNTR)
  • Probe hybridization with Micro and Minisatellite
  • Restriction Fragment Length Polymorphism (RFLP)25.
  • Preparation of vaccines: Vaccines are biological preparations effective against a particular infectious disease. Vaccines work by providing active acquired immunity against the pathogen. The current advancement in biotechnology techniques, rDNA technologies, monoclonal antibodies techniques has brought strategies for vaccine development against infectious diseases. Eg: French microbiologists Albert Calmette and Camille Guerin in the year 1921 developed BCG (Bacillus Calmette-Guerin) vaccine against tuberculosis. In India, the first BCG vaccination drive was carried out in the year 19484.
  • Production of other biopharmaceuticals: Microorganisms such as bacteria, algae, viruses, fungi, yeast are one of the major sources for the production of biopharmaceuticals. Secondary metabolites obtained from microorganisms are reported to have significant therapeutic activity. With the development and advancement in biotechnology, rDNA technology these microorganisms can be easily manipulated to increase the production of the secondary metabolites. Biopharmaceuticals obtained from microorganisms are used as pharmaceutical excipients or as a therapeutic drug6. Few examples of the biopharmaceuticals obtained from microorganisms are listed in table 1.

Table 1: Examples of biopharmaceuticals used in pharma industry6  

BiopharmaceuticalsSourcePharmaceutical use
Xanthan gum (polysaccharide)Xanthomonas campestrisUsed as a binder, matrix former, drug release modifier, viscosity enhancer, stabilizer, emulsifier, suspending agent, disintegrator, solubilizer, gelling agent, and bioadhesive.
LuteinGreen microalgaeAntioxidant, anti-inflammatory, and hepatoprotective effects.
PhycocyaninSpirulina (Cyanobacteria)Cosmetics, immunofluorescent techniques, antibody label.
Pyropheophytin a fucoxanthinBrown macroalgaeHepatoprotective

4) Conclusion:

  • Biotechnology is a fusion of biological sciences and technology to produce products that are beneficial for human use.
  • After 2000 biotechnology has made advancement in the field of rDNA technology, PCR, DNA fingerprinting, etc.
  • Production of transgenic plants, growth hormones, and vaccines, stem cell therapy, gene therapy are some of the significant applications of biotechnology in the pharma industry.
  • At present biotech products is one of the major sectors in the pharma industry.

5) Notations:

  • Recombinant DNA technology: also known as rDNA technology is the process of joining two separate DNA molecules with help of DNA ligase. This rDNA molecule is inserted into the target cell29.
  • DNA ligase: is a class of enzyme that facilitates the attachment of DNA strands28.
  • Prescription Drug User Fee Act (PDUFA) 1992: was a law established by the United States Congress in the year 1992. This law authorizes the FDA to collect fees from companies that produce biological drug products. The main aim of this act was to expedite the drug approval process9.
  • Agarose gel electrophoresis: Agarose gel electrophoresis is an analytical technique used to separate a mixture of macromolecules such as DNA, RNA, protiens10.  
  • Nucleic acid blotting: Nucleic acid blotting is an analytical technique used to identify a specific gene or sequence of DNA or RNA13.  
  • Southern blot analysis: is a hybridization technique used to locate a specific DNA sequence from the mixture of biological sample12.
  • Protein blotting: also known as Western blotting is an analytical technique used to identify a specific set of proteins in the biological sample11.
  • Northern blot analysis: is a hybridization technique used to locate a specific DNA sequence from the mixture of biological sample14.
  • Dot blot technique: This technique is similar to other blotting techniques however, it doesn’t provide information regarding the size of the fragment. It is mainly used to identify a specific set of proteins in the biological sample15.
  • Autoradiography: Autoradiography is a technique in which X-ray film is used to visualize the radioactively labeled fragments such as DNA16.
  • Inter Simple Sequence Repeat: also abbreviated as ISSR is one of the PCR based techniques in which the DNA segments are amplified. ISSR is more dominant, stable, and repeatable20.
  • PCR: also known as polymerase chain reaction is a technique used to make multiple copies of sample DNA using DNA polymerase enzyme22.
  • Random Amplification Polymorphic DNA: is based on the PCR principle which helps in to detect genetic variation17.
  • Amplified Fragment Length Polymorphism: abbreviated as AFLP is a PCR based technique in which there is selective amplification of the subset of digested DNA fragments to produce and compare with fingerprints of interest genome21.
  • Variable Number Tandem Repeat (VNTR): are short nucleotide sequences organized as tandem repeats at a specific location in DNA.
  • Minisatellite: A minisatellite also known as VNTR is a repetitive sequence of 10 to 60 DNA base pairs which is repeated 5 to 50 times23.
  • Microsatellite: A microsatellite is a repetitive sequence of 1 to 6 DNA base pairs which are repeated 5 to 50 times24.
  • Restriction Fragment Length Polymorphism (RFLP): is a technique used in DNA fingerprinting. With the help of RFLP specific variations in the sequence of double-stranded DNA can be identified. Restriction endonucleases are the class of enzyme used in RFLP18.
  • Restriction endonucleases are a class of enzyme that identifies a specific set of nucleotides (also known as restriction site) and cuts the DNA at that specific site18.
  • Monoclonal antibodies: Monoclonal antibodies are man-made clones of parent immune cells. These proteins act as antibodies in the human immune system26.
  • Pegylation of proteins: is a biochemical technique used to modify proteins/ peptides, antibodies using polyethylene glycol (PEG)27.

6) References:

  2. Kumar, P.S., Ketkar, P., Nayak, S. and Roy, S., 2014. Application of DNA fingerprinting tools for authentication of ayurvedic herbal medicines–A review. J Sci Innov Res3, pp.606-612.
  3. Mohammed, B. R., Malang, S. K., Mailafia, S., & Agbede, R. I. S. (2016). Application of Biotechnology towards Diagnosis and Treatment in Veterinary Medicine in Africa: Potentials and Future Developments. J Biotechnol Biomater6(245), 2.
  4. Lahariya, C. (2014). A brief history of vaccines & vaccination in India. The Indian journal of medical research139(4), 491.
  5. Tonukari, N. J., Avwioroko, O. J., & Ehwerhemuepha, T. (2010). Diverse applications of biotechnology. Scientific Research and Essays5(9), 826-831.
  6. Ramana, K. V., Xavier, J. R., & Sharma, R. K. (2017). Recent trends in pharmaceutical biotechnology. Pharm Biotechnol Curr Res1(1), 5.
  7. Evens, R., & Kaitin, K. (2015). The evolution of biotechnology and its impact on health care. Health Affairs34(2), 210-219.
  20. Vijayan, K. (2005). Inter simple sequence repeat (ISSR) polymorphism and its application in mulberry genome analysis. International Journal of Industrial Entomology10(2), 79-86.
  21. Paun, O., & Schönswetter, P. (2012). Amplified fragment length polymorphism: an invaluable fingerprinting technique for genomic, transcriptomic, and epigenetic studies. In Plant DNA Fingerprinting and Barcoding (pp. 75-87). Humana Press.
  25. Bhatia, S., & Goli, D. (2018). Introduction to Pharmaceutical Biotechnology: Basic techniques and concepts. IOP Publishing.

Enzyme Biotechnology- Methods and applications

Enzyme Biotechnology

Definition of enzyme: Enzymes are high molecular weight proteins made up of a long chain of amino acids that are linked to each other by peptide bonds. Enzymes accelerate/ catalyze the biochemical reactions without itself getting consumed. Thus, enzymes are also known as biocatalyst1,2.

Classification of enzymes: Enzymes are classified based on the type of reaction they catalyze. There is a total of 7 categories/classes of enzymes which are listed below in table 11,2.  

Table 1: Name of the enzyme class, the function of the enzyme, the general type of reaction enzyme-catalyzed, and examples. (X, Y, and Z represents chemical groups)  

Sr noEnzyme class nameFunction/ RoleGeneral type of reaction catalyzedExamples of enzyme
1TransferasesCatalyze the transfer/ exchange of certain chemical groups amongst the substrateX-Y + Z ↔ X + Y-ZMethyltransferase, acyltransferase, sulfotransferase. Glucotransferase.  
2HydrolasesPromotes the hydrolysis of reactant/ substrateX-Y + H2O → X-H + Y-OHEsterase, lipase, phosphatase, peptidase, nucleosidase 
3OxidoreductasesCatalyze the redox type of reactions.Xred + Yoxd ↔ Xoxd + YredOxidase, peroxidases, hydrolases, oxygenase.
4LyasesPromotes the removal of certain chemical groups from the substrate molecules OR promotes the reverse reaction.X-Y ↔ X + YDecarboxylase, aldehyde lyase
5IsomeraseCatalyzes the conversion reaction for isomersX-Y-Z ↔ Y-Z-XGlucose isomerase, maleate isomerase
6LigasePromotes the joining of two molecular substrates to get a single compound. Such type of reaction is characterized by the release of energy.X + Y + ATP → X-Y + ADP + PO4DNA ligase
7TranslocasePromotes the movement of ions/ molecules across the biological membrane. Ornithine translocase.
  • Recombinant enzyme technology:

Recombinant DNA technology produces recombinant DNA also known as rDNA using a specific set of enzymes called recombinant enzymes.

Some of the recombinant enzymes used in rDNA technology are mentioned below:

  1. Restriction enzymes.  Restriction enzymes are a set of enzymes that cleaves the DNA at specific sites. Nucleases belong to the restriction enzymes class that breaks the DNA molecule by breaking the phosphodiester linkage in DNA. Examples of nucleases: RNAse (cleaves the RNA) and DNAse (cleaves the DNA).

Nucleases are of two types: Exonuclease (cleaves the DNA molecule at the end) and endonucleases (cleaves the DNA molecule in the middle of the molecule).

  • Exonucleases: removes the terminal nucleotide of the DNA molecule
  • Endonucleases: cleaves the internal phosphodiester bond. There are 3 types of endonucleases types I, II, and III. Endonucleases scan the entire DNA chain and cut the DNA molecule which leads to the formation of 3’, 5’, blunt ends, or sticky ends29
  • Ligases: are a class of enzymes that join the DNA and RNA fragments together. DNA is joined by DNA ligase whereas RNA is joined by RNA ligase. DNA ligase helps in repairing the single-strand breaks in DNA.
  • Polymerases: are a set of enzymes that synthesize the long chain of polymers. DNA and RNA polymerase belong to this class.

The function of DNA polymerase: DNA polymerase helps in synthesizing the DNA molecule from deoxyribonucleotides8.

The function of RNA polymerase: RNA polymerase copies the DNA sequence into an RNA sequence during transcription process9.

  • Enzyme topography

The rate of a biological reaction depends upon the topography or the shape of an enzyme. Each enzyme with a specific shape can attach to only a specific substrate. Each enzyme will fit into only one substate molecule because each enzyme has a specific shape of the active site. For a biological reaction to occur the active site of the enzyme binds to the substrate.

Example: Acetylcholine (Ach) binds to the extracellular domain of the acetylcholine receptor30.

  • PEGylation

PEGylation is a biochemical modification process of the fusion or attachment of polyethylene glycol (PEG) polymer to macromolecules such as drugs, protein, vesicles, antibodies, and enzymes. The macromolecule is covalently or non-covalently conjugated to the PEG polymer. Due to PEGylation desired therapeutic properties are obtained in the macromolecule.

The initial research of PEGylation was done by Frank Davis Sir in the late 1970s. He demonstrated that PEGylated proteins had longer half-lives in the bloodstream and decreased immunogenicity31.

  • Advantages/Significance of PEGylation: The significance of PEGylation is listed below:
  • The in-vivo half-life of the drug, enzyme or protein is increased
  • Improved drug stability
  • Reduced dosage frequency
  • Enhanced protection from proteolytic degradation
  • The immunogenicity and antigenicity of the macromolecule are reduced.
  • Minimal loss of the pharmacological activity
  • The solubility of the macromolecule (drug, antibody, protein, etc..) is increased.
  • Helps in achieving target-specific drug delivery
  • Improves the pharmacokinetic properties of the therapeutic agent31.

The first FDA approved drug Adagen that used PEGylation was launched in the year 1990. Since then various pegylated drug products are approved by FDA which are in use.

Some of the pegylated compounds which are currently available in the market for use are listed in table 2:

Table 2: Examples of few FDA approved PEGylated products31

KrystexxaReduces the uric acid levels and aids in removing gout crystals
Adagen (pegademase)The modified enzyme used for Enzyme Replacement Therapy (ERT).
Somavert (pegvisomant)Prescription medicine for acromegaly, a disease caused by the surplus of growth hormones in the body. The goal is to have a normal IGF-1 level in the blood.
Cimzia (certolizumab)An injected prescription medication that works to prevent inflammation that may result from an overactive immune system.

Why PEG is used in therapeutic formulations?

Some of the reasons for use of PEG in the systemic and non-systemic formulations are listed below:

  • Higher solubility in both organic and inorganic solvents
  • Minimum toxicity levels
  • Lower immunogenicity
  • Non-biodegradability
  • Hydrophilic nature
  • Fast renal clearance31

Due to the reasons mentioned above PEG polymer is the most commonly used in comparison to the other polymers of similar molecular weight and size.

There are three types of PEGylation: a) first-generation and b) second generation and c) third generation PEGylation.

  1. First-generation PEGylation: First-generation PEGylation is also known as non-specific PEGylation. Amine conjugation is the most commonly used method for non-specific PEGylation. The PEG conjugates obtained from non-specific PEGylation were not uniform and resulted in the formation of positional isomers. However, there are multiple first-generation PEGylated drug products in the market which are still in use. E.g. Pegasys used in the treatment of Hepatitis C, utilizes non-specific PEGylation.
  2. Second generation PEGylation: also known as site-specific PEGylation. In this method specific site of the PEG molecule interacted with the chemical groups on the protein or therapeutic drug to obtain a PEGylated conjugated product. The various strategies for site-specific PEGylation include N-terminal PEGylation, thiol and bridging PEGylation, histidine tags, and enzymatic PEGylation.
  3. Third generation PEGylation: The main aim of third-generation PEGylation is to improve the potency and half-life, site-specificity, and lower dosage of the drug. The main strategy for third-generation PEGylation is based on electrostatic linkages between PEG and drug.

The main pathway of site-specific PEGylation is reversible conjugation or release of prodrugs. Reversible conjugation is even less inhibiting on drug activity than irreversible conjugation, used in the first-generation PEGylation. Second generation PEGylation looks to temporarily attach PEG molecules via cleavable linkages. This way, drugs can be released according to a specified schedule, in vivo via hydrolytic cleavage31.

  • Limitations of PEGylation:
  • Possibility of the formation of side products.
  • PEG has limited conjugation ability
  • Costly process
  • Steric interference of protein with the target receptor
  • Interference with protein binding site31.
  • Drug cloning

Cloning is a process through which identical copies of the cell, tissues, or organism are produced. There are three types of cloning:

  • Gene cloning: creates replicas of DNA segments.
  • Reproductive cloning: creates copies of whole animals.
  • Therapeutic/drug cloning: creates embryonic stem cells. These embryonic stem cells are used to replace the injured tissues or cells in the body by healthy tissues or cells.

Application of therapeutic cloning:

  1. Cloning is most applicable in the preclinical phase of drug discovery. Cloning helps in the discovery of various receptor types and subtypes. Muscarinic, melanocortin, and dopamine receptors are some of the receptors identified through therapeutic cloning. Examples of successful drug cloning that are now used as therapeutics are insulin, growth hormone, erythropoietin, and tissue factors.
  2. Vaccines are another area of therapeutic cloning application.
  3. Stem cell therapy for treating disorders like cancer, diabetes6.

Advantages of therapeutic cloning: Some of the advantages of therapeutic cloning are mentioned below:

  • It helps in understanding the growth and development of stem cells. Thus, it is helpful to discover new treatments, medicines for various diseases.
  • The use of pluripotent stem cells in stem cell therapy helps in treating diseases in any body organ and tissues by replacing the damaged or mutated tissues7.

Disadvantages of therapeutic cloning:

  • Costly and time-consuming process.
  • Initial research and development require experienced and skilled personnel7.
  • Gene therapy

What is gene therapy?

Gene therapy is a medical field that uses genes to treat diseases. The various strategies for gene therapy are as follows:

  1. Inserting a healthy gene into the target cell to replace the defective/faulty/mutated gene.
  2. Inactivating a defective/faulty/mutated gene
  3. Inserting a new healthy gene in the target cell to prevent diseases3.

How does gene therapy work?

In gene therapy, new genetic material is introduced inside the target cells to replace the mutated/ abnormal genes. The new gene inserted makes a useful protein or restores the function of the mutated gene. When a gene is directly inserted into the target cell it fails to show therapeutic activity. Thus, a carrier also known as a vector is used to insert the gene into the target cell. Most commonly genetically modified viruses that are non-virulent are used as vectors to introduce the new genetic material inside the target cell. The most common viruses used as vectors are adenoviruses, retroviruses3.

There are two approaches to gene therapy:

  1. Introducing the vector by the parenteral route (intravenous route) directly into the specific tissue.
  2. A sample of the patient’s tissue or cell is removed and then exposed to the vector for a defined period in the controlled conditions. The cells containing the vector are again introduced back into the patient’s body.

If the new gene introduced inside the patient’s body starts making functional proteins then it indicates that the gene therapy is successful3.

Advantages of gene therapy

  • Used to prevent and cure hereditary disorders. E.g. Cystic fibrosis
  • It helps to achieve the required pharmacological action4.

Disadvantages of gene therapy:

  • Inside the body, cells undergo constant division due to which long-lasting therapeutic effect is not obtained from gene therapy.
  • Due to the insertion of new genes, there is a possible immune response to be stimulated in the body which can be life-threatening.
  • The use of a virus as a vector in gene therapy may induce toxicity, immune response in the patient’s body.
  • Diseases which occur due to defect in a single gene can only be treated.
  • The entire therapy is costly4.

Applications of gene therapy:

Gene therapy is used to treat various disorders which are mentioned below:


Medical Biotechnology Applications in Pharma and Healthcare

A) What is a Medical Biotechnology?

Medical biotechnology or red biotechnology is the branch of biotechnology that involves the use of living cells, tissues to manufacture products that can be used as therapeutic drugs for treatment, prevention, and diagnosis of various diseases5.

B) What is biomedical science?

Biomedical science is the branch of science that combines natural science with biology. It mainly focuses on how the cells grow, divide in the body. Biomedical science also helps to develop medicines for human diseases10.

C) Techniques in medical biotechnology: The various techniques in medical biotechnology are discussed below:

1) Polymerase Chain reaction: also abbreviated as PCR. The technique was developed in the 1980s by Kary Mullis. PCR is an in vitro test performed inside a test tube using a DNA polymerase, DNA template, nucleotides, and primers to synthesize new strands of DNA. The PCR reaction is performed at a higher temperature which leads to the denaturation of the double-stranded DNA molecule into two new single strands. After which the primers bind to the specific homologous DNA sequences. The primer that is homologous to the target DNA sequence is elongated by the DNA polymerase enzyme. At the end of the PCR, a million copies of the target DNA sequence are obtained. The PCR is used to create multiple copies of target DNA and RNA both.

There are 3 main steps in PCR:

Man Looking Through A Microscope
  • Denaturation of target DNA: carried out a high temperature
  • Annealing/ Joining: Primer joins to the homologous sequence on target DNA.
  • Elongation: DNA polymerase enzyme is used for elongation purposes5.

2) Cell culture:

Cell culture is a major advancement in the field of molecular and cell biology. Cell culture is a technique in which in vivo cells are grown outside the body under controlled lab conditions. The various cells that can be grown are plant, animal, human cells, microbial planktonic cells, and fungal cells. Cell culture is a technique used for the production of enzymes, interleukins, hormones, antineoplastic agents, and vaccines.

There are three main types of cell culture which are listed below:

Scientist Using Microscope

a) Primary cell culture: In primary cells, culture cells are obtained from parental tissue and allowed them to grow under controlled conditions. Primary cell culture is of two types 1) Adherent cells and 2) Suspension cells

  1. Adherent cells are immobile cells which require attachment for growth. Example: Kidney cells
  2. Suspension cells are cells that do not require attachment for growth. Example: lymphocyte cells. In suspension cells, cells are suspended in a liquid medium. The advantage of cell suspension is that it allows the cells to be uniformly suspended in the liquid growth medium.

Advantage of primary cells: Since primary cells mimic the in vivo cells, hence they are more preferred over cell lines and are important in cell and molecular biology research1.

b) Secondary cell culture: are the cells which are obtained by subculturing from primary cells2.

c) Cell line: The secondary cells undergo continuous division to form a group of finite cells which is known as cell line2.

The cell culture can also be divided into 2 types:

  1. 2-dimensional cell culture: the cells are grown on a 2-dimensional surface.
  2. 3-dimensional cell culture: the cells are grown on a 3-dimensional surface. In this, the cells differentiate into specific cells that are essential in tissue formation. (book ref)

The main components required for culturing and growth of cells are listed below:

  1. Growth medium: Natural or synthetic medium. (depends on the type of cells)
  2. Optimum Temperature conditions  
  3. Buffer to maintain the pH.
  4. CO2 incubator.

Cell culture techniques are used to obtain stem cells for stem cell therapy5.

Laboratory Test Tubes

3) Stem cells: Stem cells are nonspecific human body cells that differentiate into specific human cells. Self-renewal and differentiation are the most significant features exhibited by stem cells.

Examples of stem cells: Embryonic stem cells (ESCs), induced pluripotent stem cells (IPSCs), cancer stem cells (CSCs), inner cell mass (ICM) of blastocyst embryos, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs)5.

Stem cell therapy promotes the repair of injured/ nonfunctional/ mutated cells and tissues. For stem cell therapy, stem cells are grown in the lab under controlled conditions. The stem cells obtained are then inserted into the disease person4.

4) Recombinant DNA technology: Recombinant DNA technology abbreviated as rDNA technology is a major advancement in the field of biotechnology. With the help of rDNA technology, single or multiple genes can be inserted into the genome of another individual. In the field of medical biotechnology, human insulin is the first major product produced using rDNA technology.

rDNA technology uses three main parameters:

1) Enzymes: various enzymes such as restriction enzymes, polymerases, and ligases are used. The function of all three enzymes are listed below:

a) Restriction enzyme: is the most important used in rDNA technology. Restriction enzymes cut the DNA molecule at a specific site called the restriction site.

b) Polymerases: help to synthesize the DNA molecule.

c) Ligases: Sticks the two strands of DNA.

2) Vectors: Vectors are the carriers of the desired gene into the host organism. The most commonly used vectors are bacteriophages and plasmids.

3) Host organism: is the target cell inside which the functional/desired gene needs to be inserted. Bacterial, fungal, and animal cells are some of the examples of the host organism.

The various techniques used to introduce the vector inside the host/ target cells are using either microinjection, gene gun, alternate cooling, and heating, etc…

The entire procedure for rDNA technology involves four steps which are listed below:

Dna Free Stock Photo - Public Domain Pictures
  • The functional DNA is cut at the specific site using restriction enzymes.
  • Amplifying the desired gene copies by PCR technique.
  • The desired gene is inserted into the vectors.
  • The vector is introduced inside the target cell5.

5) Fluorescence in situ hybridization technique: is one of the most basic techniques used to study the structure of DNA. With the help of this technique, the position of a specific DNA sequence can be determined. This technique was developed by Gall and Pardue in the year 1969. In this technique, a fluorescent probe is used. Initially, target DNA is mixed with the fluorescent probe. During the reaction, the fluorescent probe binds to the complementary sequence on the target DNA and then visualized using a fluorescent microscope technique5.

6) DNA sequencing: DNA sequencing is a technique which helps to determine the sequence of nucleotides within a DNA molecule. The technique of DNA sequencing was developed by Fred Sanger in the year 19555. Some of the most commonly used DNA sequencing techniques are listed below:

  • Chain termination method
  • Sanger sequencing method
  • Clone by clone sequencing
  • Maxam-Gilbert sequencing
  • Next-generation sequencing3

Applications of DNA sequencing:

  • It helps in understanding the structure of genes and the sequence codes for which type of proteins.
  • It helps in predicting/ identifying the genetic mutations in various diseases.
  • It helps in preparing therapeutic proteins3.

7) Genome sequencing: is a technique in biotechnology used to insert or replace a new gene or multiple genes in the human genome. Faulty, dysfunctional, or mutated genes can also be removed from the genome using this technique. For genome sequencing, specific nucleases class of enzymes are used.  Zinc finger nucleases, transcription activator-like effector-based nucleases, the CRISPR/Cas system, and engineered meganuclease reengineered homing endonucleases are the four major classes of nucleases used for genome editing. This class of enzymes causes double-strand breaks in the DNA molecule at a specific sequence. Then, the endogenous repair mechanism within cells repairs the double-stranded breaks in DNA either by homologous recombination or nonhomologous end joining5.

D) Application of medical biotechnology5, 11

Scientist Working in Laboratory

1) Vaccines: are a class of biologic products that are used to prevent various diseases. Vaccines provide acquired immunity against a particular disease. Vaccines consist of disease-causing inactivated antigen or toxins or their surface proteins which stimulate an immune response in individuals.

Examples of vaccines: BCG vaccine made from Mycobacterium bovis to prevent tuberculosis in individuals5.

2) Monoclonal antibodies: are man-made antibodies in a laboratory that neutralizes the effect of external antigens.  Monoclonal antibodies are used for the treatment of cancer, an autoimmune disease. They are also used for diagnosis and prevention of diseases. In the treatment of cancer and autoimmune diseases, the major mechanisms by which monoclonal antibodies exert pharmacological effect is through blocking the targeted molecule functions, promoting apoptosis, and modulating the cell signaling pathways5, 11.

3) Recombinant proteins: are used as drugs for treatment. In the year 1982, human insulin was the first recombinant protein used for treatment purposes. At present, there are more than 170 recombinant protein products in the market which are used as therapeutic drugs5, 11.

Example of recombinant proteins: Growth factors, interleukins, TNF, etc.

4) Stem cell therapy: Stem cell therapy is used to treat neurodegenerative, cancer, birth defects, bone degeneration, osteoarthritis, etc.

Example: Cartistem stem cell product is used for the treatment of osteoarthritis.

Since stem cells help in identifying the signals and mechanisms of abnormal cells and thus helps in proposing the new strategies to combat diseases. Thus, stem cells are used for research purposes5, 11.

5) Tissue engineering: is a branch of biotechnology that involves the study of growth and development of tissues. At present various research studies are been attempted to generate tissues, cartilages, blood vessels, etc.5, 11

6) Antibiotics: belongs to a class of antimicrobial products used for the treatment and prophylaxis of bacterial infections/ infectious diseases. Antibiotics act by either disrupting the cell wall or prevent them from multiplying. Most of the antibiotics in use are obtained from a natural source such as plants, microorganisms, etc. Antibiotics either have a bactericidal effect or bacteriostatic effect5, 11.

Prontosil was the first systemically active antibacterial drug that was discovered by Gerhard Domagk in the year1933. Penicillin’s, cephalosporins, sulfonamides, etc. are some of the other classes of antibiotics5, 11.

E) Medical biotechnology Notations:

Scientist Using Microscope
  • Embryonic stem cells (ESCs): are the stem cells obtained from the inner cell mass of embryos5.
  • induced pluripotent stem cells (IPSCs): are artificially produced stem cells obtained by promoting pluripotent properties in somatic adult cells5.
  • inner cell mass (ICM): is a class of stem cells isolated from the primordial embryo5.
  • hematopoietic stem cells (HSCs): are the stem cells that undergo division to give rise to new blood cells5.
  • mesenchymal stem cells (MSCs): are the stem cells isolated from bone marrow. They are mainly responsible to make and repair skeletal tissues, cartilage, bones5.
  • DNA: Deoxyribonucleic acid is the genetic material in all living organisms6.
  • TNF: Tumor Necrosis Factor is a substance produced by macrophages and monocytes in response to the endotoxins produce the pathogen. TNF is mainly responsible for producing inflammation and induces the destruction of tumor cells7.
  • Interleukins: is a class of in vivo proteins that stimulates inflammatory response8.
  • CRISPR/Cas system: is a genome editing system isolated from bacteria. CRISPR/Cas system helps the bacteria to remember the invading viruses9.

F) References:

  1. Cell Culture
  2. Bone Marrow Transplant
  3. Pham, P. V. (2018). Medical biotechnology: techniques and applications. In Omics Technologies and Bio-Engineering (pp. 449-469). Academic Press.
  4. DNA
  5. Rheumatoid Arthritis
  6. Interleukin
  7. Genomicresearch
  8. Biomedical Sciences Admissions
  9. Biotechnology Applications