Isolation of Pure Cultures

In natural habitats microorganisms usually grow in complex, mixed populations with many

species. This presents a problem for microbiologists because a single type of microorganism

cannot be studied adequately in a mixed culture. A pure culture which is a population of cells

arising from a single cell is needed to characterize an individual species.

German bacteriologist Robert Koch developed of pure culture techniques and within about 20

years after the development of pure culture techniques most pathogens responsible for the

major human bacterial diseases had been isolated.

There are several ways to prepare pure cultures. Pure cultures usually are obtained by

isolating individual cells with any of three plating techniques: the spread-plate, streak-

plate, and pour-plate methods.

The spread-plate and pour-plate methods usually involve diluting a culture or sample and

then plating the dilutions. In the spread-plate technique, a specially shaped (L shaped)

rod/hockey stick is used to spread the cells on the agar surface; in the pour-plate technique,

the cells are first mixed with cooled agar-containing media before being poured into a petri

dish. The streak-plate technique uses an inoculating loop to spread cells across an agar

surface.

The Spread Plate 

If a mixture of cells is spread out on an agar surface at a relatively low density, every cell

grows into a completely separate colony, a macroscopically visible growth or cluster of

microorganisms on a solid medium. Because each colony arises from a single cell, each

colony represents a pure culture. The spread plate is an easy, direct way of achieving this

result.

A small volume of dilute microbial mixture containing around 30 to 300 cells is transferred to

the center of an agar plate and spread evenly over the surface with a sterile bent-glass rod.

The dispersed cells develop into isolated colonies. The number of colonies developed should

be equal to the number of viable organisms in the sample, so, spread plates can be used to

count the microbial population.

 

The Pour Plate

The pour plate technique also can yield isolated colonies. The original sample is diluted

several times to reduce the microbial population sufficiently to obtain separate colonies when

plating. Then small volumes of several diluted samples are mixed with liquid agar that has

been cooled to about 45°C, and the mixtures are poured immediately into sterile culture

dishes. Most bacteria and fungi are not killed by a brief exposure to the warm agar. After the

agar has hardened, each cell is fixed in place and forms an individual colony. Like the spread

plate, the pour plate can be used to determine the number of cells in a population. Plates

containing between 30 and 300 colonies are counted. The total number of colonies equals

the number of viable microorganisms in the sample that are capable of growing in the

medium used. Por plates can thus be used to enumerate/count the microbial population.

Colonies growing on the surface also can be used to inoculate fresh medium and prepare pure

cultures.

 

The streak plate

Pure colonies also can be obtained from streak plates. The microbial mixture is transferred to the edge of an agar plate with an inoculating loop or swab and then streaked out over the surface in one of several patterns. After the first sector is streaked, the inoculating loop is sterilized and an inoculum for the second sector is obtained from the first sector. A similar process is followed for streaking the third sector, except that the inoculum is from the second sector. Thus, this is essentially a dilution process. Eventually, very few cells will be on the loop, and single cells will drop from it as it is rubbed along the agar surface. These develop into separate colonies.

In microbiology, streaking is one of the most fundamental techniques used to isolate pure colonies of microorganisms from a mixed sample. By spreading microbial cells across the agar surface in a defined pattern, the density of organisms is reduced, ultimately allowing pure, isolated individual colonies to be distinguished and studied.

Pure cultures are essential in accurate identification and can be used further for antibiotic testing & biochemical studies, Streaking is thus an essential skill for every microbiologist.

Major types of streaking techniques:

1. Quadrant Streaking The most common method. The plate is divided into four

quadrants and streaked progressively. Useful for isolating bacteria from mixed

cultures.

2. T-Streaking Plate is divided into three zones (T-shaped pattern). Provides better

separation than a simple streak and is widely used in clinical labs.

3. Continuous (Simple) Streaking Involves spreading the inoculum in one continuous

motion without turning the plate. Suitable when the culture is not highly dense.

4. Radiant (Star) Streaking Streaks radiate outward from a central point. Often used

for quick isolation and for demonstration purposes.

Streaking is an essential technique as it

 Helps in isolating pure colonies.

 Essential for identifying microorganisms.

 Forms the basis for antibiotic sensitivity testing and biochemical studies.

In both spread-plate and streak-plate techniques, successful isolation depends on spatial separation of single cells.

Spread/pour/streak techniques require the use of special culture dishes named petri dishes or plates after their inventor Julius Richard Petri, a member of Robert Koch’s laboratory; Petri developed these dishes around 1887. They consist of two round halves, the top half overlapping the bottom. Petri dishes are very easy to use, may be stacked on each other to save space, and are one of the most common items in microbiology laboratories.

Dilution plating and enrichment technique

A major practical problem is the preparation of pure cultures when microorganisms are present in large or very low numbers in a sample. Serial dilution can be used to dilute the sample if microorganisms are present in large numbers. Plating methods can be combined with the use of selective or differential media to enrich and isolate rare microorganisms.

Dilution Plating (Serial Dilution Method)

Dilution plating is a technique used to reduce the concentration of microorganisms in a sample by serial dilution so that individual colonies can be isolated and counted.

Principle

When a sample containing many microorganisms is serially diluted, the number of cells decreases progressively. After plating, each viable cell forms a separate colony (CFU –Colony Forming Unit).

Procedure

1. Prepare serial dilutions (e.g., 10⁻¹, 10⁻², 10⁻³, etc.).

2. Transfer a measured volume onto sterile agar plates.

3. Spread evenly (spread plate method) or mix with molten agar (pour plate method).

4. Incubate at suitable temperature.

5. Count colonies on plates with 30–300 colonies.

Calculation of Bacterial Count, CFU/ml= Number of colonies/ Dilution factor × Volume plated

Applications

 Estimation of bacterial load in water, milk, soil, etc.

 Isolation of pure colonies.

 Used in food microbiology and clinical microbiology.

Serial dilution, is a series of sequential dilutions performed to convert a dense solution into a more usable concentration. Serial dilution is thus, the process of stepwise dilution of a solution. In Microbiology, serial dilution reduces the concentration of cells in a culture to simplify the operation.

Objectives

 In serial dilution, the density of cells is reduced in each step so that it is easier to calculate the concentration of the cells in the original solution by calculating the total dilution over the entire series.

 The objective of the serial dilution method is to estimate the concentration (number of organisms, bacteria, viruses, or colonies) of an unknown sample by the enumeration of the number of colonies cultured from serial dilutions of the sample.

 By serial dilution, it is possible to obtain incubated culture plates with an easily countable number of colonies (around 30–100) and calculate the number of microbes present in the sample.

The serial dilution is performed as below:

Six test tubes, each with 9 ml of sterile diluents, which can either be distilled water or 0.9% saline, are taken.

1. A sterile pipette is taken.

2. 1 ml of properly mixed sample/culture is drawn into the pipette.

3. The sample is then added to the first tube to make the total volume of 10 ml. This

provides an initial dilution of 10 -1 .

4. The dilution is thoroughly mixed by emptying and filling the pipette several times.

5. The pipette tip is discarded, and a new pipette tip is attached to the pipette.

6. Now, 1 ml of mixture is taken from the 10 -1  dilution and is emptied into the second

tube. The second tube now has a total dilution factor of 10 -2 .

7. The same process is then repeated for the remaining tube, taking 1 ml from the

previous tube and adding it to the next 9 ml diluents.

8. As six tubes are used, the final dilution for the bacteria/cells will be 10 -6  (1 in

1,000,000).

Applications8

1. Serial dilution is used in microbiology to estimate the concentration or number of cells/organisms in a sample to obtain an incubated plate with an easily countable number of colonies.

2. In biochemistry, serial dilution is used to obtain the desired concentration of reagents and chemicals from a higher concentration.

Limitations

Even though serial dilution is a useful technique in laboratories, it faces some challenges.

Some of which are:

1. Errors and transfer inaccuracies lead to less accurate and less precise transfer. This

results in inaccuracies.

2. Serial dilution only allows the reduction of bacteria/cells but not the separation of

bacteria/cells like in other techniques like flow cytometry.

3. This technique also requires highly trained microbiologists and aseptic procedures.

Enrichment Technique

Enrichment technique is used to increase the number of a desired microorganism in a mixed culture by providing favourable growth conditions for it while suppressing others.

Specific nutrients, incubation conditions, or selective agents are used to promote growth of target organism and/or to inhibit unwanted organisms

Types

1. Enrichment Culture – Uses special media to enhance desired organism growth.

2. Selective Enrichment – Contains inhibitory substances to suppress competing flora.

Examples

 Selenite F broth for isolating Salmonella.

 Alkaline peptone water for Vibrio cholerae.

Applications

 Isolation of pathogens from stool samples.

 Environmental microbiology studies.

 Detection of low-number pathogens.

A good example is the isolation of bacteria that degrade the herbicide 2,4- dichloro phenoxy acetic acid (2,4-D). Bacteria able to metabolize 2,4-D can be obtained with a liquid medium containing 2,4-D as its sole carbon source and the required nitrogen, phosphorus, sulfur, and mineral components. When this medium is inoculated with soil, only bacteria able to use 2,4-D will grow. After incubation, a sample of the original culture is transferred to a fresh flask of selective medium for further enrichment of 2,4-D metabolizing bacteria. A mixed population of 2,4-D degrading bacteria will arise after several such transfers. Pure cultures can be obtained by plating this mixture on agar containing 2,4-D as the sole carbon source. Only bacteria able to grow on 2,4-D form visible colonies and can be sub-cultured. This same general approach is used to isolate and purify a variety of bacteria by selecting for specific physiological characteristics.

Culture Techniques

Stab Culture

A stab culture is prepared by inserting (stabbing) an inoculating needle containing microorganisms deep into a solid medium, usually in a test tube with agar.

Purpose:

 To study oxygen requirements of bacteria (aerobic vs anaerobic growth).

 To observe motility of bacteria.

 For maintenance and storage of bacterial strains.

Uses:

 Detecting motility in organisms like Escherichia coli.

 Growing anaerobic bacteria such as Clostridium tetani.

Growth Pattern:

 Growth only at top → Aerobic organism

 Growth throughout → Facultative anaerobe

 Growth at bottom → Obligate anaerobe

 

        

  

        Stab Culture                                                                      Stroke Culture

Stroke Culture (Slant Culture)

A stroke culture is made by streaking microorganisms over the surface of a solid agar slant in a test tube using an inoculating loop.

Purpose:

 To maintain bacterial cultures.

 To observe colony characteristics.

 For short-term preservation and transport.

Common Organisms Grown:

 Staphylococcus aureus

 Salmonella typhi

Growth Pattern:

 Growth appears along the slanted surface in a zigzag or continuous streak.

 Lawn Culture

A lawn culture is prepared by spreading bacteria evenly across the entire surface of an agar plate to produce a uniform, confluent growth. Used in the Kirby–Bauer antibiotic susceptibility test. Thick, uniform bacterial growth covering the whole plate surface.

Purpose:

 Antibiotic sensitivity testing.

 Bacteriophage typing.

 Testing disinfectants.

Growth Pattern:

Growth apperas as uniform/lawn like confluent growth on the surface of media.

Extra Mendelian inheritance - Incomplete and Co dominance, Multiple alleles, Lethal alleles Epistasis, Pleiotropy

 Extra Mendelian inheritance- Alternatives to Dominance and Recessiveness

 Mendel’s experiments with pea plants suggested that:

 (1) two “units” or copies exist for every gene; alleles

(2) alleles maintain their integrity in each generation (no blending); and

(3) in the presence of the dominant allele, the recessive allele is hidden and makes no contribution to the phenotype.

Therefore, recessive alleles can be “carried” and not expressed by individuals. Such heterozygous individuals are sometimes referred to as “carriers.”  Further genetic studies in other plants and animals have shown that much more complexity exists.

The alleles of the same gene interacts in such a way to produce a new character, which is known as allelic interactions.

 Incomplete Dominance 

• In incomplete dominance, neither allele is completely dominant over the other.
• The heterozygous phenotype is intermediate between the two homozygous phenotypes.
• Example: In snapdragon flowers, red (RR) and white (WW) alleles produce pink (RW) flowers. 

Co-dominance

• In co-dominance, both alleles in the heterozygote are fully expressed.
• The phenotype shows both traits simultaneously.
• Example: Human ABO blood group system, where I^A and I^B alleles are co-dominant producing AB blood group. 

Multiple Alleles

• More than two allelic forms exist for a gene in a population.
• An individual still carries only two alleles, but the gene has multiple variants.
• Example: ABO blood group gene has three alleles: I^A, I^B, and Iⁱ.

 

Lethal Alleles

• Alleles that cause death when present in homozygous condition.
• They can be dominant or recessive.
• Example: In mice, the yellow coat color allele (Y) is lethal when homozygous (YY). 

Epistasis

• Interaction between genes where one gene masks or modifies the expression of another gene.
• It affects phenotypic ratios in dihybrid crosses.
• Example: Coat color in Labrador retrievers where one gene controls pigment color and another controls pigment deposition.

 

Pleiotropy

• A single gene influences multiple, seemingly unrelated phenotypic traits.
• Example: The gene responsible for Marfan syndrome affects connective tissue, causing effects in skeleton, eyes, and cardiovascular system.

 

Incomplete dominance

 

Incomplete dominance is a form of gene interaction in which both alleles of a gene at a locus are partially expressed, often resulting in an intermediate or different phenotype.  It is also known as partial dominance.

• For eg, the pink color of flowers (such as snapdragons or four o'clock flowers (Mirabilis jalapa)

In incomplete dominance, the genes of an allelomorphic pair are not expressed as dominant and recessive but express themselves partially when present together in the hybrid. As a result F1 hybrids show characters intermediate to the effect of two genes of the parents. It occurs when neither of two alleles is fully dominant nor recessive towards each other. The alleles are both expressed and the phenotype, or physical trait, is a mixture of the two alleles.

In less technical terms, this means that the two possible traits are blended together.

F2 generation shows genotype ratio of 1RR:^:2Rr:1rr  and a phenotype ratio 1:2:1 red:pink:white.

 

  

    The phenotype of a heterozygous organism is a blend between the phenotypes of its homozygous parents. In the snapdragon, Antirrhinum majus, a cross between a homozygous white-flowered plant C^WC^W and a homozygous red-flowered plant C^RC^R will produce offspring with pink flowers C^RC^W. This type of relationship between alleles, with a heterozygote phenotype intermediate between the two homozygote phenotypes, is called incomplete dominance.

 

     

 

Self-fertilization of a pink plant would produce a genotype ratio of 1C^RC^R: 2 C^RC^W:1C^WC^W and a phenotype ratio 1:2:1 red:pink:white.

  

Examples of incomplete dominance:

-Incomplete Dominance in Animals-

1. Chickens with blue feathers are an example of incomplete dominance. When a black and a white chicken reproduce and neither allele is completely dominant, the result is a blue-feathered bird.

2. When a long-furred Angora rabbit and a short-furred Rex rabbit reproduce, the result can be a rabbit with fur longer than a Rex, but shorter than an Angora. That's a classic example of incomplete dominance producing a trait 1different from either of the parents. 

-Incomplete Dominance in Plants-

1. Wild four-o-clocks tend to have red flowers, while "pure" four-o-clocks with no coloration genes are white. Mixing the two results in pink flowers which are a result of incomplete dominance. However, mixing the pink flowers results in 1⁄4 red, 1⁄4 white and 1⁄2 pink. That 1:2:1 ratio - a quarter like one parent, a quarter like the other, and the remaining half different from either - is common in cases of incomplete dominance.

2. The fruit color of eggplants is another example of incomplete dominance. Crossing deep purple eggplants with white eggplants results in eggplants of a light violet color.

-Incomplete Dominance in Humans- 

 Incomplete dominance is rare in humans; we're genetically complex and most of our traits come from multiple genes. However, there are a few examples

1. When one parent with straight hair and one with curly hair have a child with wavy hair, that's an example of incomplete dominance.

2. Eye color is often cited as an example of incomplete dominance. In fact, it's a little more complicated than that, but hazel eyes are partially caused by incomplete dominance of multiple genes related to green and brown eye color.

3. The disease familial hypercholesterolemia (FH) is an example of incomplete dominance. One allele causes liver cells to be generated without cholesterol receptors, while another causes them to be generated normally. The incomplete dominance causes the generation of cells that do not have enough receptors to remove all dangerous cholesterol from the bloodstream.

Codominance 

A condition in which both alleles of a gene pair in a heterozygote are fully expressed. Thus, Codominance, is where both alleles are simultaneously expressed in the heterozygote. As a result, the phenotype of the offspring is a combination of the phenotype of the parents. Thus, the trait is neither dominant nor recessive.

 

1.   Individuals with type AB blood.

The best example of co-dominance is ABO blood group. ABO blood grouping is controlled by gene I which has three alleles A, B, and O and show co-dominance. An O allele is recessive to both A and B. The A and B alleles are co-dominant with each other. When a person has both A and B, they have type AB blood. Thus, a person  inheriting the alleles A and B alleles will have a type AB blood because A and B alleles  are co-dominant and therefore will be expressed together.

Other co-dominance examples are the white-spotted red flower in plants and the black-and-white- coated mammals.

    

 

In co-dominance, it does not matter whether the alleles in the homologous chromosomes are dominant or recessive. If the homologous chromosome consists of two alleles that can produce proteins, then both will be produced and forms a different phenotype or characteristics to that of a homozygote.

Co-dominance vs polygenic inheritance –Co-dominance is a different concept from polygenic inheritance. While both of them do not conform to the Mendelian inheritance, they differ in a way that in polygenic inheritance multiple genes are involved to produce cumulative effects. In codominance, different alleles of a single gene affect the resulting trait. Examples of polygenic traits in humans are height, weight, skin color, and eye color.

 

2.     MN blood groups of humans

A person's MN blood type is determined by his or her alleles of a certain gene. An L^M allele specifies production of an M marker displayed on the surface of red blood cells, while an L^N allele specifies production of N marker. Homozygotes L^ML^M and L^NL^N have only M or an N markers, respectively, on the surface of their red blood cells. However, heterozygotes L^ML^N have both types of markers in equal numbers on the cell surface. 

Mendel's rules can predict inheritance of codominant alleles. For example, if two people with L^ML^N genotypes had children, there would be M, MN, and N blood types and L^ML^M,  L^ML^N  and L^NL^N genotypes in their children in a 1:2:1 ratio.

 

Question

Pure culture techniques-Streak, Spread, pour plate methods

Isolation of Pure Cultures

In natural habitats microorganisms usually grow in complex, mixed populations with many species. This is a problem for microbiologists because a single type of microorganism cannot be studied adequately in a mixed culture. A pure culture which is a population of cells arising from a single cell is needed to characterize an individual species. 

Pure cultures are so important that the development of pure culture techniques by the German bacteriologist Robert Koch transformed microbiology. Within about 20 years after the development of pure culture techniques most pathogens responsible for the major human bacterial diseases had been isolated.

There are several ways to prepare pure cultures. Pure cultures usually are obtained by isolating individual cells with any of three plating techniques: the spread-plate, streak-plate and pour-plate methods.

The spread-plate and pour-plate methods usually involve diluting a culture or sample and then plating the dilutions. In the spread-plate technique, a specially shaped rod is used to spread the cells on the agar surface; in the pour-plate technique, the cells are first mixed with cooled agar-containing media before being poured into a petri dish.

The streak-plate technique uses an inoculating loop to spread cells across an agar surface.

 

The Spread Plate and Streak Plate

    If a mixture of cells is spread out on an agar surface at a relatively low density, every cell grows into a completely separate colony, a macroscopically visible growth or cluster of microorganisms on a solid medium. Because each colony arises from a single cell, each colony represents a pure culture.

 The spread plate is an easy, direct way of achieving this result. A small volume of dilute microbial mixture containing around 30 to 300 cells is transferred to the center of an agar plate and spread evenly over the surface with a sterile bent-glass rod. The dispersed cells develop into isolated colonies. Because the number of colonies should equal the number of viable organisms in the sample, spread plates can be used to count the microbial population.

 

    Pure colonies also can be obtained from streak plates. The microbial mixture is transferred to the edge of an agar plate with an inoculating loop or swab and then streaked out over the surface in one of several patterns. After the first sector is streaked, the inoculating loop is sterilized and an inoculum for the second sector is obtained from the first sector. A similar process is followed for streaking the third sector, except that the inoculum is from the second sector. Thus this is essentially a dilution process. Eventually, very few cells will be on the loop, and single cells will drop from it as it is rubbed along the agar surface. These develop into separate colonies. In both spread-plate and streak-plate techniques, successful isolation depends on spatial separation of single cells.

 

 

The Pour Plate

Extensively used with procaryotes and fungi, a pour plate also can yield isolated colonies. The original sample is diluted several times to reduce the microbial population sufficiently to obtain separate colonies when plating. Then small volumes of several diluted samples are mixed with liquid agar that has been cooled to about 45°C, and the mixtures are poured immediately into sterile culture dishes. Most bacteria and fungi are not killed by a brief exposure to the warm agar. After the agar has hardened, each cell is fixed in place and forms an individual colony. 

Like the spread plate, the pour plate can be used to determine the number of cells in a population. Plates containing between 30 and 300 colonies are counted. The total number of colonies equals the number of viable microorganisms in the sample that are capable of growing in the medium used. Colonies growing on the surface also can be used to inoculate fresh medium and prepare pure cultures.

These techniques require the use of special culture dishes named petri dishes or plates after their inventor Julius Richard Petri, a member of Robert Koch’s laboratory; Petri developed these dishes around 1887. They consist of two round halves, the top half overlapping the bottom. Petri dishes are very easy to use, may be stacked on each other to save space, and are one of the most common items in microbiology laboratories.

Gene therapy

 Genes are the basic physical and functional units of heredity. Genes are specific sequences of bases that encode instructions on how to make proteins. When genes are altered so that the encoded proteins are unable to carry out their normal functions, genetic disorders develops. 

Gene therapy is a technique for correcting defective genes responsible for disease development. Several approaches can be used for correcting faulty genes:

·   A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common.

·        An abnormal gene could be replaced with a normal gene through homologous recombination.

·        The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function.

·        The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

In most gene therapy studies, a "normal" gene is inserted into the genome to replace an "abnormal," disease-causing gene. A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient's target cells. Currently, the most common vector is a virus that has been genetically altered to carry normal human DNA.

Target cells such as the patient's liver or lung cells are infected with the viral vector. The vector then unloads its genetic material containing the therapeutic human gene into the target cell. The generation of a functional protein product from the therapeutic gene restores the target cell to a normal state.

Gene therapy may be classified into two types:

Germ line gene therapy

Here germ cells, (sperm or eggs) are modified by the introduction of functional genes, which are integrated into their genomes. Therefore, the change due to therapy would be heritable and would be passed on to later generations.

Somatic gene therapy

Here the therapeutic genes are transferred into the somatic cells of a patient. Any modifications and effects will be restricted to the individual patient only, and will not be inherited into later generations.

·       Some of the different types of viruses used as gene therapy vectors are Retroviruses (A class of viruses that can create double-stranded DNA copies of their RNA genomes. These copies of its genome can be integrated into the chromosomes of host cells), Adenoviruses (A class of viruses with double-stranded DNA genomes), Adeno-associated viruses (A class of single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome), Herpes simplex viruses (A class of double-stranded DNA viruses), etc.

·       Besides virus-mediated gene-delivery systems, there are several nonviral options for gene delivery. The simplest method is the direct introduction of therapeutic DNA into target cells. This approach is limited in its application because it can be used only with certain tissues and requires large amounts of DNA.

·       Another nonviral approach involves the creation of an artificial lipid sphere with an aqueous core (liposome). This liposome, which carries the therapeutic DNA, is capable of passing the DNA through the target cell's membrane.

·       Therapeutic DNA also can get inside target cells by chemically linking the DNA to a molecule that will bind to special cell receptors and get into the interior of the target cell.

·       Introducing a 47th (artificial human) chromosome into target cells. This chromosome would exist autonomously alongside the standard 46 --not affecting their workings or causing any mutations. A problem with this method is the difficulty in delivering such a large molecule to the nucleus of a target cell.

Gene Therapy in India

Gene Therapy can be a viable option for increasing the regeneration of the disease for which no other reliable treatment is available. The number of injections required for treating most of the conditions is comparatively low and costs less than the conventional alternatives, The advantages include long-term effects such as the possibility of permanent solutions for the conditions treated by gene therapy. The removal of problematic genes from the body of future parents also removes any chances of recurrence of the same condition in the next generation also.  Gene Therapy can treat Parkinson’s disease, muscular dystrophy, Kidney problems, eye diseases, neurodegenerative Diseases and Immune deficiencies.

Gene therapy in India is rapidly advancing, marked by the launch of affordable, indigenous CAR-T cell therapy for cancer (NexCAR19) from IIT Bombay in 2024.. The ICMR and DBT provide regulatory guidance, to make these costly therapies accessible and affordable for India's large patient population. The focus is on oncology, rare diseases, and genetic conditions like Muscular Dystrophy.

·        Cancer (CAR-T - Chimeric antigen receptor T cell Therapy):  India launched its first indigenous CAR-T cell therapy, NexCAR19, for B-cell malignancies, making advanced cancer treatment affordable.

T cells are the backbone of CAR T-cell therapy. Collect blood from the patient and separate out the T cells. These cells are then genetically engineered to produce special proteins on their surfaces called chimeric antigen receptors, or CARs. The CARs help the cells to bind on to specific antigens, that are present on cancer cells (and some normal cells). They enhance the T cells' ability to kill cancer cells. These modified T cells are grown, and returned to the patient as a single infusion. Currently, this entire process—from the initial blood collection to the cells being infused back into the patient—takes about 3 to 5 weeks. T cells will grow in the patient's body and, bind to cancer cells using their special receptors killing them.

·        Hemophilia A: A landmark trial showed successful gene therapy, eliminating the need for regular infusions by enabling patients to produce Factor VIII, offering a long-term solution.

·        Rare Diseases Focus: India is actively developing gene therapies for numerous rare genetic disorders, addressing a significant unmet need for conditions like Muscular Dystrophy, night blindness, and sickle cell anemia.

Disadvantages:

• Short-lived nature of gene therapy - The therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits.
• Immune response - Anytime a foreign object is introduced into human tissues, the immune system is designed to attack the invader. The risk of stimulating the immune system that reduces gene therapy effectiveness is always a potential risk.
• Problems with viral vectors – Viruses may cause toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease.
• Multigene disorders - Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. Multigene or multifactorial disorders such as these would be difficult to treat effectively using gene therapy.