Biotechnology and its Applications — NCERT Solutions

What is Gene Therapy?

Gene therapy is a technique used to correct a gene defect in an individual by:

• Inserting a functional (normal) copy of the defective gene into the target cells, OR
• Replacing a defective mutant allele with a functional one.

It is used to treat hereditary (genetic) diseases where a gene is absent or non-functional.

Illustration — ADA (Adenosine Deaminase) Deficiency:

Background:

• ADA deficiency is caused by the deletion/mutation of the gene encoding the enzyme Adenosine Deaminase.
• ADA is crucial for the functioning of the immune system (specifically T-lymphocytes).
• Without ADA, toxic metabolites accumulate and destroy T-cells, leading to Severe Combined Immunodeficiency (SCID) — the child cannot fight infections.

Steps in Gene Therapy for ADA deficiency:

$$\text{Step 1: Isolation of lymphocytes from patient's blood}$$

$$\text{Step 2: Introduction of functional ADA gene into lymphocytes using a retroviral vector (in vitro)}$$

$$\text{Step 3: Genetically engineered lymphocytes (now carrying ADA gene) are cultured and multiplied}$$

$$\text{Step 4: Engineered lymphocytes are reinfused into the patient's body}$$

Outcome: The introduced lymphocytes produce functional ADA enzyme, restoring immune function.

Limitation: This is not a permanent cure because the introduced lymphocytes have a finite life span. The patient requires periodic infusion of engineered lymphocytes.

Permanent cure possibility: If the ADA gene is introduced into cells at the early embryonic stage, it could provide a permanent cure, as all cells derived from that embryo would carry the functional gene.

Alternative treatment: Enzyme replacement therapy — injection of ADA enzyme — but it is expensive and not permanent.

Concept: This involves recombinant DNA technology — isolating the gene of interest, inserting it into a vector, transforming a host bacterium, and expressing the gene to produce the desired protein.

**Steps in Cloning and Expressing Human Growth Hormone Gene in E. coli:**

Step 1: Isolation of the gene of interest
$$\text{Human cell (source of mRNA for growth hormone)}$$
$$\downarrow \text{ Reverse Transcriptase}$$
$$\text{cDNA (complementary DNA) of growth hormone gene}$$

Step 2: Cutting the gene and vector with restriction endonuclease
$$\text{cDNA} + \text{Plasmid vector (e.g., pBR322)} \xrightarrow{\text{Same Restriction Enzyme (e.g., EcoRI)}} \text{Sticky ends generated on both}$$

Step 3: Ligation — Formation of Recombinant DNA
$$\text{cDNA (sticky ends)} + \text{Cut plasmid (sticky ends)} \xrightarrow{\text{DNA Ligase}} \text{Recombinant plasmid}$$

**Step 4: Transformation into *E. coli***
$$\text{Recombinant plasmid} \xrightarrow{\text{Heat shock / Electroporation}} \text{E. coli host cell}$$

Step 5: Selection of transformants
$$\text{Plating on selective medium (antibiotic plates)} \rightarrow \text{Only transformed colonies survive}$$

Step 6: Expression of the gene
$$\text{E. coli transcribes and translates the human growth hormone gene}$$
$$\downarrow$$
$$\text{Human Growth Hormone (recombinant) is produced}$$

Step 7: Extraction and purification
$$\text{Bacteria are lysed} \rightarrow \text{Protein is extracted and purified} \rightarrow \text{Recombinant Human Growth Hormone}$$

Diagrammatic Representation (described):

Human cell
    |
    | (extract mRNA → reverse transcriptase)
    ↓
cDNA of GH gene
    |
    | (cut with restriction enzyme)
    ↓
Gene with sticky ends
    +
Plasmid vector (cut with same enzyme)
    |
    | (DNA Ligase)
    ↓
Recombinant Plasmid
    |
    | (Transformation into E. coli)
    ↓
Transformed E. coli
    |
    | (Culture, expression)
    ↓
Recombinant Human Growth Hormone

Note: The recombinant human growth hormone produced is identical to the natural hormone and is used to treat growth hormone deficiency (dwarfism) in children.

Understanding the chemistry of oil in seeds:

Seed oils (e.g., in groundnut, soybean, mustard) are triglycerides — esters of glycerol and fatty acids. They are synthesised in seeds through specific enzymatic pathways. Key enzymes involved include fatty acid synthase, desaturases, and acyl transferases.

Method using rDNA Technology — RNA Interference (RNAi) / Gene Silencing:

Approach 1 — Antisense RNA / RNAi technology:

1. Identify the key gene(s) encoding enzymes responsible for oil (fatty acid/triglyceride) biosynthesis in seeds (e.g., the gene for fatty acid synthase or acyl-ACP thioesterase).
2. Using rDNA technology, introduce a complementary (antisense) sequence of this gene into the plant.
3. The antisense RNA produced will bind to the mRNA of the oil-synthesis gene, forming a double-stranded RNA (dsRNA).
4. This triggers RNA interference (RNAi) — the dsRNA is degraded by the RISC complex, silencing the gene.
5. As a result, the enzyme for oil synthesis is not produced → oil is not synthesised in seeds.

Approach 2 — Knockout of oil biosynthesis genes:

1. Using rDNA technology, the gene(s) encoding key enzymes in the fatty acid biosynthesis pathway can be knocked out (deleted or disrupted).
2. This prevents the formation of triglycerides in seeds.

Approach 3 — Overexpression of lipase gene:

1. Introduce a gene encoding lipase (an enzyme that breaks down triglycerides) under a seed-specific promoter.
2. The lipase will degrade the oil within the seed itself.

Most practical method: RNAi-mediated silencing of the key oil biosynthesis gene is the most widely used approach in modern biotechnology to alter oil content in seeds.

Application: This approach is already used to produce low-erucic acid canola (rapeseed) and high-oleic soybean by modifying fatty acid desaturase genes.

Golden Rice:

Definition:
Golden Rice is a variety of rice (Oryza sativa) that has been genetically modified to produce β-carotene (a precursor of Vitamin A) in the edible endosperm (grain). It appears golden-yellow in colour due to the accumulation of β-carotene, hence the name.

Why was it developed?
Vitamin A deficiency (VAD) is a major public health problem in developing countries, especially in Asia and Africa, causing:

• Night blindness and permanent blindness (especially in children)
• Weakened immune system
• Increased mortality in children

Normal rice does not produce β-carotene in its endosperm.

How was it made?
Golden Rice was developed by Ingo Potrykus and Peter Beyer in the late 1990s.

Two genes were introduced into rice:

1. Psy gene (phytoene synthase) — from daffodil (Narcissus pseudonarcissus) or maize
2. CrtI gene (carotene desaturase) — from the bacterium Erwinia uredovora

These genes enable the rice endosperm to synthesise β-carotene through the carotenoid biosynthesis pathway.

Golden Rice 2:
An improved version (Golden Rice 2) was developed using the psy gene from maize, which produces 23 times more β-carotene than the original Golden Rice.

Significance:

• Provides Vitamin A to populations dependent on rice as a staple food.
• A cost-effective, sustainable solution to Vitamin A deficiency.
• Does not require changes in dietary habits.

Controversy:
Golden Rice remains controversial due to concerns about GM food safety, environmental impact, and corporate control of food supply.

Answer: No, normal human blood does not contain active proteases and nucleases in significant amounts.

Explanation:

1. Proteases in blood:

• Blood does contain some proteolytic enzymes, but they are present in inactive forms (zymogens/proenzymes), e.g., prothrombin (inactive form of thrombin), plasminogen (inactive form of plasmin).
• These are activated only under specific conditions (e.g., during blood clotting or fibrinolysis).
• Active proteases in blood would be harmful as they would digest blood proteins and plasma proteins.
• Therefore, active proteases are NOT normally present in blood.

2. Nucleases in blood:

• Blood plasma contains very low levels of DNase and RNase activity.
• However, significant active nucleases are not normally present in circulating blood.
• If nucleases were present in blood, they would degrade the nucleic acids of blood cells and other cells.

Relevance to Biotechnology:
This is why recombinant proteins (like insulin, gene therapy vectors) can be administered intravenously — they are not immediately degraded by proteases or nucleases in the blood.

However, oral administration of protein drugs is a problem because the digestive system (stomach and intestine) contains active proteases (pepsin, trypsin, chymotrypsin) and nucleases that would degrade the protein/DNA before absorption.

Conclusion: Blood does not have significant active proteases and nucleases under normal physiological conditions, which is essential for maintaining the integrity of blood proteins and cellular DNA/RNA.

Orally Active Protein Pharmaceuticals:

Most protein-based drugs (e.g., insulin, growth hormone, interferon) are currently administered by injection (parenteral route) because proteins are degraded in the gastrointestinal (GI) tract when taken orally.

Major Problem:
The major problem with oral administration of protein pharmaceuticals is:

1. Degradation by digestive enzymes: The stomach contains pepsin (acidic pH ~2) and the intestine contains trypsin, chymotrypsin, elastase, peptidases — these proteases rapidly degrade the protein drug before it can be absorbed.
2. Acidic pH of stomach: The low pH (1.5–3.5) of gastric juice denatures and inactivates most proteins.
3. Poor absorption: Large protein molecules cannot easily cross the intestinal epithelium (poor permeability).
4. First-pass metabolism: Even if absorbed, proteins may be degraded in the liver before reaching systemic circulation.

Methods to make proteins orally active:

1. Microencapsulation / Nanoparticle delivery:

• Proteins are enclosed in biodegradable polymeric nanoparticles or liposomes that protect them from gastric acid and enzymes.
• The capsule dissolves in the intestine, releasing the protein for absorption.

2. Enteric coating:

• Coating the drug with a pH-sensitive polymer that resists gastric acid but dissolves at the higher pH of the intestine.

3. Use of protease inhibitors:

• Co-administering protease inhibitors along with the protein drug to prevent enzymatic degradation.

4. Chemical modification of proteins:

• PEGylation (attaching polyethylene glycol) — protects protein from proteolysis and increases stability.
• Cyclisation or introduction of non-natural amino acids to make proteins resistant to proteases.

5. Mucoadhesive systems:

• Formulations that adhere to the intestinal mucosa, increasing contact time and absorption.

6. Transgenic plants producing edible vaccines/proteins:

• Genes for therapeutic proteins are expressed in edible plant parts (e.g., banana, tomato) — when eaten, the protein is released in the gut.
• Example: Edible vaccines in banana for hepatitis B.

7. Absorption enhancers:

• Chemicals that temporarily increase intestinal permeability to allow protein absorption.

Conclusion:
The major problem is proteolytic degradation of the protein in the GI tract (by pepsin, trypsin, chymotrypsin) and the acidic environment of the stomach. Overcoming these barriers while maintaining protein bioactivity and ensuring sufficient absorption remains the central challenge in developing orally active protein pharmaceuticals.