This MCQ module is based on: Insulin Gene Therapy
TOPIC 47 OF 64
Insulin Gene Therapy
🎓 Class 12
Biology
CBSE
Theory
Ch 10 – Biotechnology and its Applications
⏱ ~14 min
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Biotechnological Applications in Medicine - Insulin and Gene Therapy
Recombinant DNA technological processes have made an immense impact in the area of healthcare, by enabling mass production of safe and more effective therapeutic drugs. Further, the recombinant therapeutics do not induce unwanted immunological responses, as is common in the case of similar products isolated from non-human sources.
At present about 30 recombinant therapeutics have been approved for human use the world over, and in India 12 of these are presently being marketed.
Genetically engineered insulin
Management of adult-onset diabetes is possible by taking insulin at regular time intervals. But what would a diabetic patient do if enough human insulin were not available?
One would have to isolate and use insulin from other animals. Insulin used for diabetes was indeed earlier extracted from the pancreas of slaughtered cattle and pigs. Two questions follow immediately: would insulin isolated from other animals be just as effective as that secreted by the human body itself, and would it not elicit an immune response in the human body?
Insulin from an animal source did cause some patients to develop allergy or other types of reactions to the foreign protein. Now imagine instead that a bacterium were available that could make human insulin. Suddenly the whole process becomes simple: you can easily grow a large quantity of the bacteria and make as much insulin as you need.
The structure of insulin, and the problem it created
Insulin consists of two short polypeptide chains: chain A and chain B, that are linked together by disulphide bridges.
In mammals, including humans, insulin is synthesised as a pro-hormone. Like a pro-enzyme, the pro-hormone also needs to be processed before it becomes a fully mature and functional hormone. It contains an extra stretch called the C peptide, which is not present in the mature insulin and is removed during maturation into insulin.
This is where the difficulty lay. The main challenge for the production of insulin using rDNA techniques was getting insulin assembled into a mature form.
How Eli Lilly solved it in 1983
In 1983, Eli Lilly, an American company, prepared two DNA sequences corresponding to the A and B chains of human insulin and introduced them into plasmids of E. coli to produce insulin chains. Chains A and B were produced separately, extracted, and combined by creating disulfide bonds to form human insulin.
Notice the elegance of the workaround. The natural route makes one long pro-hormone and then cuts the C peptide out — a processing step that a bacterium cannot perform. So rather than teaching E. coli to process a pro-hormone, the chemists skipped the pro-hormone altogether: make the two chains separately, and do the final assembly in a flask. When the cell cannot do a step, move that step outside the cell.
Why insulin is injected and not swallowed
Think about whether insulin can be orally administered to diabetic people. It cannot — and the reason is worth stating carefully, because it applies to every protein pharmaceutical.
Insulin is a protein. Taken by mouth it would meet the stomach's strongly acidic contents and then the proteases of the digestive tract, and would be broken down into its amino acids before any of it could be absorbed into the blood. Digesting protein is exactly what the alimentary canal is built to do. Hence insulin must be injected, so that it reaches the blood without passing through the digestive tract at all.
Gene therapy
If a person is born with a hereditary disease, can a corrective therapy be taken for such a disease? Gene therapy is an attempt to do exactly this.
Gene therapy is a collection of methods that allows correction of a gene defect that has been diagnosed in a child or embryo. Here genes are inserted into a person's cells and tissues to treat a disease. Correction of a genetic defect involves delivery of a normal gene into the individual or embryo, to take over the function of and compensate for the non-functional gene.
The first clinical gene therapy, 1990
The first clinical gene therapy was given in 1990, to a four-year-old girl with adenosine deaminase (ADA) deficiency. This enzyme is crucial for the immune system to function, and the disorder is caused due to the deletion of the gene for adenosine deaminase.
Two treatments already existed, and neither was satisfactory. In some children, ADA deficiency can be cured by bone marrow transplantation. In others it can be treated by enzyme replacement therapy, in which functional ADA is given to the patient by injection. But the problem with both of these approaches is that they are not completely curative.
How the gene therapy was performed
As a first step towards gene therapy, lymphocytes from the blood of the patient are grown in a culture outside the body. A functional ADA cDNA, using a retroviral vector, is then introduced into these lymphocytes, which are subsequently returned to the patient.
However, as these cells are not immortal, the patient requires periodic infusion of such genetically engineered lymphocytes. However, if the gene isolated from marrow cells producing ADA is introduced into cells at early embryonic stages, it could be a permanent cure.
| Approach | What is done | Limitation |
|---|---|---|
| Bone marrow transplantation | Marrow from a matched donor replaces the patient's own | Can cure in some children only; not completely curative in general |
| Enzyme replacement therapy | Functional ADA is given to the patient by injection | Not completely curative; the enzyme must keep being supplied |
| Gene therapy on lymphocytes | Patient's lymphocytes are cultured, a functional ADA cDNA is introduced using a retroviral vector, and the cells are returned | Lymphocytes are not immortal, so periodic infusion is required |
| Gene therapy at early embryonic stages | The ADA gene from marrow cells is introduced into cells at early embryonic stages | This could be a permanent cure — the cells that give rise to all the others are corrected |
📐 Activity: why the treatment has to be repeated
The 1990 patient received her own lymphocytes, corrected with a functional ADA gene. Within months, the treatment had to be repeated, and it continued to be repeated.
Predict: what property of the treated cells makes repetition necessary, and what single change to the target cell would end it?
The property. As these cells are not immortal, the patient requires periodic infusion of such genetically engineered lymphocytes. A mature lymphocyte has a limited lifespan and does not renew itself indefinitely, so the corrected population dies out and the deficiency returns.
The change that would end it. Correct a cell that does keep dividing and giving rise to new lymphocytes. The chapter names the goal directly: if the gene isolated from marrow cells producing ADA is introduced into cells at early embryonic stages, it could be a permanent cure. Correcting a stem cell, rather than its short-lived product, corrects every cell that stem cell will ever make.
Why that is not simply done. Intervening at an early embryonic stage means altering cells that will give rise to the whole individual, including the germ line, so the change would be passed to descendants. That is why the chapter's last section is about ethical issues and why the Indian Government set up the GEAC. The biology is clear; the decision about using it is not a biological question alone.
🎯 Interactive: Compare the Treatments
Four ways of dealing with a missing protein. Choose one to see how it works and where it falls short.
🎯 Competency-Based Questions
Q1. Why is a recombinant therapeutic protein superior to the same protein isolated from a non-human source? Give three distinct reasons using insulin as the example.
(i) No unwanted immunological response. Recombinant therapeutics do not induce unwanted immunological responses, as is common in the case of similar products isolated from non-human sources. Insulin from an animal source caused some patients to develop allergy or other types of reactions to the foreign protein. Recombinant human insulin is made in bacteria, yet its structure is absolutely identical to that of the natural human molecule, so the body has nothing to react against.
(ii) Supply. Insulin was earlier extracted from the pancreas of slaughtered cattle and pigs, so the quantity available depended on the number of animals slaughtered. With a bacterium that makes human insulin, you can easily grow a large quantity of the bacteria and make as much insulin as you need. This is what makes mass production of therapeutic drugs possible.
(iii) Effectiveness and safety. The chapter asks directly whether insulin isolated from other animals would be just as effective as that secreted by the human body itself. An animal protein differs slightly in sequence and therefore in activity. Recombinant products are also free from the risk of infection observed in products isolated from non-human sources, since they are made in a defined culture rather than harvested from tissue.
The scale of the change. About 30 recombinant therapeutics have now been approved for human use the world over, 12 of them presently marketed in India.
Q2. Explain what the C peptide is, why it made recombinant insulin production difficult, and precisely how Eli Lilly got round the problem.
What the C peptide is. In mammals, including humans, insulin is synthesised as a pro-hormone. Like a pro-enzyme, a pro-hormone must be processed before it becomes a fully mature and functional hormone. Pro-insulin contains an extra stretch called the C peptide, which is not present in mature insulin and is removed during maturation into insulin.
Why it was a problem. The main challenge for the production of insulin using rDNA techniques was getting insulin assembled into a mature form. If you simply express the human insulin gene in a bacterium, what you get is pro-insulin — a single chain with the C peptide still in place. A bacterium has none of the processing machinery that a pancreatic cell uses to excise the C peptide and fold the remainder correctly, so the product would not be functional insulin.
How Eli Lilly solved it, in 1983. They prepared two DNA sequences corresponding to the A and B chains of human insulin and introduced them into plasmids of E. coli to produce the insulin chains. Chains A and B were produced separately, extracted, and then combined by creating disulfide bonds to form human insulin.
The general principle. Insulin consists of two short polypeptide chains, A and B, linked by disulphide bridges. Since the C peptide is discarded anyway, there was no need to make it at all. Rather than teaching the bacterium a processing step it cannot perform, that step was taken out of the cell and done chemically.
Q3. What is gene therapy? Illustrate using the example of adenosine deaminase deficiency.
Definition. Gene therapy is a collection of methods that allows correction of a gene defect that has been diagnosed in a child or embryo. Genes are inserted into a person's cells and tissues to treat a disease. Correction of a genetic defect involves delivery of a normal gene into the individual or embryo, to take over the function of and compensate for the non-functional gene.
The disease. Adenosine deaminase deficiency is caused due to the deletion of the gene for adenosine deaminase, an enzyme crucial for the immune system to function. The first clinical gene therapy was given in 1990 to a four-year-old girl with this disorder.
What was available before. In some children ADA deficiency can be cured by bone marrow transplantation; in others it can be treated by enzyme replacement therapy, in which functional ADA is given to the patient by injection. The problem with both approaches is that they are not completely curative.
The procedure. As a first step towards gene therapy, lymphocytes from the blood of the patient are grown in a culture outside the body. A functional ADA cDNA, carried by a retroviral vector, is introduced into these lymphocytes, which are subsequently returned to the patient.
The limitation, and the ideal. As these cells are not immortal, the patient requires periodic infusion of such genetically engineered lymphocytes. If, however, the gene isolated from marrow cells producing ADA were introduced into cells at early embryonic stages, it could be a permanent cure.
Q4. Why must insulin be injected rather than taken as a tablet, and what would have to be overcome to make an orally active protein pharmaceutical?
Why it must be injected. Insulin is a protein, consisting of two short polypeptide chains linked by disulphide bridges. The alimentary canal is built precisely to dismantle proteins: the stomach is strongly acidic, which denatures the protein and breaks the bonds holding its shape, and the proteases of the stomach and intestine then hydrolyse it to its constituent amino acids. Nothing recognisable as insulin would survive to be absorbed, and even if it did, an intact protein of this size crosses the intestinal wall very poorly.
The major problem to be overcome. Two problems, in fact, and both must be solved together: protecting the protein from acid and from proteolytic digestion, and then getting a large hydrophilic molecule across the intestinal epithelium into the blood.
Approaches that are pursued. Enclosing the protein in an acid-resistant, enzyme-resistant coating or capsule that releases it in the intestine; encapsulating it in liposomes or polymer nanoparticles; co-administering protease inhibitors; chemically modifying the protein so that proteases no longer recognise it while its activity is retained; and using absorption enhancers to improve uptake across the gut wall. Avoiding the gut altogether — nasal, inhaled or transdermal routes — is the other line of attack.
The general point. This is why almost every recombinant therapeutic protein, from insulin to growth hormone to clotting factors, is given by injection. The difficulty is not making the protein; it is delivering it.
Q5. Enzyme replacement therapy supplies the missing protein, and gene therapy supplies the missing gene. Explain why the second is considered a fundamentally different kind of treatment, and what it would take for it to be a permanent cure.
What each one treats. Enzyme replacement therapy treats the consequence: functional ADA is given to the patient by injection, so the enzyme is present for as long as the injected molecules last, and then must be given again. Gene therapy treats the cause: it delivers a normal gene into the individual to take over the function of and compensate for the non-functional gene, so the patient's own cells make the enzyme continuously.
Why that is a difference in kind. A protein is a product with a finite lifetime; a gene is an instruction that is copied every time the cell divides. Correcting the instruction means the correction is inherited by the cell's descendants, which no amount of supplying the product can achieve. This is why the chapter notes that both bone marrow transplantation and enzyme replacement therapy, although they can cure or treat ADA deficiency, are not completely curative.
Why the 1990 therapy still was not permanent. The corrected cells were mature lymphocytes, and as these cells are not immortal, the patient requires periodic infusion of such genetically engineered lymphocytes. Correcting a short-lived cell gives a short-lived correction.
What a permanent cure requires. The gene must be placed in a cell that will keep producing corrected descendants. The chapter states the condition: if the gene isolated from marrow cells producing ADA is introduced into cells at early embryonic stages, it could be a permanent cure. In other words, correct the stem cell or the embryo, not the finished cell — which is exactly why gene therapy raises ethical questions that enzyme replacement does not.
🧠 Assertion–Reason Questions
For each pair, decide whether both statements are true and whether the reason correctly explains the assertion.
Assertion (A): Recombinant human insulin does not cause the allergic reactions that animal insulin sometimes did.
Reason (R): Recombinant therapeutics are identical to human proteins and so do not induce unwanted immunological responses.
Both A and R are true, and R is the correct explanation of A.
Insulin from an animal source caused some patients to develop allergy or other types of reactions to the foreign protein. Human insulin is made in bacteria, yet its structure is absolutely identical to that of the natural molecule, so the immune system has no foreign sequence to respond to.
Assertion (A): Chains A and B of insulin were produced separately in E. coli and combined afterwards.
Reason (R): The main challenge in producing insulin by rDNA techniques was getting it assembled into a mature form.
Both A and R are true, and R is the correct explanation of A.
In mammals insulin is made as a pro-hormone containing an extra C peptide, which is removed during maturation. A bacterium cannot carry out that processing, so in 1983 Eli Lilly prepared two DNA sequences corresponding to chains A and B, produced the chains separately, extracted them, and combined them by creating disulfide bonds.
Assertion (A): The 1990 ADA gene therapy patient needed the treatment repeated periodically.
Reason (R): The retroviral vector failed to introduce the functional ADA cDNA into her lymphocytes.
A is true but R is false.
The vector worked: a functional ADA cDNA was successfully introduced into the cultured lymphocytes, which were then returned to the patient. Repetition was necessary for a different reason — these cells are not immortal, so the corrected population dies out and periodic infusion of genetically engineered lymphocytes is required. Introducing the gene into cells at early embryonic stages could instead be a permanent cure.
Frequently Asked Questions - Recombinant Insulin and Gene Therapy
Why are recombinant therapeutics better than products isolated from non-human sources?
Recombinant DNA technological processes enable mass production of safe and more effective therapeutic drugs, and the recombinant therapeutics do not induce unwanted immunological responses, as is common in the case of similar products isolated from non-human sources. They are also free from the risk of infection seen with products harvested from tissue. About 30 recombinant therapeutics have been approved for human use the world over, and 12 of these are presently being marketed in India.
What was the problem with insulin extracted from cattle and pigs?
Insulin used for diabetes was earlier extracted from the pancreas of slaughtered cattle and pigs. Insulin from an animal source caused some patients to develop allergy or other types of reactions to the foreign protein, and the supply was limited by the number of animals slaughtered.
What is the structure of insulin?
Insulin consists of two short polypeptide chains, chain A and chain B, that are linked together by disulphide bridges.
What is the C peptide and why did it complicate recombinant insulin production?
In mammals including humans, insulin is synthesised as a pro-hormone which, like a pro-enzyme, needs to be processed before it becomes fully mature and functional. Pro-insulin contains an extra stretch called the C peptide, which is not present in mature insulin and is removed during maturation. The main challenge for producing insulin by rDNA techniques was getting insulin assembled into this mature form, since a bacterium cannot perform that processing step.
How was human insulin produced by recombinant DNA technology?
In 1983, Eli Lilly, an American company, prepared two DNA sequences corresponding to the A and B chains of human insulin and introduced them into plasmids of E. coli to produce insulin chains. Chains A and B were produced separately, extracted, and combined by creating disulfide bonds to form human insulin.
Can insulin be taken orally?
No. Insulin is a protein, and taken by mouth it would be denatured by the acidic stomach contents and then hydrolysed to amino acids by the proteases of the digestive tract before it could reach the blood. It therefore has to be injected.
What is gene therapy?
Gene therapy is a collection of methods that allows correction of a gene defect diagnosed in a child or embryo. Genes are inserted into a person's cells and tissues to treat a disease, and correction of a genetic defect involves delivery of a normal gene into the individual or embryo to take over the function of and compensate for the non-functional gene.
What causes ADA deficiency and how was the first gene therapy performed?
Adenosine deaminase deficiency is caused by the deletion of the gene for adenosine deaminase, an enzyme crucial for the immune system to function. The first clinical gene therapy was given in 1990 to a four-year-old girl with this disorder. Lymphocytes from the blood of the patient were grown in a culture outside the body, a functional ADA cDNA was introduced into these lymphocytes using a retroviral vector, and the cells were returned to the patient.
Why is the lymphocyte gene therapy for ADA deficiency not a permanent cure?
Because these cells are not immortal, so the patient requires periodic infusion of such genetically engineered lymphocytes. If the gene isolated from marrow cells producing ADA were introduced into cells at early embryonic stages, it could be a permanent cure.
How is ADA deficiency treated without gene therapy, and why are those treatments inadequate?
In some children ADA deficiency can be cured by bone marrow transplantation, and in others it can be treated by enzyme replacement therapy, in which functional ADA is given to the patient by injection. The problem with both of these approaches is that they are not completely curative.
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