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Study On Gene Therapy

Published on Sep 12, 2023


The term disease broadly refers to any condition that impairs normal function, and is therefore associated with dysfunction of normal homeostasis. When the functioning of one or more organs or systems of the body is adversely affected, characterised by various signs and symptoms, we say that we are not healthy, i.e., we have a disease.

Health can be defined as a state of complete physical, mental and social well-being. When people are healthy, they are more efficient at work. This increases productivity and brings economic prosperity. Health also increases longevity of people and reduces infant and maternal mortality.

Based on the cause diseases can be broadly classified as:


These are diseases caused due to invasion of a foreign parasitic organism. They are temporary because the immune system of organisms can fight such pathogens (disease causing organisms) to a certain extent hence helping in prevention of the disease. The immune system can also be aided with the use of several drugs. Apart from easy treatment they can also be easily prevented

Lifestyle Diseases

Lifestyle diseases (also sometimes called diseases of longevity or diseases of civilization interchangeably) are diseases that appear to increase in frequency as countries become more industrialized and people live longer. They can include Alzheimer's disease, asthma, and obesity. Diet and lifestyle are major factors thought to influence susceptibility to many diseases. Drug abuse, tobacco smoking, and alcohol drinking, as well as a lack of exercise may also increase the risk of developing certain diseases, especially later in life. These diseases can be prevented completely by living a healthy lifestyle.

Genetic Disorders

A genetic disorder is an illness caused by one or more abnormalities in the genome, especially a condition that is present from birth (congenital). They are medical disorders related to gene mutation. Genetic disorders are heritable, and are passed down from the parents' genes. Other defects may be caused by new mutations or changes to the DNA. In such cases, the defect will only be heritable if it occurs in the germ line. The same disease, such as some forms of cancer, may be caused by an inherited genetic condition in some people, by new mutations in other people, and by non-genetic causes in still other people. These diseases are totally random and difficult to prevent as they are not caused by external agents. Also as their root cause lies in the genome of the organism their cure was thought to be impossible until the breakthrough research unlocking the secrets of DNA leading to the development of biotechnology and hence gene therapy.

Gene Therapy

We can think of a medical condition or illness as a "broken window." Many medical conditions result from flaws, or mutations, in one or more of a person's genes. Mutations cause the protein encoded by that gene to malfunction. When a protein malfunctions, cells that rely on that protein's function can't behave normally, causing problems for whole tissues or organs. Medical conditions related to gene mutations are called genetic disorders.

So, if a flawed gene caused our "broken window," can we "fix" it? What are our options?

1. Stay silent: ignore the genetic disorder and nothing gets fixed.

2. Try to treat the disorder with drugs or other approaches: depending on the disorder, treatment may or may not be a good long-term solution.

3. Put in a normal, functioning copy of the gene: if you can do this, it may solve the problem!

If it is successful, gene therapy provides a way to fix a problem at its source. Adding a corrected copy of the gene may help the affected cells, tissues and organs work properly. Gene therapy differs from traditional drug-based approaches, which may treat the problem, but which do not repair the underlying genetic flaw.

Targets for Gene Therapy

But now a question arises, which disorders or diseases can we target using gene therapy? Many disorders or medical conditions might be treated using gene therapy, but others may not be suitable for this approach. For a disease to be targeted by gene therapy it must satisfy the following conditions:

1. The condition must result from mutations in one or more genes

2. To treat a genetic flaw, the knowledge of which gene(s) to pursue is absolutely necessary. Also a DNA copy of that gene available in the laboratory. The best candidates for gene therapy are the so-called "single-gene" disorders - which are caused by mutations in only one gene.

3. To design the best possible approach, knowledge about how the gene factors into the disorder is required. For example:

 Which tissues are affected?

 What role does the protein encoded by the gene play within the cells of that tissue?

 Exactly how do mutations in the gene affect the protein's function?

4. Adding a normal copy of the gene should fix the problem in the affected tissue. This may seem like obvious, but it's not. What if the mutated gene encodes a protein that prevents the normal protein from doing its job? Mutated genes that function this way are called dominant negative and adding back the normal protein won't fix the problem.

5. The gene delivery to cells of the affected tissue must be possible. It depends on:

 How accessible is the tissue? Is it fairly easy (skin, blood or lungs), or more difficult to reach (internal organs)?

 What is the best mode of delivery?

The techniques of biotechnology have made it possible to isolate the required gene in the laboratory and also deliver the gene.

Isolation of Gene of Interest

The first step is to find and isolate the gene that will be inserted into the genetically modified organism. Finding the right gene to insert usually draws on years of scientific research into the identity and function of useful genes. Once that is known the DNA needs to be cut at specific locations to isolate the gene of interest. This can be done by using restriction enzymes also known as molecular scissors which cut DNA at specific sites containing palindromic DNA sequences. But in order to cut the DNA with restriction enzymes, it needs to be in pure form, free from other macro-molecules.

Isolation of DNA

Since the DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids. This can be achieved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus). Genes are located on long molecules of DNA intertwined with proteins such as histones. The RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease. Other molecules can be removed by appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol. This can be seen as collection of fine threads in the suspension.

Cutting of DNA

Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme, at the optimal conditions for that specific enzyme. The cutting of DNA by restriction endonucleases results in the fragments of DNA. These fragments can be separated by a technique known as gel electrophoresis. Since DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix. The separated bands of DNA are analysed for the required gene and then it is cut out from the agarose gel and extracted from the gel piece. This step is known as elution.

Multiplication of Gene (PCR)

PCR or polymerase chain reaction is then used to create multiple copies of the gene of interest. In this reaction, multiple copies of the gene (or DNA) of interest is synthesised in vitro using two sets of primers (small chemically synthesised oligonucleotides that are complementary to the regions of DNA) and the enzyme DNA polymerase. The enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as template. If the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times, i.e., 1 billion copies are made.

Gene Targeting

Gene delivery is one of the biggest challenges in the field of gene therapy.

Gene Delivery includes:

1. TARGETING the right cells.

2. ACTIVATING the gene. A gene's journey is not over when it enters the cell. It must go to the cell's nucleus and be "turned on," meaning that its transcription and translation are activated to produce the protein product encoded by the gene. For gene delivery to be successful, the protein that is produced must function properly.

3. INTEGRATING the gene in the cells. The gene must stay put and continue working in the target cells. If so, it must be ensured that the gene integrates into, or becomes part of the host cell's genetic material, or that the gene finds another way to survive in the nucleus without being rejected.

4. AVOIDING harmful side effects. Anytime an unfamiliar biological substance is introduced into the body, there is a risk that it will be toxic or that the body will mount an immune response against it. If the body develops immunity against a specific gene delivery vehicle, future rounds of the therapy will be ineffective.

Choosing the Best Vector

There is no "perfect vector" that can treat every disorder. Like any type of medical treatment, a gene therapy vector must be customized to address the unique features of the disorder. We have learnt the lesson, of transferring genes into plants and animals from bacteria and viruses, which have known this for ages – how to deliver genes to transform eukaryotic cells and force them to do what the bacteria or viruses want.

Part of the challenge in gene therapy is choosing the most suitable vector for treating the disorder. Some vectors commonly used are:


Usually when we think of viruses, we think of them causing diseases such as the common cold, the flu, and HIV/AIDS. When faced with the problem of gene delivery, scientists looked to viruses. Why reinvent the wheel if there's a perfectly good one out there? If we can modify viruses to deliver genes without making people sick, we may have a good set of gene therapy tools.

General advantages of viral vectors:

-They're very good at targeting and entering cells.

 -Some viral vectors might be engineered to target specific types of cells.

 -They can be modified so that they can't replicate and destroy the cell.

General drawbacks of viral vectors:

 A virus can't "expand" to fit a piece of genetic material larger than it is naturally built to carry. Therefore, some genes may be too big to fit into a certain type of virus.
 Viruses can cause immune responses in patients, resulting in two potential outcomes:

 Patients may get sick.

 A patient's immunity to a virus may prevent him from responding to repeated treatments.

However, modern viral vectors have been engineered without most of the proteins that would cause an immune response.

Non-Viral Vectors

Although viruses can effectively deliver genetic material into a patient's cells, they do have some limitations. It is sometimes more efficient to deliver a gene using a non-viral vector, which has fewer size constraints and which won't generate an immune response.

Non-viral vectors are typically circular DNA molecules, also known as plasmids. In nature, bacteria use plasmids to transfer genes from cell to cell.

Scientists use bacteria and plasmids to easily and efficiently store and replicate genes of interest from any organism.

Vectors used at present, are engineered in such a way that they help easy linking of foreign DNA and selection of recombinants from non-recombinants.

These are not the only way to introduce alien DNA into host cells. In a method known as micro-injection, recombinant DNA is directly injected into the nucleus of an animal cell. In another method, suitable for plants, cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA in a method known as biolistics or gene gun.

Cystic Fibrosis

Cystic fibrosis (CF), also known as mucoviscidosis, is an autosomal recessive genetic disorder that affects most critically the lungs, and also the pancreas, liver, and intestine. It is characterized by abnormal transport of chloride and sodium across an epithelium, leading to thick, viscous secretions, preventing the cilia from clearing debris which cause symptoms such as coughing, poor digestion and increased vulnerability to infection.

CF is caused by a mutation in the gene for the protein cystic fibrosis transmembrane conductance regulator (CFTR) gene on chromosome 7. Most commonly, the mutation in the CFTR gene is a three-base-pair deletion. This protein is required to regulate the components of sweat, digestive fluids, and mucus. CFTR regulates the movement of chloride and sodium ions across epithelial membranes, such as the alveolar epithelia located in the lungs. Since all of the cells of a CF patient have the defective protein, large quantities of thick, sticky mucus build up throughout the lungs and other organs. This results in the severity of symptoms seen in CF patients.

Is It A Good Target For Gene Therapy?

To check this some questions must be answered:

 Does the condition result from mutation? Yes.

 Is the biology of the disorder known? Yes.

 Will adding a normal copy of the gene fix the problem in the affected tissue? Yes. While the mutated CFTR gene encodes a non-functional ion channel protein, it will not prevent a normal CFTR channel protein from working properly. Therefore, adding a normal copy of the CFTR gene should fix the problem

 Is it feasible to deliver the gene to the cells of the affected tissue? Yes, in part. Treating the lungs of patients with CF might be feasible, since the lung surfaces are exposed to the air and somewhat easy to reach. Because the digestive system is less accessible, however, it might be a more difficult region to treat.
Hence we can conclude that it is a perfect disease to be treated by gene therapy.


Some the factors that have kept gene therapy from becoming an effective treatment for genetic diseases are:

• Short-lived nature of gene therapy -

Before gene therapy can become a permanent cure for any condition, 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. Patients will have to undergo multiple rounds of gene therapy.

• 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 in a way that reduces gene therapy effectiveness is always a potential risk. Furthermore, the immune system's enhanced response to invaders it has seen before makes it difficult for gene therapy to be repeated in patients.

• Problems with viral vectors -

Viruses, while the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient --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 especially difficult to treat effectively using gene therapy.

Recent Upcoming


CRISPR stands for clustered regularly interspaced short palindromic repeats. These RNA sequences serve an immune function in archaea and bacteria, but in the last year or so, scientists have seized upon them to rewrite genes. The RNA sequence serves as a guide to target a DNA sequence in, say, a zygote or a stem cell. The guide sequence leads an enzyme, Cas9, to the DNA of interest. Cas9 can cut the double strand, nick it, or even knock down gene expression. After Cas9 injures the DNA, repair systems fix the sequence - or new sequences can be inserted.


It isn't the first or only method of gene repair therapy that’s been developed, but the CRISPR technology, says Ramesar, is so special because, unlike previous methods which were more laborious and could only target one kind of cell in the body, it appears to be a "one size fits all delivery", adaptable for different tissues. The procedure also seems relatively simple to perform.

Exciting as the development may be, CRISPR won’t be delivering instant cures just yet.

Ramesar says, from his initial impressions of the literature, that it would seem that localised, accessible abnormal tissue (as in the retina or skin) could be targeted more easily.

Conditions affecting the body more systemically, however, such as certain developmental syndromes, or central nervous system disorders, might be problematic in terms of getting the repair technology into enough of the target cells in that tissue to make an effective difference.

"It may also depend on the stage one attempts to carry out the therapy, in terms of the patient’s age and level of advancement of the disease," says Ramesar.


Although early clinical failures led many to dismiss gene therapy as over-hyped, clinical successes since 2006 have bolstered new optimism in the promise of gene therapy. These include successful treatment of patients with the retinal disease Leber's congenital amaurosis, X-linked SCID, ADA-SCID, adrenoleukodystrophy, chronic lymphocytic leukaemia (CLL),acute lymphocytic leukaemia (ALL),multiple myeloma, haemophilia and Parkinson's disease. These recent clinical successes have led to a renewed interest in gene therapy, with several articles in scientific and popular publications calling for continued investment in the field.


1. Wikipedia

2. Science daily