GENE THERAPY

BYB.E.N

13th JULY 2015

IntroductionClasses of gene therapyStrategies for gene therapyMethods used for gene therapyFactors that have kept gene therapy ineffective for treatment of genetic diseasesDevelopments in gene therapy researchEthical concerns

IntroductionGene therapy is a technique that involves insertion of normal genes to correct defective genes responsible for disease development.

It is the use of DNA as a pharmaceutical agent to treat disease.

It derives its name from the idea that DNA can be used to supplement or alter genes within an individual's cells as a therapy to treat disease

The most common form of gene therapy involves using DNA that encodes a functional, therapeutic gene in order to replace a mutated gene.

Other forms involve directly correcting a mutation or using DNA that encodes a therapeutic protein (rather than a natural human gene) to provide treatment.

Gene therapy was first conceptualized in 1972, with the authors urging caution before commencing gene therapy studies in humans.

On September, 1990, the first gene therapy was performed by Dr. William French Anderson on a four-year-old girl, Ashanti DeSilva, at the National Institutes of Health, Bethesda, A girl was treated for ADA-SCID. Since then, over 1,700 clinical trials have been conducted using a number of techniques for gene therapy.

APPROACHES FOR CORRECTING FAULTY GENES A normal gene may be inserted into a non-specific location within the genome to replace a nonfunctional gene. This approach is most common

An abnormal gene could be swapped for 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 of a particular gene could be altered (the degree to which a gene is turned on or off)

A) Somatic Gene TherapyIn somatic gene therapy, the therapeutic genes are transferred into the somatic cells or body of a patient. Any modifications and effects will be restricted to the individual patient only and will not be inherited by the patient's offspring or later generations.

Somatic gene therapy represents the mainstream line of current basic and clinical research, where mRNA is used to treat a disease in an individual.

In germ line gene therapy, Germ cells, i.e., sperm or eggs, are modified by the introduction of functional genes, which are integrated into their genomes.

This would allow the therapy to be heritable and passed on to later generations (offsprings).

Although this should, in theory, be highly effective in counteracting genetic disorders and hereditary diseases, many jurisdictions prohibit this for application in human beings for a variety of technical and ethical reasons

Pronuclear (PI) Injection

(3) Strategies for Gene Therapy1. Ex vivo, which means outside the body Cells from the patients blood or bone marrow are removed and grown in the laboratory.

They are then exposed to a virus carrying the desired gene. The virus enters the cells, and the desired gene becomes part of the DNA of the cells.

The cells are allowed to grow in the laboratory before being returned to the patient by injection into a vein or bone marrow.

2. In vivo, which means inside the body No cells are removed from the patients body. Instead, vectors are used to deliver the desired gene to cells in the patients body.

3. In Situ Vector is placed directly into the affected tissues.

List of Common Genetic Disorders

Disorder Mutation Chromosome Angelman syndromeDCP15Color blindness P XCystic fibrosis P 7Down syndrome C21Haemophilia PXKlinefelter's syndrome CX

Two methods are involved(i) Non-viral mediated gene delivery method(ii) Viral mediated gene delivery method

(I) Non-viral mediated gene delivery method (i)Direct injection of therapeutic DNA into target cellsThis approach is limited in its application because it can be used only with certain tissues and requires large amounts of DNA

(ii) LiposomesCreation of an artificial lipid sphere with an aqueous coreIn this method, the liposome, which carries the therapeutic DNA, is capable of passing the DNA through the target cell's membrane

(iii) Inorganic nanoparticles.Chemically linking the DNA to a molecule that will bind to special cell receptors. Once bound to these receptors, the therapeutic DNA constructs are engulfed by the cell membrane and passed into the interior of the target cell

(iv) Researchers also are experimenting with 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 potential method is the difficulty in delivering such a large molecule to the nucleus of a target cell.

There are other several methods for non-viral gene therapy, including, electroporation, the gene gun, sonoporation, magnetofection etc.

E.g In 2003 a University of California, Los Angeles research team inserted genes into the brain using liposomes coated in a polymer called polyethylene glycol. The transfer of genes into the brain is a significant achievement because viral vectors are too big to get across the blood-brain barrier. This method has potential for treating Parkinson's disease.

Gene gun OR microprojectile bombardmentbiolisticsTiny DNA-coated particles are shot into the cells

The Gene GunPDS1000 Microparticle Delivery SystemHelium chamberRupture diskMacrocarrierDNA coated gold particleStopping screenFocusing deviceTarget tissue

This method employs viruses as vehicles to transport the desired genes to the target cells (These are recombinant viruses sometimes called biological nanoparticles)

Different types of viruses are used as vectors for gene therapyRetroviruses A class of viruses that can create double-stranded DNA copies of their RNA genomes

Adenoviruses - A class of viruses with double-stranded DNA genomes that cause respiratory, intestinal, and eye infections in humans

Adeno-associated viruses - A class of small, single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19

Herpes simplex viruses A class of double-stranded DNA viruses that infect a particular cell type, e.g. Neurons

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

Scientists have manipulated the virus genome to remove disease-causing genes and insert therapeutic genes.

When target cells are infected with the viral vector. The vector then unloads its genetic material containing the therapeutic human gene Then, generation of a functional protein product from the therapeutic gene restores the target cell to a normal state.

(i) Short-lived nature of gene therapy - Problems with integrating therapeutic DNA into the genome and the mortal nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy (ii) Immune response - Anytime a foreign object is introduced into human tissues, the immune system is designed to attack the invader

Furthermore, the immune system's enhanced response to invaders it has seen before makes it difficult for gene therapy to be repeated in patients

(iii) Problems with viral vectors -Though viruses are the carriers of choice in most gene therapy studies, there some possible potential problems to the patient; toxicity, immune and inflammatory responses and the fear that the viral vector, once inside the patient, may recover its ability to cause disease.

(iv) Multigene disorders -Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy -Unfortunately, some of the most commonly occurring disorders, such as heart disease, HBP, 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.

If the DNA is integrated in the wrong place in the genome, for example in a tumor suppressor gene, it could induce a tumor

This has occurred in clinical trials for X-linked severe combined immunodeficiency (X-SCID) patients, in which hematopoietic stem cells were transduced with a corrective transgene using a retrovirus and this led to the development of T cell leukemia in 3 of 20 patients.

Since gene therapy is relatively new and at an experimental stage, it is an expensive treatment to undertake.

This explains why current studies are focused on illnesses commonly found in developed countries, where more people can afford to pay for treatment.

It may take decades before developing countries can take advantage of this technology.

Scientists currently know the functions of only a few genes. Hence, gene therapy can address only some genes that cause a particular disease.

Worse, it is not known exactly whether genes have more than one function, which creates uncertainty as to whether replacing such genes is indeed desirable.

Generally, Gene therapy is very promising

Sickle cell was successfully treated in mice. (2002) Thalassaemia, cystic fibrosis and some cancers were successfully treated (2002).

Leber's congenital amaurosis, a type of inherited childhood blindness caused by a single abnormal gene. The procedure has already been successful at restoring vision for dogs. This is the first trial to use gene therapy in an operation to treat blindness in humans.

Gene Therapy cured deafness in guinea pigs (Feb 2005)- A gene, called Atoh1, which stimulates the hair cells' growth, was delivered to the cochlea by an adenovirus

"Bubble boy disease" more accurately called severe combined immunodeficiency, or SCID results from a malfunctioning gene in the bone marrow, which produces immune cells.

Because the immune system is so severely weakened, any infection or disease could result in death. The children are therefore forced to live very sheltered and isolated lives.

11 children with bubble boy disease.1 child did not respond to treatment8 children cured 2 children came down with leukemia

Slimming Gene discovered that regulates Body fat The scientists therefore have hopes in new medicines with which obesity could be fought. Their research bas been published in 'The EMBO Journal' (doi: 10.1038/emboj.2009.305).

THERAPY IN NEWS

Some Questions to ConsiderWhat is normal and what is a disability or disorder, and who decides?

Are disabilities diseases? Do they need to be cured or prevented?

Does searching for a cure demean the lives of individuals presently affected by disabilities?

Ethical Considerations contWho should pay?Preliminary attempts at gene therapy are exorbitantly expensive. Who will have access to these therapies? Who will pay for their use? Gene therapy currently cost up to $200,000 per treatment.

Unforeseen consequencesIs it possible that eliminating defective genes fromour genome may have unforeseen consequences

Religious ObjectionsMajority of the world believes in some type of higher power.

The majority of religions states that everyone should be happy with what they have.

Do we have the right to play GOD?

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