The biology of human gene therapy is very complex, and there are many techniques that still need to be developed and diseases that need to be understood more fully before appropriate use of gene therapy.

The public policy debate surrounding the possible use of genetically engineered material in human subjects has been equally complex. Major participants in the debate have come from the fields of biology, government, law, medicine, philosophy, politics, and religion, each bringing different views to the discussion.

On September 14, 1990 researchers at the U.S. National Institutes of Health performed the first (approved) gene therapy procedure on four-year old Ashanti DeSilva. She had a rare genetic disease called ‘severe combined immune deficiency’ (SCID).

Therefore, she lacked a healthy immune system and was vulnerable to every germ. Children with this illness usually develop overwhelming infections and rarely survive to adulthood. Ashanti was kept alone to avoid contact with people outside her family and remained in the sterile environment of her home.


In Ashanti’s gene therapy procedure, doctors removed white blood cells from the child’s body, grew the cells in the laboratory, inserted the missing gene into the cells, and then infused the genetically modified blood cells back into the patient’s bloodstream.

The therapy strengthened Ashanti’s immune system. She no longer had recurrent colds, and was allowed to attend school, and was immunized against whooping cough. This procedure was not a cure. The white blood cells treated genetically only work for a few months, and the process needs be repeated every few months.

In May 2006 a team of scientists from the San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET) in Milan (Italy) reported a breakthrough for gene therapy. They developed a way to prevent the immune system from rejecting a newly delivered gene.

Similar to organ transplantation, gene therapy has been plagued by the problem of immune rejection. So far, delivery of the good gene has been difficult because the immune system does not recognise the new gene and rejects the cells carrying it.


To overcome this problem, the HSR-TIGET scientists utilized a newly uncovered network of genes regulated by molecules known as microRNAs.

The scientists used this natural function of microRNA to selectively turn off the identity of their therapeutic gene in cells of the immune system and prevent the gene from being found and destroyed.

The researchers injected mice with the gene containing an immune-cell microRNA target sequence. They found that the mice did not reject the gene when vectors without the microRNA target sequence were used.

This work will have important implications for the treatment of haemophilia and other genetic diseases by gene therapy.


In March 2006 an international group of scientists announced the successful use of gene therapy to treat two adult patients for a disease affecting myeloid cells. The study is believed to be the first to show that gene therapy can cure diseases of the myeloid system.

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, 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.

1. Types of Gene Therapy in Humans :


There are more than 3000 genetic diseases in humans. Only a few of them are treatable. Example of some of the genetic diseases is: phenylketonuria, haemophgilia A, cystis fibrosis, severe combined immunodeficiency (SCID), emphysema, hyper-ammonemia, Duchenne muscular dystrophy, sickle cell anaemia, hypercholesterokemia, etc.

There are two types of gene therapies, somatic cell gene therapy and germ line gene therapy. Somatic gene therapy includes rectification of faulty genes of somatic cells, while germ line gene therapy refers to correction of faulty genes found in germ cells e.g. eggs, spermocytes and embryos. However, no germ line gene therapy experiments are currently proposed or likely to be proposed in the near future.

Somatic cell gene therapy is at preliminary stage of development. Some of phase I trials for somatic cell gene therapy approved during 1992 are as below:

1. Gene factor IX was transferred into fibroblast in case of haemophilia B.


2. Gene adenosine deasminase was transferred in T cell/stem cells.

3. Gene HSV thymidine kinase was transferred in cytotoxic T lymphocytes in AIDS patients.

Various strategies for implementing somatic cell gene therapy are grouped into the three broad categories: ex vivo gene therapy, in vivo gene therapy and antisense therapy.

(a) Ex Vivo Gene Therapy:


Ex vivo gene therapy. It involves the following steps:

1. Isolation of cells with the defective gene from a patient (A).

2. Growing the isolated cells in culture (B).

3. Transfixion of isolated cells with a remedial gene constructs (C).

4. Selection, growth and testing of transected cells (D).

5. Transplantation or Transfusion of transected cells back into the patients (E).

(b) In Vivo Gene Therapy:

Direct delivery of a remedial gene into the cells of a particular tissue of a patient is called in vivo gene therapy. A cloned and expressible gene is delivered into a cell of a tissue of a patient with a genetic disease. The promoter (P) is tissue specific and the remedial gene encodes a protein that corrects the genetic defect of the patient.

2. Vectors in Gene Therapy:

Viruses attack their hosts and introduce their genetic material into the host cell. This genetic material contains basic instructions to produce many copies of the viruses by using the cellular machinery of the host.

The host cell will can out these instructions and produce additional copies of virus. This result in infection of more This Apache baby has under-gone gene cells. Some types of viruses actually physically insert therapy, some of his bone marrow cells were their genes into the host’s genome for example retroviruses.

Doctors and molecular biologists realized that some viruses could be used as vehicles to carry good genes into a human cell. First, a scientist would remove the genes in the virus that cause disease.

Then they would replace those genes with genes encoding the desired effect (for instance, insulin production in the case of diabetics). This procedure must be done in such a way that the genes which allow the virus to insert its genome into its host’s genome are left intact.

Moreover, numerous problems exist that prevent gene therapy by using viral vectors such as: trouble preventing undesired effects, ensuring the virus will infect the correct target cell in the body, and ensuring that the inserted gene doesn’t disrupt any Vital genes already in the genome.

However, this basic mode of gene introduction currently shows much promise and doctors and scientists are working hard to fix any potential problems that could exist.

(a) Retroviruses:

RNA is the genetic material in retroviruses, while the genetic material of their hosts is DNA. When a retrovirus infects a host cell, it introduces its RNA together with some enzymes into the cell. This retroviral RNA molecule produces a DNA copy from its RNA molecule before it can be considered for part of the genetic material of the host cell.

The process of producing a DNA copy from an RNA molecule is termed reverse transcription. It is carried out by an enzyme called ‘reverse transcriptase’. The viral DNA is incorporated into the genome of the host cell. This process is done by another enzyme called ‘integrase’.

Now the genetic material of the virus is incorporated into host DNA and has become part of the genetic material of the host cell i.e. the host cell is now modified to contain a new gene. If this host cell divides later, its descendants will all contain the new genes.

Gene therapy trials to treat (SCID) were restricted in the U.S A. when leukemia was reported in three of eleven patients treated in the French Therapy X-linked SCID (XSCID) gene therapy trial. Ten XSCID patients treated in England have not presented leukemia to date.

They were found to have similar success in immune reconstitution. Gene therapy trials to treat SCID due to deficiency of the Adenosine Deaminase (ADA) enzyme continue with relative success in the U.S.A., Italy and Japan.

(b) Adenoviruses:

Adenoviruses are such viruses that carry double-stranded DNA as their genetic material. They cause respiratory (especially the common cold), intestinal, and eye infections in humans. They introduce their DNA molecule after infecting the host. Their genetic material is not incorporated into the genetic material of host cells.

But their DNA is left free in the nucleus of the host cell. Within the host’s cell, instructions in this extra DNA molecule are transcribed just like any other gene. The extra genes are not replicated when the cell is about to undergo cell division. Therefore, the descendants of that cell will not have the extra gene.

The absence of integration into the host cell’s genome should prevent the type of cancer that has been found in the SCID trials. Vector system based on this virus has shown real promise in treating cancer. Really, the first gene therapy product to be licensed is an adenovirus to treat cancer.

(c) Adeno-associated Viruses (AAV):

Adeno-associated viruses belong to parvovirus family. They are small non-pathogenic viruses having a genome of single stranded DNA. These viruses can insert genetic material at a specific site on chromosome 19. There are a few disad­vantages in using AAV.

Most people treated with AAV will not build an immune response to remove the virus and the cells that have been successfully treated with it. Several trials with AAV are on­going to treat muscle and eye diseases.

Clinical trials have also been initiated where AAV vectors are used to deliver genes to the brain. This is possible because AAV viruses can infect non-dividing (quiescent) cells, for example neurons in which their genomes be expressed for a long time.

3. Antisense Therapy:

There are several regulatory steps in the pathway from gene to protein. The process of gene expression consists of the transcription of antisense DNA (acting like template) to single stranded mRNA which becomes a sense strand followed by mRNA binding to cellular factors (ribosomes) where specific proteins dictated by the genes are made (translated).

Many diseases occur due to inappropriate expression of host DNA or foreign DNA/RNA (from viruses) and production of disease-associated proteins. The concept of antisense therapy was initiated by Zamecnik and Stephenson (1978).

The objectives of antisense therapy were: (i) to design an oligonucleotide which can bind to an mRNA of a gene, (ii) to inhibit its translation, (iii) to prevent protein expression, (iv) to turn off activity of the gene, and (v) to exert a therapeutic effect.

Thus antisense therapy is the in vivo treatment of a genetic disease. It is done by the two methods-antisense oligonucleotide strategy and antisense gene strategy. In both of the cases production of a specific protein inside a specific cell type is reduced significantly.

In the first approach, to block the production of undesirable protein, a short DNA of about 10- 20 nucleotides are synthesised which are complementary to certain conserved regions of host DNA/ viral DNA or RNA. This synthetic DNA is the mirror image (antisense) to a portion of the mRNA (sense). Then the endogenous mRNA and exogenous antisense RNA hybridise.

The binding activity inhibits the production of disease-associated proteins. In second approach, the activity of antisense oligonucleotide is enhanced by involving an enzyme, RNase H which mops up the unwanted copies of mRNA strand.

The RNase H dismantles mRNA strand which consists of antisense oligonucleotides dismantling the mRNA leaving the antisense nucleotides as such untouched. This antisense nucleotide may bind to another mRNA one after another and so on.