In Vivo gene therapy methods

In this In vivo gene therapy methods post we have briefly explained about In vivo gene therapy, vectors, and example.

In Vivo Gene Therapy

The direct delivery of the therapeutic gene (DNA) into the target cells of a particular tissue of a patient constitutes In vivo gene therapy. Many tissues are the potential candidates for this approach. These include liver, muscle, skin, spleen, lung, brain and blood cells. Gene delivery can be carried out by viral or non- viral vector systems.

The efficiency of the uptake of the therapeutic gene by the target cells

Intracellular degradation of the gene and its uptake by nucleus

The expression capability of the gene

in vivo gene therapy

In Vivo Gene Therapy Methods

Vectors in In Vivo Gene Therapy

Many viral vector systems have been developed for gene delivery. These include retroviruses, adenoviruses, adenoassociated viruses and herpes simplex virus.

Retrovirus vector

Replication defective retrovirus vectors that are harmless are being used. A plasmid in association with a retrovirus, a therapeutic gene and a promoter is referred to as plasmovirus.

The plasmovirus is capable of carrying a DNA (therapeutic gene) of size less than 3.4 kb. Replication defective virus particles can be produced from the plasmovirus. As such, for the delivery of genes by retroviral vectors, the target cells must be in a dividing stage. But majority of the body cells are quiescent.

In recent years, viral vectors have been engineered to infect non-dividing cells. Further, attempts are on to include a DNA in the retroviral vectors (by engineering env gene) that encodes for cell receptor protein. If this is successfully achieved, the retroviral vector will specifically infect the target tissues.

Adenoviral vector

Adenoviruses (with a DNA genome) are considered to be good vectors for gene delivery because they can infect most of the non-dividing human cells. A common cold adenovirus is a frequently used vector. As the target cells are infected with a recombinant adenovirus, the therapeutic gene (DNA) enters the nucleus and expresses itself.

However, this DNA does not integrate into the host genome. Consequently, adenoviral based In vivo gene therapy required periodic administration of recombinant viruses. The efficiency of gene delivery by adenoviruses can be enhanced by developing a virus that can specifically infect target cells. This is possible by incorporating a DNA encoding a cell receptor protein.

Adeno-associated virus

Adeno-associated virus is a human virus that can integrate into chromosome 19. It is a single-stranded, non-pathogenic small DNA virus (4.7 kb). As the adeno-associated virus enters the host cell, the DNA becomes double- stranded, gets integrated into chromosome and expresses.

Adeno-associated viruses can serve as good vectors for the delivery of therapeutic genes. Recombinant viruses are created by using two plasmids and an adenovirus (i.e., helper virus) by a special technique.

Some attempts were made to use therapeutic genes for the treatment of the human diseases-hemophilia (for production of blood clotting factor IX) and cystic fibrosis (for synthesis of cystic fibrosis trans membrane regulator protein) by employing adeno-associated viruses.

Non-Viral Systems

There are certain limitations in using viral vectors in In vivo gene therapy. In addition to the prohibitive cost of maintaining the viruses, the viral proteins often induce inflammatory responses in the host. Therefore, there is a continuous search by researchers to find alternatives to viral vector systems.

Pure DNA constructs

The direct introduction of pure DNA constructs into the target tissue is quite simple. However, the efficiency of DNA uptake by the cells and its expression are rather low.

Consequently, large quantities of DNA have to be injected periodically. The therapeutic genes produce the proteins in the target cells which enter the circulation and often get degraded.


The lipid-DNA complexes are referred to as lipoplexes or more commonly liposomes. They have a DNA construct surrounded by artificial lipid layers. A large number of lipoplexes have been prepared and used. They are non-toxic and non-immunogenic.

The major limitation with the use of lipoplexes is that as the DNA is taken up by the cells, most of it gets degraded by the lysosomes. Thus, the efficiency of gene delivery by lipoplex is very low. Some clinical trials using liposome-CFTR gene complex showed that the gene expression was very short-lived.

DNA-molecular conjugates

The use of DNA-molecular conjugates avoids the lysosomal breakdown of DNA. Another advantage of using conjugates is that large-sized therapeutic DNAs (> 10 kb) can be delivered to the target tissues.

The most commonly used synthetic conjugate is poly-L-lysine, bound to a specific target cell receptor. The therapeutic DNA is then made to combine with the conjugate to form a complex This DNA molecular conjugate binds to specific cell receptor on the target cells.

It is engulfed by the cell membrane to form an endosome which protects the DNA from being degraded. The DNA released from the endosome enters the nucleus where the therapeutic gene is expressed.

Human artificial chromosome

Human artificial chromosome (HAC) which can carry a large DNA one or more therapeutic genes with regulatory elements is a good and ideal vector.

Studies conducted in cell cultures using HAC are encouraging. But the major problem is the delivery of the large-sized chromosome into the target cells. Researchers are working to produce cells containing genetically engineered HAC.


Cystic fibrosis

Cystic fibrosis (CF) is one of the most common (frequency 1: 2,500) and fatal genetic diseases. It is characterized by the accumulation of sticky, dehydrated mucus in the respiratory tract and lungs. Patients of CF are highly susceptible to bacterial infections in their lungs and most of them die before reaching the age of thirty.

Gene Therapy

As the defective gene for cystic fibrosis was identified in 1989, researchers immediately started working on In vivo gene therapy for this disease.

Adenoviral vector systems have been used, although the success has been limited. The major drawback is that the benefits are short-lived, since the adenoviruses do not integrate themselves into host cells.

Multiple administration of recombinant adenovirus caused immunological responses that destroyed the cells.

By using adeno-associated virus vector system, some encouraging results were reported in the gene therapy of CF.

In the phase I clinical trials with CF patients, the vector persisted for about 70 days and some improvement was observed in the patients.

Some researchers are trying to insert CF gene into the developing fetal cells (in experimental animals such as mice) to produce CFTR protein. But a major breakthrough is yet to come.

Further Readings