GENE THERAPY METHODS

Gene therapy can be defined as treatment of specific genetic disorders, both recessive as well as dominant, by addition or replacement of normal copy of the gene responsible for the disease or by inhibiting the translation of unwanted gene.

TYPES OF GENE THERAPY

1.                  In vivo gene therapy: Introduction of genes directly into target tissue to correct a disease.

2.                  Ex vivo gene therapy: Defective cells are taken out and normal copy of DNA is introduced in vitro. The rectified cells are again implanted in patient.

3.                  Germ line gene therapy: Introducing DNA into sells that can form part of a human germ line and be passes onto successive generations. For ethical, safety, and technical reasons, human germ line gene therapy is not being examined experimentally at this time.

4.                  Somatic cell gene therapy: At present, all research on human gene therapy is directed toward correcting genetic defects of somatic cells, i.e., cells that do not contribute to the next generation (non sex cells).

5.                  Enhancement gene therapy: This type of gene transfer is done for the improvement of a specific trait in animals; for example introduction of growth hormone gene to increase height. It is being carried out in laboratory and farm animals

6.                  Eugenics: Novel genes can be introduced in humans to alter or improve complex traits such as intelligence and personality. This type of therapy is not being attempted because it is far beyond our technical capabilities, and due to bioethics.

7.                  Bone Marrow Gene Therapy: Individual with genetic diseases that respond to bone marrow transplantations are likely candidates for ex vivo gene therapy. The reason that bone marrow transplantation works as a therapeutic procedure for a diverse set of diseases is that bone marrow contains totipotent embryonic stem cells at a frequency of 10-4 to 10-5. These cells can divide and differentiate into various important cell types, including B and T lymphocytes, macrophages, red blood cells, platelets, and bone cells (osteoclasts). In the patient, genetically engineered totipotent stem cells would continuously provide a missing cell type or gene product.

 Genetic Diseases That Can Be Treated By Bone Marrow Transplantations

ADA deficient Severe Combined Immunodeficiency

Non-ADA deficient SCID

Adrenoleukodystrophy

Osteoporosis

Chronic granulomatous disease

Purine nucleotide phosphorylase deficiency

Hunter disease

Reticular dysgenesis

Granulocyte actin deficiency

Sickle cell disease

Gaucher disease

Thalassemia

Infantile agranulocytosis

Wiskott-Aldrich syndrome

METHODS OF GENE TRANSFER

A number of strategies using viral and non-viral vector systems have been devised to deliver a therapeutic gene to target tissues such as skin, muscle, lung, brain, colon, spleen, liver, and blood cells within the human body.

Attributes of ideal in vivo gene delivery system:

  1. High efficiency of uptake of the therapeutic gene by the target cells.
  2. Transportation of the therapeutic gene to the nucleus of the target cell with a minimum of intracellular degradation.
  3. Sustained expression of the therapeutic gene at a level that alleviates the disease.

 VIRAL VECTORS

Retrovirus Vector System

5’LTR                                                                                                                   3’LTR

 

 

gag

pol

env

 

 

Genetic Map of Typical Retrovirus

5’LTR                                                                                                                   3’LTR

 

 

Gene X

 

 

Neor

 

 

Gene X = Remedial Gene

Genetic Map of Retrovirus Vector

a)      Replication defective retrovirus vectors:  These are retroviral vectors which are defective of their replication and cannot cause any apparent adverse effects. Moreover their safety features are continuously being upgraded.

Ex: plasmovirus

Plasmovirus: These are vectors derived from replication defective retroviruses. In plamovirus vectors the gag and pol genes are under the control of 5’ promoter and the therapeutic gene and env gene are controlled by cytomegalovirus promoter. It is very unlikely, that this construction will recombine to form replication-competent retroviruses. The DNA carrying capacity of plasmovirus is only 3.5 kb, they can be used for several cDNA and anticancer genes which range from 0.5 2 kb.

b)      Pseudotyped Retroviral Vectors:  a recombinant retroviral vector genome that is packaged in the enveloped protein of another virus will have the binding specificity and infection spectrum that are determined by the envelope protein. In this way, we can specify the cell type that should be infected. This phenomenon is called pseudotyping.

 Adenovirus Vectors 

Live adenoviruses have been used as vaccines in US military personnel without any major side effects. These can infect a wide range of cells. Adenoviruses can be used in gene therapy of lung disorders because of their natural tropism for infecting respiratory epithelium.

 Adeno-Associated Virus (AAV) Vectors

AAV is a non-pathogenic parvovirus of human. It has aroused an interest as a vector. It requires a helper virus co-infection for viral replication. An outline of introduction of ADA gene into bone marrow cells / lymphocytes is given in fig;

 

NON-VIRAL METHODS OF GENE TRANSFER

PHYSICAL METHODS: The various physical methods of gene transfers are given below:

Microinjections:  it is very tedious method. However, it is used in oocytes, eggs, and embryos. Chamberlain et al (1993), microinjected the DNA containing 427 K gene into the zygote of mutant mice. The transplanted genes worked properly and produced necessary protein and thus, prevented the diaphragm.

Macro injections:  Direct injection of DNA into skeletal muscle has created a lot of excitement and led to the possibility of using gene as vaccines and this led to the concept of DNA vaccines and genetic immunization (Rangarajan & Padmanaban, 1996).

Gene gun: It was developed originally for transformation of plant cells, but also used in animal systems. It is also called as biolistic gun or gene bombardment gun.

Electroporation:  In this technique, cells and gene to be transferred are taken in a container with two electrodes. High voltage for short duration is pulsed. The shock creates pores in cell membrane which aids transformation.

Magic bullet:  It is a drug delivery system through a cell loaded with an appropriate treatment molecule which upon injection in to a patient migrates through circulatory system directly to the diseased site.

CHEMICAL METHODS

Sometimes chemicals are used to make easy delivery of DNA into recipient cell.

Using detergent mixture:  Certain charged chemical compounds like calcium phosphate, dextran or lipids are mixed with functional cDNA of desired function. The mixture is introduced near the vicinity of recipient cells. Thereafter, it is spread to the interior of the body organ. The chemical mixed with cDNA disturbs the cell membrane, widens pore size and allows the cDNA to pass into the cell.

Receptor-mediated gene delivery:  It is an attractive strategy. It takes advantage of normal physiological pathway. The receptors are exclusively present on hepatocytes. They bind to certain glycoproteins lacking the terminal asialic acid. The concept of delivering gene into specific tissue was examined extensively with liver asialoglycoprotein receptors.

SUCCESS OF GENE THERAPY:

Success of gene delivery depends on gene delivery mechanism as well as on the choice of target tissue. Factors affecting success are as follows;

  1. Cell types capable of dividing in vitro should be selected,
  2. function of gene product determines the target tissue,
  3. Synthesis unwanted gene products can be inhibited using antisense RNA technology.
  4. Regulations regarding gene therapy laid down by local governing authority.
   
 
BIOTECH PORTAL