14. SUMMARY

• Gene therapy is a new approach for treating many human diseases.  It involves transferring normal gene that either replace or supplement the defected gene. Therefore the introduced gene will function normally forming the biologically active product to alter the course of the pathologic processes.

• The human traits are transmitted following the basic genetic rules.  These are controlled by genes which are located on either the autosome or on the sex chromosomes. The genes are found in two different forms either dominant or recessive. The mode of inheritance of any characters depends on the nature of the gene and its location.  Some traits are controlled by one gene i.e. monofactorial, while others are controlled by more than one gene i.e. multifactorial.

• Recent development of molecular biology has explained the nature of gene expression to show the phenotype. The gene is chemically composed of deoxyribonucleic molecule (DNA). It acts through its control to form protein.  The process of gene expression depends on two major processes, namely the transcription and translation. The process of transcription involves the synthesis of three types of RNA. They are messengers RNA that carry the genetic information i.e. genetic code which is represented by the type, number and sequence of bases. The transfer RNA carries the different amino acids. The ribosomal RNA combines with the protein to form the ribosomes where protein synthesis takes place.

• The process of translation involves translating the genetic code to determine the type and the sequence of amino acids in the protein.

• Molecular biology has helped in manipulating the genes, and a new technology, appears that is the recombinant DNA technology.  This involves cutting the DNA at a specific site using enzymes known as restriction enzymes. Ligate the DNA fragment i.e. the gene of interest in vehicle known as vector to form a chimerical DNA, which is the recombinant DNA. The vectors used could be either bacterial plasmid or virus. The recombinant DNA can then be introduced to a host such as bacteria. When bacteria divides, the recombinant DNA replicate.  Therefore we increase the number of copies of the transferred gene and also its product. This is known as gene cloning.  Gene cloning is very important in gene therapy.

 • There are two main types of gene therapy. Somatic gene therapy where we introduce the gene into somatic cells. The other is the germline gene therapy, which involves introducing the gene into the germ line.

 • Gene therapy can be carried either outside the human body that is known as ex-vivo gene therapy. Following transfer of the genetic material to the cells, the cells are implanted back into the host. The other type is the in-vivo gene therapy that is introducing the manipulated gene directly to the host.

 • There are different types of target cells used in gene therapy such as bone marrow cells, skin cells, live cells, T cells and stem cell.

 • The major steps in gene therapy is isolating the gene and transfer it using a number of delivery systems. The delivery systems are known as vectors.  There are viral vectors and non-viral vectors.  The viral vectors generally used are the retrovirus, Adenovirus, Adenoassociated virus and Herpes Simplex Virus.

 • The retrovirus is an animal virus composed of protein coat and RNA as genetic material. It has a unique life cycle. When it enters the cell, a DNA copy is formed from it using the enzyme reverse transcriptase.  It is then integrated with host genome randomly. To use the retrovirus as a vehicle for gene therapy requires the removal of all the genes that are responsible for viral replication.  It only infects replicating cells.

 • Adenovirus is not enveloped, and its genome is a double stranded DNA.   When infection it does not integrate with the host genome. The adenoviral vector used in gene therapy constructed of two parts, the first is the viral DNA vector and the second is a packaging cell line. It is capable of infecting a broad range of cell types, dividing and non-dividing.  Its advantage is ability of inserting large size DNA and is not integrative. However, its disadvantage that its expression is transient so the therapy would need to be replaced.

 • Adenoassociated viruses requires coinfection with either adenovirus or herpes simplex virus. Its genome is DNA and can be integrated to specific sites on chromosome number 19 in human. Its advantage, that they are not pathogenic, infecting non-dividing cells and shows longer lasting expression.  But it cannot incorporate genes larger than 5kb.

 • Herpes simplex virus (HSV) infects the nervous system. A number of HSV vectors have been developed. They are constructed by deleting al least one of the essential regulatory genes making the virus less cytotoxic.

 • A number of non-viral vectors are developed for gene transfer.  Among these are calcium phosphate, where plasmid DNA are precipitated with a buffer of phosphate and calcium chloride.

 • The DNA can be introduced to the target cells by either microinjection or electroporation and Naked DNA injection.

 • Non-viral techniques used for both in vitro and in vivo gene therapy applications are the liposomes, polycation conjugation. The liposomes are spheres of lipid, and could surround the DNA. The other type is polycation conjugation. This method involves the conjugation of DNA and receptor binding molecules that leads its way to the target cell.  Then it enters inside the cell.

 • One of the most important aspects of gene therapy is the ability to target a specific gene to a specific cell type and its regulation of its expression.  This could be achieved either by making use of cell properties such as its rate of cell division. This is possible to use vectors that only attack the dividing cells. The other is to activate the expression of the gene using a receptor. The third way is to target the cell by a liposome that are prepared to incorporate a specific proteins onto their surface, and combined with DNA. This liposome can recognize a specific cell to deliver the gene.

 • Mice are developed to be used as a model for testing the gene therapy techniques. They have to be prepared in a way to generate genetically manipulated mice.

 • There are large numbers of inherited human diseases are subjected for gene therapy.  Most of them are caused by single gene defect and they are well defined. The candidate disease should have no effective conventional treatment e.g. sickle cell anemia, cystic fibrosis, Huntington’s disease, Duchenn’s muscular dystrophy and many others.

 • Recently, gene therapy was used to treat other diseases such as lung diseases, e,g. cystic fibrosis, non heritable diseases such as HIV, and cancer diseases.

 • Gene therapy could be considered as a future means of treating many genetic diseases and acquired diseases. Despite the great progress in the clinical trials but the practical reality is still out of reach. Some of the difficulties in gene therapy involves the discovery of new efficient vectors and expression of the transferred gene.

 • Gene therapy raises some important ethical issues specially on the germline gene therapy.