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Essay on Gene Targeting and Gene Therapy
Essay Contents:
- Essay on the Meaning of Gene Targeting and Gene Therapy
- Essay on the Applications of Gene Targeting
- Essay on the Scope of Gene Therapy
- Essay on the Types of Gene Therapy
1. Meaning of Gene Targeting and Gene Therapy:
Gene targeting is a form of in vivo site-directed mutagenesis involving homologous recombination between a targeting vector containing one allele and an endogenous gene represented by a different allele.
Gene therapy is a technique of gene targeting which is an important tool of genetic engineering or recombinant DNA technology.
Following two types of targeting vectors are used:
i. Integration vectors (end-in-vectors):
Where cleavage within the homology domain stimulates a single crossover resulting in integration of the entire vector.
ii. Trans-placement vectors (end-out vectors):
Where linearization occurs outside the homology domain or a double cross-over or gene-conversion event within the homology domain replaces part of the genome with the homologous region of vector.
The most efficient gene transfer vectors are based on mammalian viruses, specifically retroviruses, adenoviruses and the adeno-associated viruses (which integrate into the genome and may therefore mediate permanent correction of genetic defects) and the herpes viruses (which are neurotropic and remain episomal).
More direct approaches of gene transfer include injection (into muscles) and microbailistic techniques (in which tungsten or gold particles coated with DNA are fired into cells at high velocity using a gene gun). A transfer procedure based on the packaging of DNA in liposomes is also popular in cancer gene therapy.
Aerosols are used to introduce recombinant viral vectors into the lungs (they are internalized by receptor-mediated endocytosis) and are used for the treatment of cystic fibrosis.
2. Applications of Gene Targeting:
Gene targeting has the following applications in genetic engineering:
i. Gene knockout or targeted disruption:
It can be achieved by inserting a cassette (i.e., a segment of exchangeable genetic information) anywhere in the integration vector or within the homology domain of trans-placement vector. This cassette is usually a dominant selectable marker, such as bacterial neo gene, which allows selection of targeted cells.
ii. Allele replacement:
One allele is replaced by another e.g., to investigate the effects of a subtle mutation.
iii. Gene knock-in:
It is a novel application where one gene is replaced by another (non-allelic) gene.
v. Gene therapy:
In this case, a non-functional mutant allele is replaced by a normal allele.
3. Scope of Gene Therapy:
Gene therapy can involve genetic modification of cells in a living patient (in vivo gene therapy) or the genetic modification of cultured cells which are then returned to the patient {ex vivo gene therapy).
In vivo gene therapy involves both genetic modification of target cells (somatic transgenesis) and therapeutic use of DNA as an epigenetic treatment (without changing the nucleotide sequence). The therapy can be used to improve or correct conditions caused by human gene mutuation or to prevent infectious diseases (e.g., by interfering with the life cycle of a virus).
i. Gene augmentation therapy (GAT):
In this technique, DNA is added to the genome to replace a lost function. Its aim is to correct a loss of function effect, e.g., caused by a deletion, by adding functional alleles. Transferred genes may be stably integrated into the genome (in which case there is the potential to permanently correct the defect, especially if stem cells are transformed) or may be maintained episomally (in which case there is an inevitable decay in the maintenance of gene expression and treatment may need to be repeated). Several GAT clinical trials are currently underway including cystic fibrosis, adenosine deaminase deficiency and familial hypercholesterolemia.
ii. Gene inhibition therapy at the nucleic acid level:
It involves treatment of disease by targeted correction (i.e., allele replacement) or gene knock-out to remove the mutant allele. A novel approach which may play a role in gene therapy in the future is targeted correction at the RNA level by using ribozymes (Box 53.1) or RNA editing enzymes to correct pathogenic mRNA.
Use of antisense RNA:
An alternative approach which is presently undergoing clinical trials for the treatment of several types of cancer, is the use of nucleic acids to inhibit gene expression. The introduction of antisense genes allows the stable and permanent expression of antisense RNA (see Box 53.3), which binds to (mutant) mRNA and prevents translation (it may also target the mutant mRNA for degradation).
Furthermore, increasing use is being made of antisense constructs containing ribozymes which degrade the mRNAs for epigenetic gene therapy (i.e., therapy which does not involve changes in the genome).
iii. Gene inhibition therapy at the protein level:
Targeted inhibition of gene expression can also take effect at the protein level, by expression within a cell of genetically engineered antibodies (intrabodies) which bind to and inactivate mutant proteins.
A novel approach is to use degenerate oligonucleotides to identify specific oligonucleotide sequences which interact with proteins. These oligonucleotides are called aptamers and can be used to inactivate specific mutant proteins.
a. Antisense RNA (or micRNA = mRNA interfering complementary RNA):
Antisense RNA is complementary to mRNA and can form a duplex with it to block protein synthesis. Naturally occurring antisense RNA is found in many systems but predominantly in bacteria and is termed mRNA – interfering complementary RNA.
b. Ribozyme:
RNA molecule which can catalyze chemical reactions (RNA enzymes).
4. Types of Gene Therapy:
Gene therapy can be performed at two different levels:
(i) Patient therapy:
In patient therapy, cells with healthy genes may be introduced in the affected tissue, so that the healthy gene overcomes the defect without affecting the inheritance of the patient.
Patient therapy includes the following steps:
(1) Identification of a defective gene;
(2) Isolation or synthesis of normal healthy gene;
(3) Isolation of cells of the tissue, where the normal healthy gene has to function;
(4) Introduction of healthy gene into the cell.
During early 1990s, in the routine exercise of patient therapy any gene is isolated and this isolated gene is either directly injected into the cell or be carried by a virus (vector) to which it is linked by recombinant DNA technique.
After entering the cell, the gene might become a part of nuclear DNA or remain free in cytoplasm like extra-chromosomal DNA. However, in each case RNA is synthesized only at the rate of few copies per cell in comparison to normal cells where thousands of copies are made.
Table 53.2. List of human genetic diseases for which genes have been isolated and cloned for gene therapy.
(ii) Embryo therapy:
In embryo therapy the genetic constitution of embryo at the post- zygotic level is altered so that the inheritance is altered.
This technique involves the following steps:
(i) In vitro fertilization of the egg;
(ii) Insertion of normal gene into embryo at post-zygotic level, either with viruses or directly by microinjection; and
(iii) Integration of inserted gene in host DNA, where it may or may not function.