Whatever is the type of therapy, the gene has to be delivered into the target cell either via a viral vector or directly by a conventional non-viral method.
Gene delivery by viral vectors
This is the most commonly used method of gene delivery into mammalian cells several mammalian viruses have proved to transfer genes into mammalian cells
Among these, retroviruses; adenoviruses; and Herpes simplex virus are the vectors of choice.
These are viruses with RNA as the genetic material. These viruses infect mammalian cells very efficiently and following infection, the RNA changes into a double stranded DNA catalyzed by the enzyme, reverse transcriptase. This double stranded DNA integrates into the host cell chromosome at a single site.
The integrated DNA replicates with every cycle of host cell DNA replication. However, these viruses have two demerits. They infect the actively dividing cells only and may silence the tumor suppressor genes and activate proto-oncogene.
Adenoviruses are DNA viruses, which infect upper respiratory tract epithelium, cornea and gastro-intestinal tracts. These viruses are not highly pathogenic. Unlike retroviruses, these infect a wide variety of non-dividing cells from different locations.
Thus, it is a vector of choice for lung genetic disorder, Cystic Fibrosis therapy. The inserted DNA does not integrate into the host cell DNA and hence, there is little risk of mutation of the host cell genes. Despite all these, these viral vectors have disadvantages. Firstly, these may induce inflammatory immune response and secondly, the remedial gene may express transiently because they do not integrate into the host cell DNA.
Herpes simplex virus
This virus infects neurons of the central nervous system. The DNA does not integrate into the genome of the neuron and hence, there is no long term expression of the remedial genes. This vector is used for the therapy of neurological disorders such as Parkinson’s disease and brain tumors.
Safety concerns about recombinant viral vectors
The recombinant viral vectors used for gene delivery to target cells are engineered so that the pathogen city is disabled, i.e. the genes necessary for viral replication are deleted.
These viruses are known as replication-incompetent cells. However, among all viral vectors used, retroviruses may, on occasions, integrate into the host cell genome and either activate cellular proto-oncogenes or suppress tumor suppressor genes, which may result in cancer.
Adenoviruses are generally non- integrating, but repeated injection may provoke a severe inflammatory responses. Recently, a severe inflammatory response has been reported in a patient participating in a gene therapy clinical trial for cystic fibrosis.
Similarly, a patient died at the University of Pennsylvania in 1999, while he was participating in a clinical trial for the therapy of ammonia transcarboamylase deficiency. In this case also, there was a severe immune response. These incidents have compelled the scientists to think over some non-viral methods of gene delivery into the target cells so that the foregoing incidents do not occur.
Several conventional non-viral methods of gene delivery into cells are already available. Important ones among these are direct injection, microinjection, electroporation and particle (microprojectile) bombardment etc. Direct injection has proved very effective in Duchene Muscular Dystrophy (DMD) therapy. A dystrophin minigene is intra-muscularly injected in the mouse model and the result is being investigated.
In the particle bombardment, DNA coated tungsten particles are fired into the target cells from a microprojectile gun. These methods are comparatively safe. However, the only demerit with these is a low level of gene expression. Lipofection is another effective method.
DNA fragment enclosed in double-layered lipid vesicles are targeted to the host cells. The lipid bilayer fuses with the one of the plasma membrane, consequently delivering the DNA fragment into the interior of the cell.
There is an alternate method to the ones discussed above. This method is known as receptor-mediated endocytosis. Endocytosis refers to taking something into the cell. Most macromolecules, as such, cannot cross the plasma membrane barrier of the cell and move in.
They bind to some receptors present on the outer side of the plasma membrane and then the molecule-receptor complex moves in. DNA fragments do not have receptors.
When tagged to a macromolecule having a receptor, the DNA fragments move into the interior of a cell. The movement is facilitated by the macromolecule-receptor complex. This phenomenon is known as receptor mediated endocytosis.
Other methods of gene therapy
The methods, discussed above, rely on substituting a mutant gene with a correct remedial gene and monitoring its expression at a high level. However, other methods have been discovered, which switch off the expression of a mutant gene in vitro. Some of these methods are discussed below.
Triple helix therapy
A gene is a fragment of double stranded DNA. The first step of gene expression (transcription) of a mutant gene is switched off by binding a complementary polynucleotide by Hoogsteen hydrogen bonds to the template strand. This results in the formation of a triple helix.
In this method, the transcribed product, the mRNA is blocked by a complementary ribonucleotide, known as an antisense RNA. There are two strategies for antisense RNA therapy. In the first strategy, the target cells are treated with a set of antisense oligonucleotide sequences to the mRNA.
The antisense sequence binds to the mRNA and inhibits its translation. In the second strategy, the target cells are transected by a vector, which carries the cloned antisense sequence of the target gene. The transcript of the antisense sequence binds to the mRNA by complementarity and thus inhibits its translation.
A ribozyme is a catalytic RNA that catalytically degrades or inactivates an RNA molecule. The transcribed mRNAs from mutant genes are catalytically degraded by some ribozymes, so that the translation of the defective polypeptide is inhibited. Ribozymes, potential for this application come from plant pathogens, specifically viruses. Ribozymes have been engineered to cleave the RNA of the HIV in a sequence specific manner. These are more stable than antisense RNA. An example of a ribozyme cleaving a target RNA is depicted in the.
Infra-cellular antibody (Intrabody) therapy
Antibodies are extra-cellular. They are either present in extra-cellular fluid or found attached to the lymphocyte membrane. Such antibodies do not have a means to interact with the intra-cellular harmful proteins or antigens. However, genes have been engineered to synthesize antibodies, which remain inside the cell.
Such antibodies, known as intrabodies, will interact with the viral antigens and inactivate them. Success has been achieved in designing intrabodies against the HIV surface antigen, gpl20 and thereby reducing its potentiality for infection.
Ethics forbids the germ cell gene therapy. Presently, gene therapy is confined to somatic cells only. The change in the genotype of somatic cells is for one generation only. It does not perpetuate through generations.
There is no significant threat of changing the gene pool of the population, since the manipulated gene is confined to the individual and with the death of the individual, the journey of the gene stops. Even then, every effort has to be made to ensure the safety of the patient, since, the technology used in somatic gene therapy is far from perfect. Patients, who are selected for such treatment, have life threatening diseases, for which no effective conventional cure is available. Still some people raise serious concerns about this therapy.
Whatever is the point of view, each new development in scientific research meets with skepticism. As time progresses and knowledge expands, attitude changes and things once considered unacceptable become a part of the normal life. Presently, nobody knows about how this will be viewed by our posterity, but attempts to suppress research in the pretext of one thing or other will never be successful.
A gene therapy. Protocol has to be tested on animal models and humans for its safety and efficacy before it is made public.
A protocol must be approved by appropriate regulatory agencies such as Food and Drug Administration (FDA) and Recombinant DNA Advisory Committee (RAC) within the National Institute of Health (NIH) before it is conducted on human. All NIH funded clinical trials have been reviewed by an RAC panel of 20 scientists and non-scientists for safety and risk assessment.
Following its success on animal models, several rounds of pre-clinical trials are conducted before its full commercialization. As in June, 1995, the RAC has approved 106 clinical trials involving 567 patients, out of which 51 trials were for cancer.
Several companies, the world over, have developed gene therapy protocols for conventional genetic disorders, AIDS and cancer. Targeted Genetics Corp. (TGC), Seattle has developed an ex vivo protocol for HIV gene therapy, under phase I trial. Viagene, SanDiego has had both ex vivo and in vivo protocols for AIDS and cancer, under phase I trial. Genvec, Maryland has developed a radiation based protocol for cancer therapy.
Transkaryotic Therapies, Inc, Cambridge has developed a safe non-viral method of transferring genes into patients. Skin fibroblasts are transected by genes by electroporation and reintroduced subcutaneously for expression of the polypeptide product.
Whatever may be the case, the gene therapy practice, speculated as the therapy of the future quite a few years ago has now become a reality? Expectedly, this will become the recommended therapy and will take over the conventional methods of therapy in the years to come.