Gene Expression and Gene Therapy Mediated by Recombinant Virus
Evaluating the effects of overexpressing a particular gene on a specific cellular process and therapeutically delivering a gene to an animal or patient suffering
from a specific disease are highly desirable approaches in the era of modern genomics and proteomics. Although with a variety of DNA transfection techniques
being available it has become relatively easy to overexpress a gene in vitro (in cultured cells), the success of this approach is often limited by poor transfection
efficiency. This limitation is far greater with primary cell cultures, which are extremely difficult to transfect by current DNA transfection protocols. However, the use
of a virus-based vector readily circumvents the limitation, achieving nearly a 100% efficiency in terms of the percentage of cells which successfully express the
exogenously introduced gene. This amazing efficiency is perhaps not unexpected given the fact that viruses have evolved to aggressively introduce their DNA
into our cells with high efficiency. It is only fair now for us to introduce our DNA into viruses for our own benefits!
The most widely used viral vectors for gene delivery are adenoviruses (and adeno-associated viruses) and retroviruses. Two salient features distinguish these
two viral systems: the adenoviral genome remains epichromosomal in all cells (except eggs) after infection whereas the retroviral genome integrates randomly
into the host chromosome. Although the integration of retroviral DNA into the host genome translates to a more sustained gene expression than that mediated
by adenoviruses, the DNA integration event may interfere with certain host gene functions (such as activation or inactivation of cellular oncogenes). Another
difference is that retroviruses can only infect replicative cells. Thus the adenoviral vector is the system of choice for the study of gene expression in primary non-
replicative or quiescent mammalian cells.
The human adenovirus serotypes 2, 5, and 12 have been the most extensively studied, and these viruses have been valuable research tools since the dawn of
molecular biology. In order to make room for insertion of a foreign gene into the viral genome for the purpose of gene expression, the adenoviral E1 and E3
genomic regions are deleted, thus allowing for the insertion of a foreign DNA fragment up to 7.5 kilo-bases in length. Since the E1 region is normally required for
adenoviral replication, the recombinant adenovirus derived from this genetic engineering becomes replication deficient in the recipient cell or animal host. This
means from a practical point of view that the use of the recombinant adenovirus in research or clinical tests is relatively safe (require a BL-1 facility).
Considerable efforts have been made in recent years to take advantage of the superb gene delivering capacity of the recombinant adenovirus system in animal
and human gene therapy procedures, and encouraging results have been obtained from these trials. Targeted disorders included muscular dystrophy, Gaucher’
s disease, Alzheimer’s disease, Parkinson’s disease, myocardial ischemia, Wilson’s disease, nephrotic syndrome, cystic fibrosis, and etc. Initially, gene therapy
was applied to individuals suffering from the effects of genetic disorders caused by gene mutations; but it has since been applied to many diseases which do not
have a primary genetic defect. Thus, many forms of cancer as well as chronic diseases have been targeted for treatment by adenovirus-mediated gene therapy.
It is this type of gene therapy that is particularly applicable to neuromusclular degenerative diseases and tissue ischemia, both of which involve pathologic cell
loss. For example, genes encoding growth or trophic factors for various tissue and cell types can be systemically or locally delivered to an individual with the viral
vector to promote cell survival.
Aside from these direct in vitro and in vivo applications of the viral vector system, a new type of gene delivery referred to as “ex vivo” gene therapy emerges
from recent interests in stem cell-based therapy. In this stem cell engineering approach, stem cells are isolated, amplified, and infected in vitro with recombinant
viruses so as to overexpress a potentially therapeutic protein of interest. These genetically engineered stem cells are then transplanted back to the host where
they are designed to amend and heal the broken tissue. This combined synergistic cell-virus therapy may achieve more efficient gene expression than that
mediated by viral infection alone if the stem cells become integrated with the tissue after transplantation. However, tumorigenic potentials of transplanted stem
cells should not be overlooked in this type of therapeutic approach.
Biomedical Research Service
& Clinical Application