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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