Cardiovascular gene transfer (gene therapy should only be used when we have data concerning the therapeutic potential of an agent) has a lot of promise in inducing angiogenesis, reducing restenosis, preventing graft failure after CABG, reducing cholesterol and preventing coronary atherosclerosis, preventing coronary thrombosis and plaque rupture. However, all these promises will only be kept if significant advances in vector technology, delivery modalities, and most importantly a better understanding of the biology of these processes. In addition, investigators have to exercise restraints not to report anecdotal and uncontrolled study results, which may set the stage for unrealistic expectations for these treatment strategies, where even modest successes might be viewed as failures. It is unlikely that the gene transfer vectors of today will be the therapeutic agents of tomorrow, rather, continued advances will give us the ideal vector, molecule, and target, with the option to regulate its expression. With these boundaries, gene transfer will open the door to a revolutionary treatment strategy that promises to benefit all patients with coronary artery disease.
Click here for a slide presentation on basics of gene therapy
Non-viral vectors: When naked DNA
comes into contact with the cell membrane, only a small amount will enter the
cell, leading to relatively low gene-transfer efficiency. Therefore, a carrier
or a virus vector is generally used to increase transfection efficiency and
achieve adequate expression of the therapeutic molecule. Plasmid or liposomal
complexes are the most commonly used carrier molecules. However, plasmid a small
fraction of plasmid DNA enters the nucleus, where it persists in an episomal
location (not integrated into the genome), resulting in limited duration transgene
expression in both proliferating and non-proliferating cells. Although transgene
expression has been reported to be as long as 3-4 weeks, in most cases the expression
is far shorter in duration. The production and scale up of plasmid and liposomal
complexes is relatively easy, but low transfection efficiency, short duration
of and low levels of transgene expression limit this approach, which may not
be sufficient to achieve therapeutic concentrations of the protein product.
Phospholipid formulas such as 1, 2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-dodecanoyl/
1, 2-dioleoyl-sn-glycero-3- phosphocholine and cationic polymers such as polyL-ornithine
with galactose and the fusigenic peptide mHA2 (Galactose-polyLornithine-mHA2)
can improve the transfection efficiency.In addition, EBV-based expression plasmids
may prolong gene expression at therapeutic levels, and can efficiently and repeatedly
re-transfect immunocompetent hosts.
Viral vectors: Adenoviruses enter
cells via specific receptors with subsequent lysosomal degradation releasing
viral DNA into the cytoplasm, which makes its way to the nucleus, remaining
in the extra chromosomal location. Replication-deficient adenoviruses are produced
in vitro in specific packaging cells that complement gene products (E1, E3)
deleted from the viral genome to prevent in vivo replication. Adenoviruses have
the advantage of easy production in high titers, relatively high transduction
efficiency and the ability to express in both proliferating and non-proliferating
cells. However, both first and second-generation adenoviral vectors are associated
with a significant local inflammatory reaction that eventually extinguishes
transgene expression. Circulating anti-adenoviral antibody, common for some
adenoviral subtypes, greatly reduce duration and magnitude of expression. Newer
encapsidated (gutted) adenoviruses may produce less inflammatory reactions.
These modifications also increase the capacity of these adenoviruses allowing
them to carry full-length genes such as the dystrophin gene.
Recombinant adeno-associated viruses (AAV) are promising candidates as gene vectors, as they transduce non-dividing cells and permit lasting transgene expression in a wide spectrum of tissues. However, AAV are difficult to produce, and they have a small expression cassette. Newer procedures for high throughput production, screening and characterization of AAV vectors may circumvent the scalability problem and the size limitation may be overcome by using a dual vector approach.
Retroviruses enter cells via specific receptors, following which viral RNA is reverse transcribed to DNA that is integrated into the cellular chromosomal architecture, leading to stable, prolonged, and high expression of the therapeutic transgene. Replication deficient retroviruses are produced in vitro in specific packaging cell lines containing retroviral genes (G, P, E) that have been deleted from the retroviral genome. Retroviruses can only transduce dividing cells, thus limiting their target cell population. The second limitation of retroviruses is their low titer. In addition, the efficient insertion of genes by retroviruses is often complicated by transcriptional inactivation of the retroviral long terminal repeats (LTRs) and by the production of replication-competent retroviruses. Solutions to these and other difficulties are being resolved by using modular vectors, in which the desirable features of different vector systems are combined. Examples of synergistic vectors include virosomes (liposome/virus delivery), adeno-retro vectors, and MLV/VL30 chimeras. The development of lentivirus vectors has allowed efficient gene transfer to quiescent cells and the development of pseudotyping has increased viral titers. Other gene transfer vectors are being developed to circumvent all the pitfalls of currently available vectors.
DELIVERY OF GENE TRANSFER VECTORS:
Although a tremendous amount of investigation has been performed to design and perfect gene transfer vectors and even more work to identify potential target molecules for gene transfer, little has been done with regards to how to deliver the best gene in the best vector to where it should be expressed. The vasculature and the myocardium are among the easiest targets for gene transfer because of ease of access and the need for only transient expression. Although it was thought that getting the vector in contact with its target would be sufficient, a whole series of steps were being taken for granted. The vasculature and the myocardium are subject to rapid blood flow, which leads to wash out of the vectors after only brief period of contact with target cells. Local and intramyocardial delivery result in significant systemic re-circulation, exposing non-target organs to the vector. Intramyocardial delivery using the endocardial catheter-based approach or the epicardial open chest approach appear to be similar in terms of transfection efficiency, thus obviating the need for open thoracotomy. However, the equipment used to deliver these vectors may result in inactivation of the viral and plasmid particles, which has not been adequately studied. For example, catheter based delivery appears to result in significant inactivation of adenoviral particles, which is proportionate to the dwell time in the catheter. Catheter-based plasmid delivery result in a significant reduction in the transfection efficiency of plasmid vectors, which is proportional to the injection speed and pressure. Of note, all these experiments were performed well after clinical trials with these vectors.