Transient Expression Enhancers
James Williams, Ph.D., vp for R&D at Nature Technology, discussed his company’s development of antibiotic-free plasmids as vectors for DNA vaccines.
Antibiotic selection is a standard approach to manipulating vector growth and isolation, but it has the disadvantage of requiring continuing exposure to the antibiotic in order to retain the plasmid, which may be cost prohibitive when large-scale projects are required.
Moreover, regulatory agencies recommend elimination of antibiotic-resistance markers in gene-based therapeutic protocols. To deal with this ruling, Nature Technology has taken advantage of the SacB gene, which codes for the enzyme Levansucrase. The activity of this enzyme is lethal in the presence of sucrose for most gram-negative bacteria. By adding sucrose to the media and modifying the plasmid to encode a 150 bp antisense RNA that represses expression of a host strain chromosome-encoded SacB gene, it is possible to dampen expression of the SacB gene and select for retention of the plasmid, thus avoiding the use of antibiotics.
Nature Technology’s NTC8385 antibiotic-free gene medicine vector complies with guidance for plasmid size and composition and it provides increased antigen expression. The system uses a chimeric promoter, composed of a portion of the human T-cell lymphotropic virus type-1 and a CMV enhancer. This combination increases translation of transiently expressed RNA but does not increase integration of gene expression, which could have serious side effects for the host.
“The NTC antibiotic-free, gene medicine vectors increased transgene expression with no effect on integrated gene expression,” Dr. Williams said. “The end result of the adoption of our technology is improved manufacturing yields and increased expression of the gene of interest.”
Minimally Invasive Electroporation
A major impediment to successful DNA vaccination has been the failure to achieve sufficient uptake of the DNA by host tissue. This roadblock caused the collapse of a number of clinical trials a decade ago. The problems have now been resolved through improvements in transfection techniques, specifically electroporation. Inovio Biomedical has aggressively pursued development of a minimally invasive electroporation device, according to Kate Broderick, Ph.D., manager, R&D.
With the Inovio platform, DNA is delivered via a standard needle and syringe, electrodes are applied to the tissue of the host, and short pulses of electricity are delivered, causing channels to open briefly in the cell membranes, allowing entry of the vector carrying the genes for the antigen of choice. Whereas, earlier electroporation studies were conducted with muscle tissue as the target, extensive work has shown that the skin is a more attractive alternative tissue. Gene-expression levels, as well as immune responses using the minimally invasive dermal device yields potent cellular and humoral responses, Dr. Broderick said.
She described how a DNA vaccine delivered with Inovio’s minimally invasive electroporation instrument provided 100% protection in mice against a lethal influenza challenge. The same approach generated a greater than 1:40 hemagglutination inhibition antibody titer response in guinea pigs and macaques.
According to Dr. Broderick, “our results suggest that the minimally invasive dermal device may offer a safe and efficient method to administer DNA vaccinations in a prophylactic clinical setting.”
The Inovio electroporation technique behaves as an adjuvant and can induce significant immune responses, including both antibody and T cells, she explained. The procedure is tolerable to the patient without anesthetics against the delivery mechanism, so it can be employed for repeated administration.
In addition to electroporation, DNA vaccines may be delivered using a custom-built syringe, according to Matti Sallberg, Ph.D., at Karolinska Institute and one of the founders of Tripep.
The Tripep device consists of three to 10 needles in close proximity. The needles are bored with holes on the sides in such a way that they spray inward toward one another, thus the tissue is blasted and overloaded with DNA from all directions. The device is simple and improves DNA uptake in a larger animal, Dr. Sallberg said. It works with commercial syringes, is disposable, and can be mass produced.
Dr. Sallberg also discussed studies on a DNA vaccine for human HCV, referred to as ChronVac-C. Delivered by Inovio’s electroporation platform, it produced a transient reduction in the viral load that lasted for two to ten weeks.
The concept is directed toward activation of host T-cell response by presenting the viral antigens in a unique manner that activates CD4+ and CD8+ T cells. Using a gene gun to deliver to the skin, or injection or electroporation to the muscle cells, the vectors move from the injection site to the dendritic cells where they stimulate activation signals.
So far, the procedure has been used in a Phase I trial with encouraging results. The study tested the safety and the HCV-specific immune response. Working under the supposition that the T cells in chronic HCV infection are dysfunctional, the procedure is aimed at improving activation of T cells by the vaccination procedure.
“We feel the current status of the work is cautiously promising,” Dr. Sallberg stated. “There are many HCV-based vaccine approaches that are in the clinic, and some may have an immediately beneficial effect when combined with appropriate standards of care.”
DNA vaccine technology has made impressive strides in recent years as products come on the market, although, so far, only in the animal vaccine field. “There are already five different animal vaccine products available. We’re there with small animal DNA vaccine technology, and with a number of late-phase human trials in progress, we look forward to approval of a range of new human vaccines, including flu, HPV, and several cancer vaccines.”