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Feature Articles : Jan 1, 2013 (Vol. 33, No. 1)

Tackling Transfection Tasks

  • Neil McKenna, Ph.D.

The discovery in the early 1990s that administration in mice of plasmid DNA encoding both viral and nonviral antigens induced antibody responses held out the prospect of achieving broad immunogenicity using DNA vaccines without the safety issues associated with a replicating pathogen.

Despite this promise, the efficacy of first-generation DNA vaccines against HIV, HPV, and hepatitis was compromised by low antibody titers and sporadic immune cell responses. In addition, DNA vaccines face broader challenges encountered by other classes of vaccines such as manufacturing, scale-up, and purification, as well as vaccine resistance.

A number of companies are developing processes and technical platforms designed to circumvent these issues, with particular emphasis on improving the efficiency of delivery and uptake of vaccines by target cells, referred to as transfection, which can be confounded by differences in tissue and cell type accessibility between individuals, in addition to other factors.

Many of these companies were present at last month’s DNA Vaccines conference in San Diego. The meeting was sponsored by International Society of DNA Vaccines and organized by BioConferences International, a Mary Ann Liebert company.

“Vaccines have saved more lives than any other invention in human history”, explained J. Joseph Kim, Ph.D., president and CEO of Inovio Pharmaceuticals. “Conventional vaccines have been successfully used against the lower hanging fruit in terms of disease, and more complex diseases such as cancer represent both opportunities and challenges for DNA vaccines."

According to Dr. Kim, one of the biggest hurdles for DNA vaccines has been inducing sufficiently high immune responses for effective vaccination. “There are two issues at stake,” he said. “The first is to make plasmids more ‘people-friendly’ to optimize their expression once in the cell, and the second is to optimize the rapid transfer of the DNA across the cell membrane to avoid degradation by endonucleases.

Inovio’s approach in this area includes informatically assisted optimization of the coding sequence to expand immunogenicity across multiple targets and boost expression levels of immunogens in target cells, and to improve the delivery of the plasmid DNA into host cells to minimize exposure to extracellular nucleases. The development of one of Inovio’s candidate high-grade cervical dysplasia vaccines, VGX-3100, encoding the HPV16 and HPV18 E6/E7 antigens, incorporated a combination of these features to increase vaccine antigen immune potency.

“The VGX-3100 vaccine was developed using our SynCon platform and includes highly efficient leader and Kozak sequences,” said Dr. Kim. “We also introduced an endoproteolytic cleavage site to improve protein folding and cytotoxic T lymphocyte processing.”

In addition, Inovio has developed an electroporation-based delivery system that, according to Dr. Kim, allows for more efficient and safe targeting of the antigenic sequence to the target cell. “A low voltage electrical field is applied at the site of vaccine injection, causing the cell membranes to transiently realign into a more porous state,” he explained, adding that electroporation is the most efficient and safe mode of delivery of nucleic acids, requiring no additional chemicals, preservatives, or adjuvants.

Looking to the future, “Electroporation is not a static technology,” continued Dr. Kim. “We are developing new devices, referred to as surface electroporators, that sit on the surface of the skin, and a piezo-electric-based system that doesn’t even require contact with the skin.”

Immune Escape

Annie DeGroot, M.D., CEO and CSO of EpiVax, highlighted immune escape as an important issue in the optimizing the amount of information that DNA vaccines deliver. Immune escape is thought to arise when an infectious agent or, in the case of cancer, a tumor, acquires a genetic profile distinct from that toward which a DNA vaccine was initially targeted, allowing it to escape the immune pressure of the vaccine.

“In response to the problem of immune escape, our approach at EpiVax has been to combine multiple key antigens or specific epitopes within these antigens, in a single recombinant vaccine,” noted Dr. DeGroot.

“We use in silico methods to optimize a gene sequence for improved vaccine expression,” added Lenny Moise, Ph.D., director of vaccine research at the company. “They include codon optimization and gene analysis, including 5´ end secondary structure, cryptic splice sites, human genome homology, bacterial promoters, eukaryotic promoters, inverted repeats, palindromes, tandem repeats, and nucleosome positioning.”

Similar to Inovio, Epivax’ transfection method is to rely on electroporation to enhance uptake to improve vaccine immunogenicity. “There are about 30 Phase I and II clinical trials using electroporation of DNA vaccines, more than any other delivery technology. It represents the most promising DNA vaccine delivery method with a path to the clinic,” said Dr. Moise.

Range of Technologies

Aldevron was the first company to produce a DNA vaccine used outside an experimental setting, a West Nile Virus DNA vaccine that has been used to protect endangered species.

“There are many reasons why so few cells are transfected by DNA vaccines,” said Michael Chambers, president and CEO of Aldevron. “One potential hurdle related to DNA vaccine manufacturing includes residual E. coli impurities like colanic acid,” referring to an exopolysaccharide common to many enterobacteria.

“These can cause toxicity and overall negative effects that hurt transfection efficiency,” he pointed out. In addition to impurities in the vaccine formulation, plasmid methylation patterns can also play a role in low transfection and expression. Other hurdles include issues related to vector size, regional micro environments around the DNA injection site, and the inherent toxicology of most compaction/transfection reagents.

“Aldevron has developed or in-licensed advanced manufacturing and purification systems to increase in vivo transfection efficiencies,” said Chambers, citing the use of Nature Technology’s HyperGro plasmid fermentation technology that enables them to produce large amounts of ultrapure DNA vaccines.

“Aldevron is also working with companies like Sekris Biomedical to develop new host strains that enable scientists to fine-tune the methylation patterns of the plastids they use for immunization,” he added.

Finally, Aldevron’s genetic immunization and antibody service allows our clients to try multiple delivery technologies like Ichor’s TriGrid electroporation system and Pharmajet’s needle-free delivery system to find the optimal way to administer their vaccines,” he noted.

Complex Formulations

Since cells prohibit entry to large molecules like DNA, unique delivery and formulation technologies must be fine-tuned to optimize the transfection and delivery of DNA vaccines.

“Technologies for delivering DNA vaccines include liposomes, polymers, or electroporation,” noted Magda Marquet, Ph.D., co-founder and co-chairperson of Althea Technologies. “However, these methods are often cumbersome for patients to receive, and present difficulties in manufacturing and scale up.”

Althea is pursuing complex formulations, such as polymers and liposomes, to overcome the obstacle of DNA vaccine delivery by delivering DNA directly to cells. Some formulations have challenging requirements.

Dr. Marquet cited one case in which aseptic processing was required throughout the formulation since product could not be sterile filtered prior to filling. “Althea successfully scaled up the process and brought in GMP formulation equipment that allowed the liposomal complex to be processed aseptically and filled into vials,” she explained.

Martin Schleef, Ph.D., CEO of the PlasmidFactory, echoed Chambers’ concerns over the high standard of purity required of DNA for delivery in humans, and cited a recent study (Woodell et al., J. Gene Med), which demonstrated that chromosomal bacterial DNA, which is typically present in kit-grade plasmid DNA preparations, is a significant contaminant leading to low efficacy and severe toxic effects.

“We have developed a manufacturing technology to avoid such contamination,” said Dr. Schleef. In addition, he highlighted plasmid topology as an important parameter in determining transfection efficiency. Covalently closed circular (ccc), or supercoiled, DNA adopts a compact form due to internal tensions in the DNA molecule that is optimal for transfection efficiency.

“Ideally the proportion of ccc-DNA in the preparation should be 95% or higher,” said Dr. Schleef, “and we use a manufacturing technology to obtain pure preparations of this form.”

PlasmidFactory has also developed a capillary gel electrophoresis-based analytical tool to quantify the major plasmid topologies in a given plasmid preparation, namely ccc-DNA, oc (open circular)-DNA, and linear DNA. In addition to fine-tuning the conformation of DNA for transfection, stripping plasmids of genes encoding resistance to antibiotics or other selection markers is an important step for preparing DNA for transfection.

“We have designed strategies to remove such genes from the bacterial backbone of the plasmids used, in addition to the origin of replication, resulting in a simply circular and extremely small DNA for vaccination, the minicircle,” said Dr. Schleef.

According to Dr. Schleef, PlasmidFactory has obtained all relevant patents for this technology and supplies researchers in gene therapy and DNA vaccination worldwide with this safe, nonviral vector. PlasmidFactory is also tackling the issue of scaling of plasmid preparations to the amounts required for more demanding applications, e.g., large clinical trials.

“We are developing technologies for ultra-large scale production of plasmid DNA (e.g., kg scale) by use of fed-batch and large scale lysis to obtain pure ccc-plasmid-DNA,” he said.

Critical Factor

As both Chambers and Dr. Schleef have pointed out, the purity of a plasmid preparation is a critical determinant of successful vaccine transfection or delivery.

“If you don’t start with pure DNA, you can forget about reproducible and effective transfection,” said Bill Kuhlman, vp North America for BIA Separations. “Removal of endotoxins and genomic DNA is commonly achievable at small scale, but producing DNA vaccines at commercial scale requires process steps that can efficiently produce tens of grams of highly pure supercoiled plasmid DNA per run.”

BIA Separations specializes in separations, with particular emphasis on monolithic HPLC columns. Rather than traditional bead-based separation columns, monolithic columns are composed of an organic or inorganic substrate and multiple highly permeable and porous channels that afford a large surface area to the stationary phase.

“We’ve achieved homogeneity of greater than 97% supercoiled DNA with greater than 99% removal of host DNA, protein, and RNA at development through industrial scales,” Kuhlman pointed out. BIA Separations has recently introduced large-scale disposable Monolith columns allowing 48 grams of super coiled DNA to be purified in a single run, while maintaining the purity and recovery seen at small scale.

In addition to manufacturing and delivery, another important consideration for DNA vaccines, like any other candidate therapeutic, is safety. The potential integration of DNA vaccines into the host cell genome is of concern due to the possibility of insertional mutagenesis resulting in the inactivation of tumor suppressor genes or the activation of oncogenes in the host genome.

According to Dr. Marquet, “DNA vaccines have been proven to be safe in multiple studies, with no integration of the DNA into the host chromosomes ever being observed.”

Her assessment is reinforced by Dr. Kim from Inovio Pharmaceuticals. “We have vaccinated or treated over 600 patients with good safety and tolerability profiles,” he said. “Adverse events are typically mild to moderate, and any injection sites resolve without sequelae.”