June 1, 2016 (Vol. 36, No. 11)

Gene Therapy Must Breach Cellular Ramparts without Reenacting the Fall of Jericho in Miniature

Immunotherapies against cancer, vaccines against new viruses (or old viruses in new places), and attempts to resolve pathogenic single-cell defects—all are looking to incorporate transfection technology. This technology, which encompasses various means of introducing nucleic acids into cells, holds great promise, but it also poses serious challenges.

These challenges have been most prominent in attempts to deploy gene therapy, which has had difficulty addressing the problem of difficult-to-transfect cells, organs, and whole animals.

Such transfection challenges have been taken up by several biotechnology companies. For example, MaxCyte, Lonza, and Mirus Bio offer electroporation as a method that leaves behind little residue. Electroporation, it happens, is not suitable for use with all animals, so other methods are needed. One alternative technique is viral infection. It is being employed by companies such as GenVec.

Another challenge, one particularly relevant to viral transfection, is the potential for stimulating an immune response. This potential negative can be a real positive, however, in the right context, which happens to be vaccine development. Harnessing viral  transfection approaches to power vaccination mechanisms is a specialty of Thermo Fisher Scientific. The company also offers traditional chemical-based transfection that works in the whole animal.

Together, the companies mentioned thus far offer technologies that cover all the main modes of therapeutically relevant transfection: chemical-based transfection, nonchemical methods (chiefly electroporation), and viral transduction. Hybrid approaches, too, are being developed, as will become clear in the following sections.


MaxCyte’s electroporation platform is GMP compliant and seamlessly scalable from research to commercial scale. “Our flow electroporation system allows a million to several hundred billion cells to be transfected in less than 30 minutes in a fully automated closed system,” expounds Madhusudan V. Peshwa, Ph.D., CSO. “Our system is both ISO 9000 and FDA compliant, so it can easily accommodate the needs of therapeutic level manufacturing.

“We routinely collaborate with pharmaceutical and biotech companies to develop ex vivo engineered immune and stem cell therapies. Sometimes the goal is to rapidly manufacture vaccines and high-yield biological drugs such as antibodies. Other times, we enable customers to create large-scale high-titer production of viral vectors using suspension and adherent cells. Not only does this aid with therapeutic development, but it can also accelerate drug discovery applications.”

This ability to process cells in large or small numbers allows scientists at the bench to develop a process protocol using flow electroporation for small cell numbers. Scaling up this process is then seamless without the usual headaches of maintaining quality, since the transfection process remains the same.

“This reduction in risk is very attractive to our partners. Ensuring consistency from research, to the clinic, to commercial scale, and from run to run, donor to donor, and patient to patient is critical to efficiently shepherding therapies through the development process,” asserts Dr. Peshwa.

While chemical and lipid approaches work well in research with established cell lines, there are often unanticipated consequences of transfecting primary cells. Additionally, these traditional chemical means of transfecting cells are difficult to scale up to industrial production.

Electroporation, the nonviral approach employed by MaxCyte, allows the biology of the transfected cells to remain in a more natural state at any scale. By preventing unintended consequences during the transfection process, MaxCyte’s technology can avoid creating roadblocks while solving the need for high transfection efficiency.

“These therapeutic interventions need to be virtually guaranteed to have minimal unintended negative impact on the cells being modified—all the biology must be the same during each stage of the process,” insists Dr. Peshwa. “In addition, our flow electroporation system can be used in an automated manner. It is already routinely used in Japan for commercial therapeutic treatments and is in various stages of adoption throughout the world.”

Another consideration is where the transfection takes place. Are cells harvested from a patient transported to another city for processing? What happens when a package is lost, or delayed, or experiences fluctuations in temperature? The platform offered by MaxCyte offers the opportunity to develop novel therapeutics through on-site processing of patients’ cells, bypassing the major logistic and COGS (cost of goods sold) issues of other methods.

Engineering cells is critical to development of cell-based therapeutics. MaxCyte’s electroporation system can avoid unintended modification of cell phenotype while meeting the key needs of cell engineering challenges: efficiency, consistency, portability, and scalability, according to the company.

Viral Transductions

Viral vectors are especially useful for whole-animal tranfection or for use with tissues that may be difficult to reach via chemical or electroporation approaches. GenVec specializes in the use of viral vectors, specifically the adenovirus, to deliver genes for therapeutic purposes.

“On first blush, the problems associated with the immune response to viral vectors seems to be problematic,” explains Doug Brough, Ph.D. “However, we have found that this can be used to stimulate an immune response to foreign materials, such that adenoviruses could be employed to deliver vaccines.”

Indeed, GenVec has thoroughly studied the adenovirus and generated several different “flavors” of adenoviruses. When exploring adenoviruses in other species, such as gorilla and monkey species, investigators can use vectors that are designed to avoid the pre-existing immunity that the general human population has to adenoviruses.

“The large library of vectors offered by GenVec is called the Adenoverse™,” informs Dr. Brough. “We have deleted large sections of the adenovirus to limit innate toxicity associated with treatment with adenovirus. In addition to preventing a deleterious immune response to the vector, this allows us to fit up to 12 kb into the virus.”

By utilizing a variety of approaches, GenVec has worked with a number of companies on different applications, nucleic acid therapeutics, and gene-editing technologies. Modified adenoviruses can deliver zinc finger approaches, both in vitro and in vivo.

An example of one such collaboration is found with the Novartis clinical trial to regenerate sensory cells into the inner ear. “By infecting cells in the inner ear with a gene encoding a key regulatory protein, new mechanosensory cells can be generated to replace those lost due to injury or inherent genetic conditions,” explains Dr. Brough.

Other joint projects can be found with partnerships in entities to treat cancer and develop vaccines. “When the time comes to commercialize a technology, GenVec uses a cell line previously approved by the FDA for these applications,” clarifies Dr. Brough.

“Another obvious advantage of using adenoviruses is that they can readily transfect whole animals. For example, a devastating disease in farm animals, called foot and mouth disease, is perpetuated by a variety of virotypes. Our system could allow the quick swapping in of whatever strain is causing an outbreak and allow a custom made vaccine to be delivered quickly,” describes Dr. Brough.


Lonza offers an advanced form of electroporation called Nucleofection™, which uses cell-type-specific transfection solutions coupled with a more nuanced pulse-delivery system that allows the transfection of many different cell types, especially normal primary human cells.

“Rather than using standard electroporation buffer solutions, we use cell-type-specific transfection solutions,” says Gregory Alberts, Ph.D., a global subject matter expert at Lonza. “This allows us to stabilize the pores generated during pulse delivery. We speculate that the pores formed in the cell membrane during standard electroporation close quickly. Our technology stabilizes the pores formed by electroporation and permits material to diffuse into the cell and, more specifically, into the nucleus of the cell.”

The Nucleofection approach utilizes many different substrates: DNA, mRNA, siRNA, peptides, proteins, and small molecules. Transfection efficiencies for siRNAs and mRNAs are very good, better than 90%. Small peptides usually transfect with around 80% efficiency, and efficiencies for plasmid DNA are anywhere from 50% to 90%, depending on cell type. Even large substrates, larger proteins such as antibodies or bacterial artificial chromosomes (BACs), can enter the target cells at reasonably effective transfection efficiencies.

“Nucleofection is surprisingly flexible,” declares Dr. Alberts. “It has been used to transfect all kinds of primary human cells. It has been used in iPSC (induced pluripotent stem cell) generation, the transfection of CRISPR and other genome-editing substrates, as well as in the transfection of more exotic targets such as the Plasmodium family of parasites that cause malaria. Other similar organisms can also be transfected with Nucleofection to enable research on tropical diseases.”

The Nucleofection platform is scalable. For example, the high-throughput Nucleofection system can handle 96-well and 384-well formats. Often this system will be employed in a core screening facility. Scientists at the bench can use the lower-throughput 4D Nucleofector to optimize assay conditions. Because the benchtop 4D Nucleofector uses the same transfection conditions, works with the same cell numbers, and delivers the same performance as the higher throughput devices, when it is time to move up to a larger scale, the assay does not require re-optimization.

“The continuity of the system allows comparison of apples to apples as you scale the project up or down,” illustrates Dr. Alberts.

“We are in the process of beta testing a new large volume transfection device that will be able to transfect 200 million to 1 billion cells in a format ranging from 1 to 20 mL. Preliminary results show that the device transfects primary human cells or cell lines at the same levels as the other Lonza Nucleofector devices,” continues Dr. Alberts. “This product will be released for research purposes only, although because of the ability of Nucleofection to transfect primary human cells so well, there will undoubtedly be interest in using this device in more clinically orientated applications.”

Dr. Alberts envisions that Nucleofection could play a role in innovative and personalized cancer therapies, such as chimeric antigen T Cell (CAR-T) therapy, as well as other cell-based therapies requiring the transfection or genomic modification of primary human cells.

“The ability of Nucleofection to easily and effectively transfect primary human T cells will attract attention in gene therapy approaches to treat disease. Lonza’s approach to potential gene-
therapy applications also has a very small footprint. This is crucial because ‘leftover’ machinery from other transfection techniques can lead to unintended biological consequences,” concludes Dr. Alberts.

Scientists at Lonza have evaluated the company’s Nucleofection technology’s ability to transfect dissociated adult rat aortic smooth muscle cells (AoSMCs). Cryopreserved rat AoSMCs were thawed and cultured for seven days in 24-well culture plates, and cells were transfected in adherence using AD1 4D-Nucleofector Y solution. Twenty-four hours post transfection, cells were fixed and analyzed. Actin is shown in red; GFP, in green.


Mirus Bio develops and manufactures novel transfection formulations, which allow for high-efficiency and low-toxicity delivery of many different types of nucleic acid molecules. Many of the formulations are free of animal-derived components. “Animal free” is an important quality for preclinical and clinical applications.

The CRISPR system requires the delivery of a guide RNA (gRNA) and expression of the Cas9 endonuclease, which can be in the form of protein, mRNA, or DNA. Mirus Bio offers transfection solutions to support effective delivery of all the different Cas9-encoding molecules. When using chemical transfection methods for the delivery of Cas9 protein, investigators can use much lower levels of protein if it is precomplexed with the gRNA.

“Some hard-to-transfect cell types yield higher cleavage efficiencies with the transfection of the Cas9 protein complexed within the RNP (ribonucleoprotein) complex,” states Laura Juckem, Ph.D., R&D group leader at Mirus Bio. “The Mirus TransIT-X2® Dynamic Delivery System effectively delivers RNP complexes and allows lower concentrations of Cas9 protein to be used compared to electroporation.”

Another challenge facing companies is the move from adherent to suspension cultures to accommodate the large amount of material needed for clinical trials. This is especially true for the production of recombinant lentivirus and adeno-associated virus (AAV). “We work closely with our clients to ensure that their transfections are successful and that changes in their workflow still yield high quality product,” explains Dr. Juckem.

“For cell-based therapy, the CHO-gro® Expression System was developed to give high biotherapeutic protein yields in suspension CHO cells. This optimized system promotes high density cell growth and enables researchers to obtain sufficient protein to perform pre-clinical studies and initial characterization analyses,” concludes Dr. Juckem.

This image, from a Mirus Bio presentation describing the high-efficiency transfection of stem cells, indicates that somatic cells such as adult fibroblast cells can be transfected or transduced via several methods with a combination of transcription factors. Once the cells have been reprogrammed to a pluripotent state, they can be guided to different fates through growth factor addition and/or transfection of selection markers driven by cell-type-specific promoters. The image emphasizes that transfection reagents can be added to a stem cell workflow at several points.

Whole-Animal Approaches

Thermo Fisher Scientific stays abreast of customer needs to generate more biologically relevant data using primary cells, rather than immortalized cell lines. Primary cells are traditionally more difficult to transfect with chemical methods. However, data generated from primary cell cultures tends to provide answers that translate better into in vivo whole-animal models.

Three-dimensional cell culture models, which yield more pertinent results than cell two-dimensional cell cultures, are more difficult to transfect with traditional methods.

“Transfection of primary cultures with DNA is challenging. Using siRNA (small inhibitory ribonucleic acid), mRNA (messenger RNA), and protein directly is more readily accepted by primary cells, as these compounds need only be delivered to the cytoplasm and not the cell nucleus,” states Xavier de Mollerat du Jeu, Ph.D., director of R&D at Thermo Fisher Scientific.

“Delivery to the nucleus happens as the cells divide,” continues Dr. de Mollerat “Since primary cells do not divide as readily as cell lines, we can circumvent the problem by delivering molecules to the cytoplasm rather than the nucleus.”

Continuing the quest for more biologically relevant systems in whole animals, “we have found that in vivo use of Invivofectamine® 3.0 can effectively knock down expression of a proteins by 90% in liver cells,” articulates Dr. de Mollerat. “This in vivo use of Invivofectamine gives scientists a more effective model of what treatments look like in the whole animal.”

Another application of transfection technology is the development of vaccines. The timeline for the development of vaccines can be shortened immensely by using transfected mRNAs to express antigens against which the body can develop an immune response. Given the number of novel viruses found in the world such as Zika and chikungunya, having the ability to rapidly develop vaccines will greatly improve world health.

With the continued development of immune therapies targeting cancers, T cells are often targeted with specific receptors. “We have worked with companies interested in developing these therapies,” elucidates Dr. de Mollerat. “We work closely with companies to address the questions of how to manufacture these treatments, make these treatments in large scale, and make them available worldwide.”

Invivofectamine 3.0 is a reagent provided by Thermo Fisher Scientific for in vivo RNAi delivery. According to the company, a single injection results in significant knockdown at day 1 and for up to 3 weeks. This finding emerged from a study in which the reagent was injected into the tail of a rodent at various doses (1, 0.5, and 0.25 mg/kg). Serum was collected on days 2, 5, 9, 16, 23, and 30, and serum was analyzed for FVII protein knockdown by a chromogenic assay. The company noted that higher amounts of siRNA in the injected complexes resulted in longer-lasting knockdown over the range tested.

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