October 15, 2018 (Vol. 38, No. 18)
The past decade has seen an exapansion of the tools available for genome engineering, which has allowed genomes to be interrogated at an uprecedented depth and accuracy.
Among these tools, transfection methodologies have become virtually indispensable for any area of life sciences. Initially viewed merely as a methodology, transfection has recently evolved into a vibrant field at the interface of several disciplines, and while novel tools are entering the research arena, the ones developed decades ago continue to be improved upon and integrated into new applications.
Baculovirus as a Delivery Tool
“The need for large DNA cargos is becoming limiting in the field of gene therapy, especially T-cell therapy, and while baculovirus could solve that problem, it requires major engineering to get there,” says Imre Berger, Ph.D., chair in biochemistry at the University of Bristol and Wellcome Trust senior investigator.
In comparison with other viruses used for gene delivery, such as lentiviruses, baculoviruses infect only very specific insect cells and are incapable of replicating in human cells. In this regard, they act like a transfection reagent. Another distinction is that while lentiviruses will integrate into the target genome, where they bring external viral DNA as a result, baculoviruses deliver only their DNA cargo, which then carries out an intended function without integrating into the host genome. Typically, the introduction of foreign DNA into viruses is constrained by the size of their rigid capsids.
“This is why, when using a virus as a vehicle to smuggle DNA into human cells, researchers are restricted by capacity,” notes Dr. Berger. A key characteristic that made baculovirus a valuable tool in cell biology is that its flexible, tube-shaped shell can expand to allow DNA to be added into its genome. “That means unprecedented DNA cargo that encodes for functionality, and baculovirus is astonishingly well suited to deliver this,” emphasizes Dr. Berger.
Currently, the DNA cargo capacity of baculoviruses is not known. “It is at least 100 kb or more at the moment, but it could be, potentially, a lot more,” predicts Dr. Berger.
Several decades ago, a novel baculovirus technology, BacMam, was developed to deliver DNA into mammalian cells. “BacMam is a great delivery tool that has become a very popular system and has a good economic and safety profile,” says Tom Kost, Ph.D., former drug discovery scientist at GlaxoSmithKline (GSK). During his time at GSK, Dr. Kost was among the pioneers who introduced BacMam for a variety of gene delivery applications. “Step by step, using this technology, researchers have been able to bring more functionality to cell types that were considered to be refractory to transfection, such as primary neurons,” states Dr. Berger.
More recently, Dr. Berger and colleagues developed MultiBac, an optimized baculovirus tailored for simultaneously expressing many genes encoding for multiprotein complexes in insect cells, the original host of this virus. Based on this groundbreaking development, they then implemented MultiBacMam, a multigene delivery system characterized by very large DNA cargo and high transduction rates in mammalian cells and tissues. By integrating MultiBacMam with bimolecular fluorescence complementation (BiFC), Dr. Berger’s team and Roche developed a very efficient system for characterizing protein-protein interaction (PPI) modulators. A screen for compounds that target the CDK5-p25 PPI, which is involved in a range of diseases including cancer, led to the identification of new compounds that efficiently inhibit this interaction, indicating the potential for new drugs.
“As we do more and more transcriptomics, we need to be able to manipulate cells by introducing genes and RNA much more easily,” says James H. Eberwine, Ph.D., the Elmert Bobst Professor of Systems Pharmacology and Translational Therapeutics and co-director of the Penn Genome Frontiers Institute at the University of Pennsylvania.
A key effort in Dr. Eberwine’s lab focuses on understanding the cellular and molecular basis of neuronal functioning. One of the technologies that Dr. Eberwine and colleagues developed, laser light–induced phototransfection, introduces transient pores in cell membranes to allow the carefully controlled entry of the exogenous genetic material. “We use phototransfection extensively to transfect RNAs, siRNAs, and proteins into cells, and we are now also using it in live slice preparations,” informs Dr. Eberwine.
Using phototransfection, Dr. Eberwine and colleagues spatially controlled the delivery of Elk1 mRNA into discrete regions of neurons in vivo to study their functions. Phototransfection is not limited by the cell type or the nature of the genetic material, and delivery can be targeted to a specific cell type in a population or to a specific region of a cell. Light of any wavelength can be used to introduce RNA or DNA into cells, but the shorter the wavelength, the more damage it will cause. “If phototransfection is not performed properly, it can damage or kill the cell,” cautions Dr. Eberwine.
In a recent study that proposed to examine transcriptome function in vivo, Dr. Eberwine and colleagues engineered a multifunctional photoactivatable mRNA molecule known as the TIVA (transcriptome in vivo analysis) tag. A cell-penetrating peptide transported the TIVA tag into the cell, and the disulfide bond linking them was subsequently cleaved in the cytoplasm. After the selective photoactivation of the TIVA tag, the light-activated hairpin oligonucleotide, which is the mRNA capturing moiety of the molecule, annealed to the polyA tail of the cellular mRNA target. This provided the first noninvasive approach to capture mRNA molecules in their native environment.
Empty Vectors, Stressed Cells
“When we compared DNA gene transfection methodologies, we found that all of them can change the genomic landscape of the cells,” says Henry H. Heng, Ph.D., professor of molecular medicine, genetics, and pathology at Wayne State University. While off-target activities and artifacts of siRNAs and shRNAs methodologies have been extensively studied, those resulting from transfection with an empty plasmid have been understudied in comparison. “It makes sense that there would be effects, because all treatments cause stress in cells, and when enough stress occurs in a system, the stochastic genomic alterations increases. This promotes the system to evolve in a different direction,” explains Dr. Heng. Recently, Dr. Heng and colleagues showed that the transient or permanent transfection of cancer cells with an empty vector can lead to genomic, transcriptomic, and phenotypic changes.
“Initially people assumed that if they compare a specific gene transfection with a control, the phenotype that is observed must be from the gene, but quite often that is actually not the case,” points our Dr. Heng. Along with colleagues, he proposed that manipulations during transfection, which include exposure to chemicals, overexpression of transgenes, or changes in culture conditions, can lead to genetic, epigenetic, and phenotypic changes. What all these factors have in common is their potential to cause stress to the biosystem. “A cell line whose genome is unstable will be particularly sensitive even if the transfection is performed very carefully, and the population genomic structure will change,” notes Dr. Heng.
These perturbations are explained by the genome theory of somatic evolution. For a long time, it was assumed that a phenotype is shaped by interactions between a fixed genotype and a variable environment. “But when a transfection experiment is performed, the phenotype is a spectrum that depends on where the gene may integrate into the genome, and the heterogeneity of karyotype. Furthermore, the procedure of DNA manipulation can trigger changes in the genomic landscape, resulting in new genome systems,” emphasizes Dr. Heng. To describe a new type of inheritance at the somatic cell level, Dr. Heng and colleagues introduced the term “fuzzy inheritance.”
Dr. Heng’s group recently discovered that transmission of genetic information is often different from the traditional Mendelian mechanisms. “We use ‘fuzzy inheritance’ to convey the fact that genetic coding refers not to the particular type of change but to an array of changes,” states Dr. Heng.
An Integrated Approach
“We have been working with suspension bioreactors for over 10 years and our group has largely used them to expand stem cells,” says Derrick E. Rancourt, Ph.D., professor of oncology, biochemistry and molecular biology, and medical genetics at the University of Calgary. A major effort in Dr. Rancourt’s lab is focusing on developing methodologies to scale up the production and differentiation of human induced pluripotent cells in bioreactors. “We found that the bioreactor environment promotes cellular reprogramming,” says Dr. Rancourt.
Historically, the biomanufacturing of cell therapy products has involved two successive steps: 1) establish stable cell lines by transfection, and 2) large-scale production of the cells. In a recent proof-of-concept study, Dr. Rancourt and colleagues described a one-step integrated approach in which the genetic modification step was combined with large-scale expression. Primary human fibroblasts are transfected in a continuous stirred-suspension bioreactor, and the processing platform allows key variables, such as microcarrier type, agitation rate, or dissolved oxygen concentration, to be adjusted. One of the unique features of this approach is the nonviral transfection of cells on microcarriers in the bioreactor.
Dr. Rancourt and colleagues examined nine microcarrier types—which differ in their charge, size, surface coating, and surface area—and found that each of them affects cell attachment, proliferation, and transfection efficiency in different ways. “The biggest challenge is to try to optimize transfection on microcarriers,” notes Dr. Rancourt. Transfection in the stirred-suspension bioreactor was more efficient than in static planar cultures, and cells grew faster and more uniformly. “It would be ideal if we could eliminate the requirement for first generating the genetically modified cells in a static culture dish and do everything directly in a bioreactor, so that we can eliminate the need for all human intervention,” says Dr. Rancourt.
Using Lentivirus-Derived Nanoparticles as Delivery Tools
“We had the idea that if we could manipulate a virus to bring a protein into cells, that would be an interesting tool for protein delivery,” says Jacob G. Mikkelsen, Ph.D., professor of biomedicine at Aarhus University. Previously, Dr. Mikkelsen and colleagues used lentivirus-derived nanoparticles to deliver nucleases and DNA transposases fused to the N-terminus of the Gag or Gag/Pol polypeptides. “Our idea was to manipulate the virus and fool it into bringing the protein together with its own viral proteins into the cell, but we were uncertain whether the virus would allow this extra uptake of foreign protein,” adds Dr. Mikkelsen.
More recently, investigators in Dr. Mikkelsen’s lab revealed the functionality of lentivirus-derived nanoparticles in which the piggyBac transposase was fused to the C-terminus of the integrase protein. “It is all about trying to find a good balance where the virus accepts this extra fusion protein,” notes Dr. Mikkelsen. Protein uptake in these nanoparticles was rapid and led to high enzymatic activity of the transposase. “If we look inside the cells where the virus delivers the protein, we don’t have massive amounts of proteins, but we see activity, which means that even from the small amount of protein that we deliver with a virus, some gets into the nucleus,” Dr. Mikkelsen points out.
A key difference between DNA transfection and the use of lentivirus-derived nanoparticles as delivery tools emerged when Dr. Mikkelsen and colleagues compared the number of transposon copies that were inserted using the two approaches. “Our observation is not straightforward to explain, but whereas we normally notice a lot of variation with DNA transfection, we see only one insertion per cell with the virus system,” clarifies Dr. Mikkelsen. “We believe this is an indication that variation in uptake between cells is much reduced using a virus-based approach.”
An advantage of introducing less cargo into the cell is the improved safety profile. “Something we are interested in is to see if we can use our viral delivery system to co-deliver a protein and genetic information into a cell at the same time,” says Dr. Mikkelsen. For example, Cas9 protein and a gRNA expression cassette could be introduced into a cell together. “Then we would have a genome editing bullet that delivers both components that are needed for CRISPR editing, and this could provide an alternative to other ways of doing it,” suggests Dr. Mikkelsen.