January 1, 2014 (Vol. 34, No. 1)

Kathy Liszewski

Whether one is elucidating biological mechanisms, correcting a faulty gene, or modulating gene expression, artificially introducing nucleic acids into cells requires an efficient and nontoxic delivery method.

Transfection (placing nucleic acids into cells by nonviral methods) and transduction (utilizing viral vectors) are fundamental tools for getting the payload into the cell of interest.

At the recent meeting of the Association for Research in Vision and Ophthalmology (ARVO), researchers described cutting-edge approaches for delivering the goods such as lipid-mediated short interfering (siRNA) knockdown of mRNA, custom-built viral nanomaterials, and engineering stem cell sheets for corneal transplants.

Noncoding RNAs (ncRNAs) have achieved a kind of superstar status in the fields of genetics and molecular biology, according to Alfred Lewin, Ph.D., professor of molecular genetics and microbiology at the University of Florida College of Medicine: “These tiny RNA molecules do not encode proteins or stable RNAs such as ribosomal RNA or transfer RNA. Rather, they influence the transcription and translation of messenger RNA.

“Our lab is interested in utilizing ncRNAs such as siRNAs and ribozymes to regulate gene expression as a treatment for dominantly inherited diseases, such as retinitis pigmentosa, and viral infections.”

Dr. Lewin’s lab performed studies transfecting siRNAs to knockdown the messenger RNA for CNGB1, which codes for a subunit of the cyclic GMP gated channel in rod photoreceptor cells of the retina. “Our ultimate goal is to use a mouse model of retinitis pigmentosa and assess whether knockdown of CNGB1 or of another protein, transducin, in rods will reduce their degeneration and preserve function of neighboring cone photoreceptors.

“We tested whether siRNAs for CNGB1 could knockdown function in situ using the easily transfectable human cell line HEK293 cells. We co-transfected two plasmids, one with the mRNA for CNGB1 tagged with luciferase, and one with a corresponding siRNA designed to knockdown expression of CNGB1. We used various ratios of both along with Lipofectamine 2000 (Life Technologies), a lipid-mediated transfection reagent. The readout was lowered luciferase activity.”

According to Dr. Lewin, the lipid-based transfection allows an easy, efficient, and low-toxicity means for co-transfection into a variety of cells. Cationic lipids are amphiphilic molecules with positively charge polar head groups that interact with the negatively charged phosphates of DNA.

“Transfecting into cultured cells allows us to screen a variety of siRNAs to find the ones that will cleave the target efficiently,” remarked Dr. Lewin. “With these data, we can decide on the best siRNA to use for subsequent animal work. We will then employ viral vectors in a mouse model based on what we found in the cell culture system.”


Researchers are exploring the use of lipid-mediated siRNA knockdown of mRNA and custom-built viral nanomaterials as two promising methods for delivering nucleic acids into cells. [Biogeek/iStock Photo]

Virus-Inspired Nanoparticles

Buttressed by 50 years of research on the flu virus, researchers are now evaluating and harnessing it for viral delivery of therapeutic payloads. “We can rebuild the flu virus from the ground up,” remarked Benjamin tenOever, Ph.D., professor of microbiology at the Icahn School of Medicine at Mount Sinai. “We view viruses as a nanomaterial that can be engineered to deliver cargo into a cell without causing disease.

“Influenza A virus (IAV) is composed of eight viral segments encoding only ten major proteins. By eliminating two segments, we render it noninfectious and capable of delivering either coding or noncoding RNAs, thereby generating a tailor-made therapeutic designed specifically to address a medical need.”

In a recent study, Dr. tenOever’s IAV-based vector system was engineered to deliver microRNAs for the treatment of glaucoma. This study, which was headed by Pedro Gonzalez, Ph.D., associate professor in ophthalmology and pathology at Duke University, ultimately evaluated how well the system accomplished in vivo delivery. “MicroRNAs offer the potential to help treat glaucoma,” said Dr. Gonzalez. “Yet the safe and efficient delivery to the ocular meshwork of cells is a significant challenge.”

The microRNA-loaded designer IAV system was evaluated in primary human trabecular meshwork cell cultures and demonstrated robust delivery by qPCR, RNA in situ hybridization, and northern blot. The system also showed successful delivery in vivo following intravitreal injection into the eye.

“Overall this approach provides a versatile virus-inspired therapeutic that can deliver small RNAs transiently to a desired tissue,” observed Dr. Gonzalez. “This provides a means for acute treatment as the genetic material (RNA) is not incorporated in the host genome. We can envision many other applications for this technology as delivery can be achieved for a broad range of tissues, but it is particularly well-suited for designing therapeutics for other viruses such as Ebola, SARS, or even IAV itself.”

“The technology could target endogenous pathways, such as the apoptotic pathway, to enhance the efficiency of virus-based cancer treatments,” added Dr. Gonzalez. “We are enthusiastic about this new RNA delivery platform and genuinely hope to see it translate into future therapeutics.”


RNA in situ hybridization of the airway epithelium. Picture displays an overlay of cellular nuclei (blue), cytoplasm (red), and RNA-virus-mediated delivery of a small RNA (green/yellow). [Mt. Sinai]

Hybrid Polymer Polyplexes

Although viral vectors can be successfully utilized for gene incorporation, they are limited by several constraints. “Successful ocular gene therapy requires efficient gene transfer and stable transgene expression, but can be limited by size of the gene,” said Daniel C. Chung, D.O., senior investigator, the Perelman School of Medicine at the University of Pennsylvania.

“Our goal was to stably express a large retinal degeneration gene using a promising technology that exploits a site-specific recombinase, the phage phiC31 integrase, and chitosan nanoparticles for gene transfer into cultured cells.” Chitosan is a modified carbohydrate polymer of chitin, a natural biopolymer derived from crustacean shells such as crabs and lobsters.

In pursuing this work, Dr. Chung collaborated with Gabriela Silva, Ph.D., and doctoral student Anna V. Oliviera, both from the department of biomedical sciences at the University of Algarve, Faro. The group developed plasmid constructs that carried the full-length cDNA for the centrosomal protein 290 (CEP290) gene, a large 8.2 transgene responsible for Leber congenital amaurosis type 10, a retinal disorder. The investigators also added an integrase attachment site and a marker gene.

“We found robust gene expression utilizing Western blot and fluorescence microscopy for up to 14 weeks with the marker gene and over 6 weeks with the large retinal degeneration gene CEP290,” said Dr. Chung. “We also determined that transfection efficiency and transgene expression was affected by the size of the polymer and the type of polyplex utilized.”

Considering the impact of these findings, Dr. Chung remarked, “This method of transfection overcomes a couple of the challenges of traditional adeno-associated, virus-mediated gene delivery. The combined strategy of utilizing polymers and integrase is more efficient than nonintegrative strategies. We can essentially overcome size drawbacks because even large genes can be easily packaged.”

“Because we are using a site-specific integrase, we can avoid random integration into the host genome, which in the past has caused significant morbidity and mortality in gene therapy trials,” added Dr. Chung. “Another advantage is that this method is nontoxic and nonimmunogenic.”

Next up for the group will be studies in an in vivo model. “We will evaluate the system in transgenic mice and also continue to develop and assess different combinations of chitosan polyplexes.”


Engineering Cell Sheets

In the past five years, corneal transplantation has seen dramatic improvements. Cells, or rather stem cell sheets, can be grown and expanded in culture and transplanted into the human eye. But one complication of transplantation can be the growth of new blood vessels. “The cornea must remain clear and resist inadvertent vascularization,” noted Mark I. Rosenblatt, M.D., Ph.D., associate professor of ophthalmology at Weill Cornell Medical College.

The presence or upregulation of angiogenic proteins, such as vascular endothelial growth factor (VEGF) can sometimes lead to blood cell proliferation and subsequent vision loss. Dr. Rosenblatt’s team examined the ex vivo gene transfer of a soluble version of the VEGF receptor (sFlt1) integrated into a lentiviral vector for ocular surface reconstruction.

“We cloned mouse sFlt1 into lentivirus and prepared a viral stock that was used to transduce primary rabbit corneal epithelial cells (RCEC),” commented Dr. Rosenblatt. “The sFlt1 essentially serves as a decoy receptor. We analyzed expression by qRT-PCR and found high-level expression of mouse sFlt1 mRNA in the transduced RCEC. Further, the sFlt1 protein was detected by immunofluorescence staining in the cytoplasm and via ELISA for up to one week.”

Lentiviruses are retroviruses that can stably deliver a high load of viral RNA into the genome of host cells. They have the unique ability to infect nondividing cells, a property that makes them one of the most efficient methods for gene delivery. They also have low immunogenicity.

“We have learned several things from our preliminary studies,” concluded Dr. Rosenblatt. “First, we can utilize the lentiviral system for high-level expression of an anti-angiogenic gene that stably incorporates into the genome of engineered cells. Second, the efficiency is very high—often one cannot approach this level of expression without viral systems. Third, an effective strategy is to employ tissue culture as a model system, verifying that there is sustained expression, before challenging the cells in a variety of ways to verify the anti-angiogenic effect.”

As delivery and other challenges are solved, genetically modified, cultivated epithelial stem cell transplants could provide the basis for everyday ex vivo corneal gene therapy to treat a variety of corneal diseases.


Coping with Hard-to-Transfect Cells

The abundance of transfection options should be encouraging, particularly to researchers working with hard-to-transfect cells. However, the prospect of actually screening any appreciable number of transfection options might give any researcher pause. Even researchers who narrow their transfection options to chemical methods—that is, transfection via lipid- or polymer-based reagents—might feel overwhelmed, or at least annoyed at having to undertake a relatively simple but time-consuming chore.

Unfortunately, transfection is not quite a science. It usually isn’t possible to predict the best set of transfection conditions. Careful scrutiny of structures doesn’t help. And delving into the literature won’t help, either. Reports comparing different transfection conditions for particular cell types are few and far between.

It might be helpful, however, to seek expert assistance or custom services. For example, custom services are available from Mirus Bio. Back in 2008, Mirus Bio’s therapeutics division was acquired by Hoffmann-La Roche. The company’s research tools division, however, remains an independent entity, and it continues to build on two decades of nonviral gene therapy research.

“Profound advances in gene delivery technology have been lacking for some time,” said Scott Hayes, Ph.D., vp, Mirus Bio. “As a result, researchers have increasingly resorted to virus-borne or physical disruption technologies to facilitate nucleic acid uptake in difficult-to-transfect cell types despite inherent limitations with each of these methods.”

One of Mirus Bio’s goals is to help researchers avoid unnecessarily harsh transfection techniques. Back in June 2013, while introducing the TransIT-X2 Dynamic Delivery System, a polymeric system for nucleic acid delivery, Dr. Hayes stated, “This system provides superior delivery of plasmid DNA and siRNA into a vast array of cell types. We feel that this technology provides distinct advantages over other means of transfection and opens new avenues for experimental design.”

Mirus Bio’s custom service, besides encompassing pDNA, mRNA, and siRNA/miRNA transfection offerings, focuses on transfection reagent development and nucleic acid labeling. With respect to reagent development, Mirus Bio may generate screens, arraying lipid/reagents according to their post-transfection luciferase activities. As for labeling, Mirus Bio uses a nonenzymatic chemical labeling method that facilitates the direct covalent attachment of either fluorescent or non-fluorescent labels to nucleic acids via a one-step chemical reaction. Mirus Bio supports applications such as FISH, microarrays, and nucleic acid visualization both in vitro and in vivo.


HEK 293 cells stably expressing eGFP were generated through transient transfection using Mirus Bio’s TransIT-2020 transfection reagent with eGFP and neomycin plasmid DNA vectors.


























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