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

Delivering Nucleic Acids into Cells

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    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]

    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.”

  • Virus-Inspired Nanoparticles

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    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]

    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.”

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