Genetic Engineering & Biotechnology News

DEC 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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22 | DECEMBER 2017 | | Genetic Engineering & Biotechnology News James E. Dixon, Ph.D., and Robin Quirk, Ph.D. Successful regenerative medicine strategies of- ten rely on exquisite control of the biological microenvironment and supplementation with extrinsic therapeutic molecules to direct cellu- lar behavior. 1,2 These macromolecules trigger intracellular signal transduction pathways, with complex cross-talk between the cascades and eventual programming of cell behaviour through transcription factors. 2–4 An issue with the strategy of delivering exogenous soluble factors to stimulate tissue regeneration is that they are often required at supraphysiological doses. This can cause many issues, ranging from cost of goods to dose-related side effects or overexuberant tissue formation. Our recent work has shown that a prom- ising alternate approach is to use recombi- nant transcription factors, which can be ex- pressed, purified, and delivered directly into the cells in order to control their behavior for regenerative medicine strategies. 5 Effective delivery is enabled by the discovery of high- synergy peptide domains which can modify stem cells that are hard to transfect using conventional methods. Limitations of CPP Delivery Technologies Various methodologies have been devel- oped to deliver therapeutic proteins intracel- lularly 6–8 with the aim of directing differentia- tion of somatic 9,10 or stem cells 11 —effectively bypassing the need for growth-factor stimu- lation, which will activate multiple cell-type pathways. Cell penetrating peptides (CPPs) can be tethered to the protein of interest 12–14 to trigger endocytosis-mediated uptake when they interact with the cell membrane. 6–8 Even though CPPs significantly increase cellular uptake, their low-level activity requires there to be a vast extracellular excess to drive en- docytosis, with the quantities required to elicit changes in cell behavior at micromolar scale. To overcome this inefficiency, we recently described a new technology based on glycos- aminoglycan (GAG)-enhanced transduction, called IntraStem. 5 It comprises a series of novel fusion proteins that couple a membrane-dock- ing peptide which bind heparan sulfate GAGs with a CPP. We show enhanced intracellu- lar transduction upon coupling CPPs with a GAG-binding peptide. Functional quantities of many cargos including fluorescent proteins, transcription factors, and enzymes have been successfully delivered using IntraStem, with significant increase in functional delivery com- pared to conventional CPP-mediated delivery and using sub-micromolar doses. Furthermore, we are able to employ con- trolled-release methodologies (for example, using poly(DL-lactic acid-co-glycolic acid– based delivery matrices, such as Locate Thera- peutics' proprietary TAOS ® platform) to deliv- er these peptides in a progressive and sustained manner to cells. 15–17 Engineering these peptides into the coding sequences of recombinant pro- teins can facilitate their enhanced delivery into a wide variety of cell types including stem cells, primary cell isolations and tissues. Programming Stem Cell Fate with IntraStem Many regenerative medicine approaches employ the use of mesenchymal stem cells (MSCs), as they can be obtained directly Using IntraStem to Deliver Therapeutics and Control Cell Fate Peptide-Based Delivery of Recombinant Proteins and Nucleic Acids Bioprocessing Tutorial Figure 3. Efficient reprogramming of fibroblasts to iPSC cells using IntraStem to transfect (A) Yamanaka factor episomes and (B) a modified cocktail of factors. Colonies were stained for alkaline phosphatase activity after culturing in defined E8 media. Figure 2. Efficient transfection of a GFP reporter gene via IntraStem peptide (A) Transfec tion of 1 μg plasmid DNA was compared to "gold standard" in vitro trans- fection reagents in terms of transfection efficiency and assessed by flow cytometry. (B) The number of GFP-positive cells with three serial transfections of IntraStem or Product B shows serial IntraStem delivery can outcompete Product B i n t e r m s o f total transfected cell numbers. Figure 1. Efficient transduction of RUNX2 (labeled with FITC) to program human mesenchymal stem cell differentiation into bone: (A) Hoechst, (B) IntraStem-RUNX2-FITC, (C) merged. Cells were transduced with 30 μg/ mL protein for 12 hours. Developed and tested with your experiments in mind High-Quality Heat Shock Proteins Explore these and more than 50 Hsp inhibitors at Pure | Native | Full Length Available in Bulk A A B C A B B

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