SBIR-STTR Award

?We are pioneering an RNA-based switch technology called structurally interacting RNAs (sxRNA) which utilizes post-transcriptional gene regulation as a reporter for miRNA detection. Normally, RNA-binding proteins (RBP) associate with a 3' stem-loop structure to facilitate translation of an upstream coding region by as much as an order of magnitude. It is possible to modify the mRNA to modulate translation of the reporter gene by controlling the binding of RBP. This is accomplished by creating a trans-acting three way junction with an additional RNA, such as a miRNA, binds and stabilizes the functional structure by base-pairing with the flanking regions of the custom designed stem-loop. Our goal with this proposal is use this selective expression capability to create homogenous populations of cells in culture, focusing specifically on stem cells. We plan to create a procedure, sold as a kit, that would eliminate unwanted cells from a differentiated cell population. We will accomplish this by using miRNA expression in the desired cells to turn on translation of an antibiotic gene thereby maintaining the desired cells. Success with this product development effort will result in a product that has broad appeal to stem cell researchers both for the benefits of increased homogeneity of stem cell cultures and potentially faster development of stem cell related lines, and position us to further enhance the technology for use in CHO cells lines for cGMP cell culturing.

Public Health Relevance Statement:


Public Health Relevance:
HocusLocus is developing a novel technology to control the expression of an mRNA. This technology can be used express an antibiotic resistance gene only in the presence of cells that express a particular miRNA. The product being developed will allow stem cell researchers to ensure the homogeneity and potentially speed up culturing of stem cells or differentiated cells by killing unwanted cells based on their miRNA expression patterns.

NIH Spending Category:
Bioengineering; Biotechnology; Eye Disease and Disorders of Vision; Genetics; Regenerative Medicine; Stem Cell Research; Stem Cell Research - Induced Pluripotent Stem Cell; Stem Cell Research - Induced Pluripotent Stem Cell - Human

Project Terms:
Adult; Amino Acid Motifs; Antibiotic Resistance; Antibiotics; base; Base Pairing; Binding (Molecular Function); Biomanufacturing; Blindness; Cell Culture Techniques; Cell Differentiation process; cell killing; Cell Line; Cell Lineage; cell type; Cells; Chinese Hamster Ovary Cell; Code; Core Facility; Coupling; Cultured Cells; Custom; Cyclic GMP; design; Detection; Development; Dihydrofolate Reductase; Disease; DNA; Ensure; Evaluation; Feedback; Gene Expression Regulation; Genes; Goals; Growth; Histocompatibility Testing; Image; in vivo; Individual; induced pluripotent stem cell; Institutes; interest; Killings; Lead; Left; Macular degeneration; Manuals; Marketing; Messenger RNA; Methods; Methotrexate; MicroRNAs; Molecular; Monitor; mRNA Expression; Mutate; nerve stem cell; new technology; novel strategies; Nucleotides; Pattern; Phase; Population; Positioning Attribute; prevent; Procedures; Process; product development; Production; protein B; Protein Binding; Proteins; public health relevance; Reporter; Reporter Genes; Research Personnel; Residual state; resistance gene; response; RNA; RNA Binding; RNA-Binding Proteins; Robotics; selective expression; Speed (motion); Staging; stem; Stem Cell Development; Stem cells; Structure; Structure of retinal pigment epithelium; success; System; targeted sequencing; Technology; Teratoma; Testing; Therapeutic Uses; therapeutic vaccine; Time; tool; Translations; Untranslated RNA; vector; Work

Over $200 billion dollars' worth of biological products, including antibodies, vaccines and proteins, are produced each year. This continues to grow worldwide at about 15%/year, making biopharmaceuticals a fairly recession-proof, growing and pro?table industry. This shift towards biopharmaceuticals re?ects a fundamental shift within the pharmaceutical industry. Biomanufacturers are under constant pressure to reduce costs by increasing productivity of their cell culturing activities. We believe, by taking advantage of post-transcriptional regulation, that we can achieve a 20%-50% improvement in productive yield, defined as titer, as well as significant reductions in workload and time. For perspective, a 25% improved titer from each cell in production could save $16 billion in biomanufacturing costs. Current biomanufacturing practice is to express a drug resistance gene as a selectable proxy for successfully transformed cells, which are then sub-cloned and screened for production of the GOI. These basic techniques have not changed in any substantial way in over 20 years, but they have two major drawbacks: (1) they require the production of the antibiotic resistant gene which competes with the GOI for scarce translational resources placing an additional metabolic burden on the cells, and (2) the selection marker or antibiotic resistance gene production is not directly coupled to the GOI giving the potential for false positives. Our protocol, PTSelect, uses post-transcriptional regulation as an alternative to using a drug resistance gene by using an siRNA coupled as an intron to the GOI. Rather than force the cells to transcribe and translate an additional drug resistance gene that serves as a proxy for GOI expression, we introduce a custom siRNA into an intron upstream of the GOI. Expression of the tethered siRNA and GOI is thus directly coupled making future selection potentially more accurate. We then use mRNA to perform selection with an mRNA has sequences that are perfectly complementary to the siRNA to induce RNA interference (RNAi) ultimately down regulating the death gene mRNA. Thus, the more the GOI is produced, the more the siRNA is also produced which results in more degradation of the mRNA. Instead of adding a chemical to select for resistance, we transfect a death gene encoding mRNA into the cells, or a fluorescent marker than identifies desired cells using fluorescence-activated cell sorting (FACS), or a cell surface marker gene mRNA coupled with magnetic-bead-Ab to perform magnetically activated cell sorting (MACS). This project will allow us to finished development of these techniques and performa a comparison study between current resistance gene products and our new PTSelect product.

Public Health Relevance Statement:
Project Narrative The current process for making a variety of biological products, such as antibodies, vaccines and proteins, has not changes in the past 20 years. We believe we can make substantial improvements in yield using our novel switching technology that lets us control protein production using a microRNA. If we are successful the cost to manufacture of these biological products will decrease.

Project Terms:
adalimumab; Adoption; Alpha Cell; Antibiotic Resistance; Antibiotics; Antibodies; base; Biological; Biological Products; Biomanufacturing; Capital; Cell Culture Techniques; Cell Death; cell killing; Cell Line; Cell Separation; Cell surface; Cell Survival; cell transformation; Cells; Cessation of life; Chemical Agents; Chemicals; Chimeric Proteins; Chromosomes; commercialization; cost; Coupled; Custom; design; Development; DHFR gene; Drug Industry; Drug resistance; Economics; Equipment; Erythropoietin; Evaluation; flexibility; Fluorescence-Activated Cell Sorting; Future; gene product; Genes; improved; Industrialization; Industry; inhibiting antibody; interest; Introns; Light; magnetic beads; Magnetism; Manufacturer Name; Messenger RNA; Metabolic; Methods; MicroRNAs; mRNA Transcript Degradation; novel; Pharmaceutical Preparations; Phase; Plasmids; Population; Post-Transcriptional Regulation; pressure; Process; process repeatability; Production; Productivity; Proteins; Protocols documentation; Proxy; Recombinants; Resistance; resistance gene; Resources; RNA Interference; Site; Small Interfering RNA; stable cell line; Surface; System; technique development; Techniques; Technology; Time; TNF gene; Transcript; Translating; Vaccines; Workload