SBIR-STTR Award

Extracellular vesicle (EV)-based therapies and vector-mediated gene therapies hold enormous promise as disease therapeutics. Both comprise nanoscale particles 30nm to 300 nm in diameter that are produced in complex and heterogeneous biological systems. To translate these materials into effective and commercially viable products requires accurate measurements of the concentration and size of the particles of interest (e.g., virus or EVs), and of any impurities at all stages of research, development and production. However, accurate tools for quantifying these basic parameters are not currently available. Most available methods are incapable of accurate measurements in the relevant size range and in such complex media as required for these applications. Other methods can take days (e.g., biological titer) and provide little or no information about particle impurities. At best these techniques create a bottleneck by requiring cumbersome and costly pre-measurement purification; at worst their measurements are misinterpreted—with important implications for patient safety. A critical unmet need therefore exists for technology that delivers fast and accurate size and concentration measurements of specific particles in complex biological mixtures. Spectradyne has commercialized Microfluidic Resistive Pulse Sensing (MRPS), an electrical technique that is uniquely suited to analyzing complex heterogeneous samples. MRPS is seeing rapid adoption in industry and academia for quantification of EVs and virus. While MRPS accurately measures all particles in a complex sample, it cannot currently distinguish the particles of interest from other similarly-sized particles in the sample. Spectradyne will develop Fluor-MRPS, a powerful new technology that adds the specificity of single-particle fluorescence measurements to the MRPS platform. This new technology will measure the size, concentration, and phenotype of single particles in solution with unprecedented accuracy, and dramatically reduce the time and cost of producing biologically derived materials such as virus- and EV- based therapeutics. To accomplish these goals, five specific aims will be met in Phase I and II of the project. In the Phase I Aims, a prototype instrument will be produced that is capable of simultaneous MRPS and fluorescence analysis of single particles. Sensitivity and throughput will be benchmarked. In the Phase II Aims, the prototype will be optimized for small particle detection, thoroughly evaluated for specificity, sensitivity, and limit of detection, and deployed for beta testing with end users in the real world. Completion of this work will yield an easy-to-use bench top instrument capable of rapid and accurate size and concentration measurements of both specifically labeled nanoparticle phenotypes and impurities in complex biological media. Fluor-MRPS will deliver significant efficiencies and powerful new capabilities in the development and production of gene therapy vectors and EV-based therapeutics.

Public Health Relevance Statement:
PROJECT NARRATIVE Extracellular vesicles (EVs) and virus-based gene therapy vectors are examples of nanoscale biological particles that hold enormous promise as disease therapeutics and biomarkers, but because they are generated in complex biological systems their accurate measurement is a significant challenge preventing the realization of their full potential. This project will develop Fluor-MRPS, a powerful new technology that accurately measures the concentration, size, and fluorescence phenotype of biological nanoparticles including EVs and viruses—directly in complex, heterogeneous biological fluids. This technology will deliver significant efficiencies in the production of gene therapy vectors, enable new discoveries in basic biology research, and allow these biological nanoparticles to be produced at significantly lower cost.

Project Terms:
Academia; Adopted; Adoption; base; Benchmarking; Biological; Biological Markers; biological systems; Biology; Caliber; Cell Culture Techniques; Complex; complex biological systems; Computer software; Concentration measurement; cost; Coupled; Custom; design; Detection; Development; Disease; Evaluation; extracellular vesicles; Feedback; Flow Cytometry; Fluorescence; gene therapy; Gene Transduction Agent; Goals; Heterogeneity; in vivo; Industrialization; Industry; instrument; instrumentation; interest; Knowledge; Label; light scattering; Liquid substance; Measurement; Measures; Mediating; Methods; Microfluidics; Microscope; Modernization; nanoparticle; nanoscale; new technology; Optical Methods; Optics; particle; Particle Size; patient safety; Phase; phase 1 designs; Phenotype; physical property; Physiologic pulse; Plaque Assay; prevent; Process; Production; prototype; Research; research and development; Resolution; Sampling; Scheme; Sensitivity and Specificity; Site; Source; Specificity; Speed; System; Techniques; Technology; Testing; Therapeutic; Time; Titrations; tool; Translating; vector; Viral Vector; Virus; Virus Replication; Work

Extracellular vesicle (EV)-based therapies and vector-mediated gene therapies hold enormous promise as disease therapeutics. Both comprise nanoscale particles 30nm to 300 nm in diameter that are produced in complex and heterogeneous biological systems. To translate these materials into effective and commercially viable products requires accurate measurements of the concentration and size of the particles of interest (e.g., virus or EVs), and of any impurities at all stages of research, development and production. However, accurate tools for quantifying these basic parameters are not currently available. Most available methods are incapable of accurate measurements in the relevant size range and in such complex media as required for these applications. Other methods can take days (e.g., biological titer) and provide little or no information about particle impurities. At best these techniques create a bottleneck by requiring cumbersome and costly pre-measurement purification; at worst their measurements are misinterpreted—with important implications for patient safety. A critical unmet need therefore exists for technology that delivers fast and accurate size and concentration measurements of specific particles in complex biological mixtures. Spectradyne has commercialized Microfluidic Resistive Pulse Sensing (MRPS), an electrical technique that is uniquely suited to analyzing complex heterogeneous samples. MRPS is seeing rapid adoption in industry and academia for quantification of EVs and virus. While MRPS accurately measures all particles in a complex sample, it cannot currently distinguish the particles of interest from other similarly-sized particles in the sample. Spectradyne will develop Fluor-MRPS, a powerful new technology that adds the specificity of single-particle fluorescence measurements to the MRPS platform. This new technology will measure the size, concentration, and phenotype of single particles in solution with unprecedented accuracy, and dramatically reduce the time and cost of producing biologically derived materials such as virus- and EV- based therapeutics. To accomplish these goals, five specific aims will be met in Phase I and II of the project. In the Phase I Aims, a prototype instrument will be produced that is capable of simultaneous MRPS and fluorescence analysis of single particles. Sensitivity and throughput will be benchmarked. In the Phase II Aims, the prototype will be optimized for small particle detection, thoroughly evaluated for specificity, sensitivity, and limit of detection, and deployed for beta testing with end users in the real world. Completion of this work will yield an easy-to-use bench top instrument capable of rapid and accurate size and concentration measurements of both specifically labeled nanoparticle phenotypes and impurities in complex biological media. Fluor-MRPS will deliver significant efficiencies and powerful new capabilities in the development and production of gene therapy vectors and EV-based therapeutics.

Public Health Relevance Statement:
PROJECT NARRATIVE Extracellular vesicles (EVs) and virus-based gene therapy vectors are examples of nanoscale biological particles that hold enormous promise as disease therapeutics and biomarkers, but because they are generated in complex biological systems their accurate measurement is a significant challenge preventing the realization of their full potential. This project will develop Fluor-MRPS, a powerful new technology that accurately measures the concentration, size, and fluorescence phenotype of biological nanoparticles including EVs and viruses—directly in complex, heterogeneous biological fluids. This technology will deliver significant efficiencies in the production of gene therapy vectors, enable new discoveries in basic biology research, and allow these biological nanoparticles to be produced at significantly lower cost.

Project Terms:
Academia; Adopted; Adoption; base; Benchmarking; Biological; Biological Markers; biological systems; Biology; Caliber; Cell Culture Techniques; Complex; complex biological systems; Computer software; Concentration measurement; cost; Coupled; Custom; design; Detection; Development; Disease; Evaluation; extracellular vesicles; Feedback; Flow Cytometry; Fluorescence; gene therapy; Gene Transduction Agent; Goals; Heterogeneity; in vivo; Industrialization; Industry; instrument; instrumentation; interest; Knowledge; Label; light scattering; Liquid substance; Measurement; Measures; Mediating; Methods; Microfluidics; Microscope; Modernization; nanoparticle; nanoscale; new technology; Optical Methods; Optics; particle; Particle Size; patient safety; Phase; phase 1 designs; Phenotype; physical property; Physiologic pulse; Plaque Assay; prevent; Process; Production; prototype; Research; research and development; Resolution; Sampling; Scheme; Sensitivity and Specificity; Site; Source; Specificity; Speed; System; Techniques; Technology; Testing; Therapeutic; Time; Titrations; tool; Translating; vector; Viral Vector; Virus; Virus Replication; Work