SBIR-STTR Award

Functional RNA molecules such as aptamers, siRNAs, miRNAs, and related compounds have enormous potential as human therapeutics and as tools for elucidating gene regulation in vivo. To reach this potential, such molecules must be highly potent and highly nuclease resistant. Unmodified RNAs typically do not meet these requirements. A variety of chemical modifications have been explored to overcome these limitations. In particular, some success has been achieved in various systems using 2'-O-methyl-ribose and phosphorothioate backbone modifications, alone or in combination. However, further improvements are highly desirable. In addition, phosphorothioate modifications are chiral, resulting in two distinct isomers at each backbone substitution. Thus, there is a need for improved chemical modifications that can be incorporated into functional RNAs. AM Biotechnologies will address these critical issues by developing 2'-O-methyl-ribonucleoside thiophosphoramidites (2'-OMe-thioamidites) to enable synthesis of phosphorodithioate 2'-OMe-RNA (PS2-2'- OMe-RNA). We have previously shown that PS2 modifications at selected backbone positions of DNA aptamers enhance binding affinity to target proteins without loss of specificity. Similarly, selected PS2 modifications in siRNAs significantly improve gene silencing activities. Thus, selected PS2-2'-OMe-RNA modifications will significantly increase binding affinity and potency of 2'-OMe-RNA aptamers, and will offer new avenues for synthesis of highly potent siRNAs. PS2-2'-OMe-RNAs will also be achiral at phosphorus, eliminating the variable biochemical, biophysical, and biological properties of diastereomeric phosphorothioate substituted RNAs. This Phase I project will: 1) develop the chemistry to produce four 2'-OMe-thioamidites (ABz, CBz, GIbu and U);2) optimize the synthesis of PS2-2'-OMe-RNAs;3) evaluate the effects of PS2-2'-OMe modifications on the binding affinity of a model RNA aptamer;and 4) evaluate the effects of PS2-2'-OMe modifications on the gene silencing activities of siRNAs targeting 2-secretase. In Phase II, AM will (a) scale reagent production up to commercial quantities and purity;(b) optimize a robust protocol for synthesis of PS2-2'OMe-RNA;(c) evaluate the effects of PS2-2'-OMe modifications on aptamers and siRNA activity in vivo;and (d) fully characterize the pharmacokinetic properties of PS2-2'-OMe-RNA. AM in Phase II may also offer for sale limited quantities of research-grade reagents for market beta testing. Upon successful completion of Phase II, AM will work with its existing industry partners to commercialize the 2'-OMe-thioamidites and enable the entire life science community to use these unique reagents in developing improved high-potency RNA drugs for a wide variety of human disease applications.

Public Health Relevance:
Functional RNA molecules such as aptamers and siRNAs have exciting potential as therapeutics for viral infections, cancer, genetic disorders, and neurological diseases. However, these potential RNA drugs require chemical modifications to achieve the necessary potency and stability. AM Biotechnologies (AM) will develop 2'-O-methyl-ribonucleoside thiophosphoramidite reagents that will allow the life science community to produce high potency, highly stable phosphorodithioate 2'-O-methyl-RNA-based drugs. The unique reagents that AM will develop under this project could have a profound impact on public health.

Thesaurus Terms:
Address;Affinity;Binding;Binding (Molecular Function);Biochemical;Biologic Sciences;Biological;Biological Sciences;Biotechnology;Chemicals;Chemistry;Communities;Cultured Cells;D-Ribose;Dna;Deoxyribonucleic Acid;Drug Kinetics;Drugs;Equipment;Exhibits;Functional Rna;Gene Action Regulation;Gene Expression Regulation;Gene Inactivation;Gene Regulation;Gene Regulation Process;Gene Silencing;Genes;Genetic Condition;Genetic Diseases;Hplc;Hereditary Disease;High Performance Liquid Chromatography;High Pressure Liquid Chromatography;High Speed Liquid Chromatography;Human;Isomerism;Loinc Axis 2 Property;Loinc Axis 4 System;Life Sciences;Man (Taxonomy);Marketing;Medication;Modeling;Modern Man;Modification;Molecular Disease;Molecular Interaction;Nervous System Diseases;Neurologic Disorders;Neurological Disorders;Non-Coding;Non-Coding Rna;Non-Polyadenylated Rna;P Element;Pharmaceutic Preparations;Pharmaceutical Preparations;Pharmacokinetics;Phase;Phosphorus;Position;Positioning Attribute;Production;Property;Proteins;Protocol;Protocols Documentation;Public Health;Rna;Rna Gene Products;Reagent;Research;Resistance;Ribonucleic Acid;Ribonucleosides;Ribose;S Period;S Phase;S Phase (Cell Cycle);Sbir;Sbirs (R43/44);Sales;Small Business Innovation Research;Small Business Innovation Research Grant;Small Interfering Rna;Solid;Specificity;Spinal Column;Spine;Synthesis Period;Synthesis Phase;System;Testing;Therapeutic;Vegfs;Vascular Endothelial Growth Factors;Vegf;Vertebral Column;Viral Diseases;Virus Diseases;Work;Yang;Aptamer;Backbone;Base;Biological Systems;Cancer Genetics;Dithiophosphoric Acid;Drug/Agent;Gene Function;Gene Product;Genetic Disorder;Hereditary Disorder;Human Disease;Improved;In Vivo;Industry Partner;Isomer;Meetings;Monomer;Nervous System Disorder;Neurological Disease;Novel;Nuclease;Phosphorodithioate;Phosphorodithioic Acid;Phosphorothioate;Public Health Medicine (Field);Resistant;Secretase;Sirna;Success;Sugar;Synthetic Dna;Synthetic Construct;Tool;Viral Infection;Virus Infection;Virus-Induced Disease

New Reagents for RNA-based Therapeutic Technologies Abstract Functional RNA molecules such as aptamers, siRNAs, miRNAs, and related compounds have enormous potential as human therapeutics and as tools for elucidating gene regulation in vivo. To reach this potential, such molecules must be highly potent and highly stable. Unmodified RNAs typically do not come close to meeting these requirements. Some success has been achieved in vitro and in vivo by using 2'-O- methyl-ribose (2'-OMe) and phosphorothioate (PS) backbone modifications, alone or in combination. However, both 2'-OMe and PS modified RNAs have limited in vivo stability and activity, which can be problematic. In addition, RNA containing PS modification(s) are chiral at phosphorus, resulting in two distinct isomers at each PS substitution. Therefore, there is a need for further improvements. In Phase I of this project, we demonstrated proof of principle for a new approach using RNA containing 2'-OMe-phosphorodithioate (MS2) modifications, prepared by using novel 2'-OMe-thiophosphoramidite (2'- OMe-thioamidite) reagents. We successfully synthesized the four 2'-OMe-thioamidites (A, C, G, and U) at small scale and used them to synthesize a variety of RNAs containing MS2 modifications. Significantly, we showed that incorporating MS2 modifications remarkably improved binding affinity toward the targeted VEGF protein more than 1000-fold, from 2 nM to < 1 pM. In addition, we showed that siRNAs containing MS2 modifications had increased gene silencing activity against multiple gene targets in cultured cells. To realize the high potential o these new reagents, Phase II of this project will focus on the following aims: (1) increase the scale of 2'-OMe-thioamidite production; (2) optimize protocols for solid-phase synthesis of MS2-RNA for in vitro and in vivo applications; (3) determine the thermal stability and structure of MS2-RNA duplexes; (4) validate the cellular binding affinity and specificity of the selected VEGF MS2-aptamers; (5) develop formulated MS2-siRNAs that provide increased potency and antitumor efficacy in a murine model of metastatic ovarian cancer. Successful completion of this project will demonstrate the value of MS2-siRNAs in vitro and in vivo, and will enable AM and its commercial partners to proceed with full commercialization of the 2'-OMe- thioamidite reagents and contribute toward the realization of effective MS2 modified RNA-based therapeutics.

Public Health Relevance Statement:


Public Health Relevance:
Functional RNA molecules such as aptamers, miRNAs, and siRNAs have exciting potential as therapeutics in areas such as viral infections, cancer, genetic disorders, and neurological diseases. However, these potential RNA drugs require chemical modifications to achieve the necessary potency and stability. AM Biotechnologies (AM) will develop 2'-O-methyl-ribonucleoside thiophosphoramidite reagents that will allow the life science community to produce highly potent, highly stable phosphorodithioate 2'-O-methyl-RNA-based therapeutics. The unique reagents that AM will develop under this project could ultimately have a profound impact on public health.

NIH Spending Category:
Biotechnology; Cancer; Genetics; Ovarian Cancer; Rare Diseases

Project Terms:
abstracting; Affinity; aptamer; Area; base; Binding (Molecular Function); Biochemical; Biological Models; Biological Sciences; biological systems; Biotechnology; Cancer Center; cancer genetics; Chemicals; commercial application; commercialization; Communities; Complex; Coupling; Cultured Cells; DNA; Drug Formulations; Drug Kinetics; experience; Functional RNA; Gene Expression Regulation; gene function; Gene Silencing; Gene Targeting; Hereditary Disease; Human; improved; In Vitro; in vitro Model; in vivo; Industry; Isomerism; Laboratories; macromolecule; Malignant neoplasm of ovary; meetings; Modeling; Modification; Molecular Conformation; Molecular Structure; monomer; Mus; nanoparticle; Nature; nervous system disorder; novel; novel strategies; nuclease; Pharmaceutical Preparations; Phase; phase 1 study; Phospholipids; phosphorodithioic acid; phosphorothioate; Phosphorus; Positioning Attribute; Production; Property; protein function; Proteins; Protocols documentation; public health medicine (field); public health relevance; Reagent; Research; Resistance; Ribonucleosides; Ribose; RNA; RNA chemical synthesis; RNA Interference; Roentgen Rays; S Phase; Small Business Innovation Research Grant; Solid; Specificity; Structure; success; sugar; synthetic construct; Technology; Therapeutic; tool; Vascular Endothelial Growth Factors; Vertebral column; Virus Diseases