SBIR-STTR Award

There is enormous potential of oligonucleotides (ON) as therapeutics, but the challenge remains how to effectively deliver ON into cells. Currently, there are no effective and reliable ways of delivery. Outer cell membranes resist the cellular uptake of charged ON, and charges-eliminating backbone modifications such as those in peptide nucleic acids (PNA) and methylphosphonates reduce but do not solve the problem, because such structural changes compromise their aqueous solubility. Use of delivery vehicles (formulations), such as virus-based delivery systems, liposomes, nanoparticles and transporter chemical groups, have not solved this problem fully and are often associated with significant side effects. Development of optimal oligotherapy for the treatment of infectious and genetic diseases still remains unrealized. ZATA Pharmaceuticals, Inc. is developing a nucleic acid technology platform that will enable the synthesis of self-neutralizing ON with enhanced intracellular penetration capabilities. In ZATA's compounds negative charges will be neutralized (not eliminated!) by formation of intramolecular ammonium/phosphate ion-pairs. The resulting modified ON (MON) should possess sufficient solubility for optimal pharmacokinetic (PK) properties and improved cell penetration. We will first synthesize novel phosphoramidite synthons containing branched amino-terminated linkers (BATLs) with positive charges at their termini, in order to neutralize negative backbone charges of the final ON. The length of each branch will allow the terminal positively charged groups to reach neighboring phosphate groups and neutralize their negative charges. Additionally, the BATLs will introduce partial hydrophobic properties to the ON backbone. Our preliminary data and computer assisted modeling indicate that introduction of those modifications will not disturb the natural Watson-Crick hybridization properties. Second, we will use these modified synthons to prepare 21-mer ribo-, and deoxyribonucleotides bearing different numbers of charge-neutralizing groups, and to test their solubility, chemical and serum stability, Watson-Crick base paring specificity and duplex stability. We will test these MONs for their intracellular uptake and mRNA knockdown experiments in C127 mouse mammary epithelial, HL-60 human lymphoblastoma, and human fibroblasts cells. This set of experiments will satisfy the main goals of Phase I: 1) validate the methods of synthesis and purification of ZATA's MONs, and 2) demonstrate their biological validity. The proposed platform ON technology will apply equally to oligodeoxy- and oligoribonucleotide derivatives. Variation of the number, site, and type of the charge-neutralizingBATLs will allow for optimal balance between hydrophobicity and water solubility of the MONs, thus maximizing intracellular penetration and minimizing non-specific binding and poor PK properties. We anticipate that this new platform may be used without the need of additional vehicles. Upon successful validation of our concept, we will continue in phase II to study and optimize the biological stability, PK properties, gene silencing properties, and therapeutic effectin disease models, alone and in combination with other compatible platforms.

Thesaurus Terms:
Acids;Address;Adverse Effects;Amino Group;Ammonium;Analog;Aqueous;Base;Base Pairing;Binding (Molecular Function);Biological;Breast Epithelial Cells;Cell Line;Cell Membrane;Cell Membrane Permeability;Cells;Cellular Membrane;Charge;Chemical Group;Chemical Stability;Chemicals;Chemistry;Computer Assisted;Custom;Data;Deoxyribonucleotides;Design;Detection;Development;Disease Model;Dna;Drug Formulations;Drug Kinetics;Epithelial;Equilibrium;Fibroblasts;Figs - Dietary;Fluorescein;Gene Silencing;Goals;Hereditary Disease;Hl60;Human;Hydrophobicity;Imagery;Improved;Infectious Disease Treatment;Inorganic Phosphate;Ions;Label;Legal Patent;Length;Lipophilicity;Liposomes;Mammary Gland;Manuals;Melting;Messenger Rna;Methods;Methylphosphonate;Modeling;Modification;Mus;Nanoparticle;Nature;Novel;Nucleic Acids;Nucleotides;Oligodeoxyribonucleotides;Oligonucleotides;Oligoribonucleotides;Penetration;Peptide Nucleic Acids;Performance;Pharmacologic Substance;Phase;Phosphoramidite;Problem Solving;Procedures;Property;Protocols Documentation;Public Health Relevance;Reporter;Research Study;Ribonucleotides;Rna;Serum;Side;Site;Small Interfering Rna;Sodium Chloride;Solubility;Solutions;Specificity;Staining Method;Stains;System;Techniques;Technology;Temperature;Testing;Therapeutic;Therapeutic Effect;Therapeutic Uses;Transfection;Uptake;Validation;Variant;Vertebral Column;Virus;Water Solubility;

There is enormous potential for oligonucleotides (ON) as therapeutics, but the challenge remains how to effectively deliver ON into cells. Cell membranes resist the cellular uptake of currently used charged ON. The application of various delivery systems has only partially solved the problem and is often associated with therapeutically unacceptable side effects. Low level cellular uptake has been the main reason of the failure of large number of ON targeting cancer, genetic-, and microorganism-mediated diseases. Specific aims for the Phase I were 1) development and validation of two new types of phosphoramidite monomers, 2) their use for the synthesis of ZATA ON with enhanced cellular uptake, and 3) demonstration that ZATA ON possess an optimal combination of properties necessary for high in vivo therapeutic activity, such as enhanced cell penetration, high efficacy toward silencing of target genes, low or lack of toxicity at therapeutic concentrations, maintenance of natural hybridization properties, stability in plasma/biological fluids, solubility n aqueous media and robust method of synthesis allowing scale-up. As demonstrated in the Progress Report section, we fully completed all Phase I specific tasks and, for the first time, have developed ON with new composition of matter that practically satisfies the complex criteria outlined herein. Particularly, novelties implemented in ZATA ON enabled a) 4 times higher cellular uptake vs. similar oligonucleotides without ZATA modifications, b) over 95% inhibition of cancer cell growth in culture with single treatment at a concentration as low as 1 µM, c) high stability in serum, and d) lack of cytotoxicity at a concentration as high as 10 µM. Our achievements can briefly be defined as a novel class of ON synthesized via standard phosphoramidite chemistry which permits facile attachment of Charge Neutralizing Groups (CNG) bearing positive charges at their termini capable of reaching the adjacent negative charges and neutralizing them. Charge-neutralization in combination with added partial hydrophobicity across the backbone of ON dramatically enhance cellular uptake and gene silencing efficacy. Our major goal for the Phase II of this technology development is further validation of ZATA ON by demonstrating their high therapeutic efficacy in vitro and in vivo (mouse) models. The main tasks for Phase II study are: 1) Optimization and scale-up of the synthesis of all four 2′-modified RNA phosphoramidites enabling the incorporation of optimal CNG (i.e. 1,3-Bis(2-(dimethylamino) ethoxy)propan-2-ol) into the backbones of our ON; 2) Synthesis and screening of over two dozen ON targeting oncogenic miR10b and miR21 in human glioblastoma and breast cancer cell lines; 3) Scale-up of the best ON drug candidate(s) and testing in vivo in mouse model using human glioblastoma xenografts as a target. Novelties developed in the Phase I study are subject to ZATA's new PCT patent application.

Public Health Relevance Statement:


Public Health Relevance:
Development of a novel class of oligonucleotides (ON) is reported. ZATA ON exhibited: 1) four times higher cellular penetration compared to currently used ON analogs, and 2) close to quantitative inhibition of cancer cell growth in culture. Proof of principle in in vivo experiments is intended.

NIH Spending Category:
Biotechnology; Brain Cancer; Brain Disorders; Cancer; Genetics; Orphan Drug; Rare Diseases

Project Terms:
Achievement; Adverse effects; analog; Animal Model; Animals; anti-cancer therapeutic; aqueous; base; Biological; Boston; Breast Cancer cell line; cancer cell; cancer genetics; Cell Culture Techniques; Cell Line; Cell membrane; Cells; Charge; Chemicals; Chemistry; Collaborations; Complex; cytotoxic; cytotoxicity; Data; Development; Disease; DNA; drug candidate; Exhibits; Failure; Formulation; Gene Silencing; Gene Targeting; Glioblastoma; Glioma; Goals; Hela Cells; Hospitals; Housing; Human; Hydrophobicity; improved; In Vitro; in vivo; Inhibition of Cancer Cell Growth; Institution; Legal patent; Licensing; Liquid substance; Maintenance; MCF7 cell; Mediating; medical schools; Methods; microorganism; MicroRNAs; Modeling; Modification; monomer; mouse model; novel; Oligonucleotides; Oncogenes; Oncogenic; Pathway interactions; Penetration; Pharmacologic Substance; Phase; phase 1 study; phase 2 study; phosphoramidite; Plasma; Postdoctoral Fellow; Preparation; Problem Solving; Progress Reports; Property; public health relevance; Reporting; Research Personnel; research study; Resources; Rivers; RNA; Safety; scale up; Scheme; screening; Serum; Solubility; Study Subject; Sugar Phosphates; System; Technology; technology development; tertiary amine; Testing; Therapeutic; Time; Toxic effect; Treatment Efficacy; tumor; tumor growth; uptake; Validation; Vertebral column; Woman; Work; Xenograft proced