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-neutralizing BATLs 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 effect in disease models, alone and in combination with other compatible platforms.
Development of new nucleic acid platform technology that enables the synthesis of self-neutralizing oligonucleotides (both oligodeoxy- and oligoribonucleotides) with enhanced cellular uptake is proposed. The modifications will result in internally neutralized oligonucleotides with enhanced stability, lipophilicity, and membrane permeability.