Trans-acting elements for intracellular delivery of nucleic acid sequences
Inventors
Beaucage, Serge L. • JAIN, Harsh V.
Assignees
US Department of Health and Human Services
Publication Number
US-10087465-B2
Publication Date
2018-10-02
Expiration Date
2035-03-13
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
Compounds of the formula (Z)x wherein: each Z is independently selected from 2′-deoxythymidinyl moiety, 2′-deoxyadenosinyl moiety, and a 2′-deoxycytidinyl moiety, x is an integer from 5-20, wherein said 2′-deoxythymidinyl moieties are connected by thiophosphate triester linkages, and 3-12 of said thiophosphate triester linkages being positively charged linkages of the formula: where n is an integer from 2 to 6; and the remainder of the thiophosphate triester linkages are neutral linkages of the formula:provided that when x is 5-6, the number of positively charged linkages is 3, when x is 7-8, the number of positively charged linkages is 3-4, when x is 9-12, the number of positively charged linkages is 3-10, and when x is 13-20, the number of positively charged linkages is 4-12.
Core Innovation
The invention relates to trans-acting elements for intracellular delivery of nucleic acids, specifically compounds of defined formulae comprising sequences of nucleosidyl moieties connected by thiophosphate triester linkages, some of which are positively charged. These compounds serve as efficient and cost effective agents for in vitro cellular transfection of neutral and charged DNA/RNA oligonucleotides and their analogues, including peptide nucleic acids (PNA) and morpholino phosphorodiamidate (PMO) oligomers. The compounds are designed to form complexes with nucleic acids, facilitating their internalization into cells and subsequent functional activity, such as splice correction of defective genes.
The background describes the problem of inefficient cellular uptake and endosomal release for therapeutic nucleic acids, limiting their clinical applicability. Existing delivery methods such as cationic lipids and cell-penetrating peptides have drawbacks including cytotoxicity, poor stability in serum, inability to deliver uncharged oligomers effectively, and technical challenges such as insolubility and conjugation requirements. Additionally, methods like dynamic polyconjugates are complex and pose difficulties in manufacturing. Thus, there exists a need for structurally simpler, more efficient, and cost-effective delivery agents capable of transfection under both serum-free and serum-containing conditions with minimal cytotoxicity.
The invention addresses these needs by providing amphipathic trans-acting phosphorothioate DNA elements with positively charged linkages that specifically recognize polyA or polyT tails on nucleic acid cargo via weak base-pairing interactions, enabling efficient cellular internalization primarily through energy-dependent macropinocytosis. These DNA-based transfection agents exhibit low cytotoxicity, can transfect both charged and uncharged nucleic acids in trans without covalent conjugation, and operate effectively in serum-containing media. The compounds are also economically and synthetically accessible through standard solid-phase DNA synthesis protocols, distinguishing them from existing cationic lipid or peptide-based carriers.
Claims Coverage
The patent includes one independent composition claim directed to novel compounds and one independent method claim directed to their use as transfection agents. The claims define the core inventive features related to specific compound structures and functional methods of nucleic acid delivery.
Compound comprising nucleosidyl moieties connected by thiophosphate triester linkages with defined numbers of positively charged linkages
The compounds have a formula (Z)x where each Z is independently selected from specified 2′-deoxynucleosidyl moieties or their 2′-O-methyl counterparts, with x ranging from 5 to 20. These moieties are connected by thiophosphate triester linkages, 3 to 12 of which are positively charged with defined formulae involving alkyl or other specified groups (R1) and an integer n from 2 to 6, while the remainder are neutral linkages.
Method of introducing nucleic acids into cells using the compounds as transfection agents
A method comprising forming a complex of the nucleic acid with the described compound, then contacting the complex with a cell to achieve intracellular nucleic acid delivery. The nucleic acids can be uncharged or negatively charged, including peptide nucleic acids (PNA), morpholino phosphorodiamidate (PMO), xeno nucleic acids (XNA), phosphorothioate DNA, plasmids, siRNA, micro RNA, and other RNA types. The method includes achieving at least 20% delivery efficiency and minimal cytotoxicity no more than 15% above medium controls.
Kit combining the compound and nucleic acid
A kit comprising one of the described compounds and a nucleic acid for use in intracellular delivery.
The claims cover novel amphipathic DNA-based compounds with defined positively charged thiophosphate linkages and methods utilizing these compounds to transfect a broad range of nucleic acids into cells with efficiency and low toxicity, providing alternatives to conventional lipid or peptide-based transfection agents.
Stated Advantages
The compounds provide efficient and cost-effective cellular transfection of both neutral and charged nucleic acids.
The DNA-based transfection elements function in both serum-free and serum-containing media with minimal cytotoxicity.
They overcome limitations of cationic lipids and cell-penetrating peptides, including avoiding conjugation requirements and maintaining solubility and functionality of nucleic acid cargo.
The compounds are synthetically accessible using standard automated solid-phase DNA synthesis, facilitating manufacturing and scalability.
Documented Applications
In vitro cellular transfection of uncharged peptide nucleic acid (PNA) and morpholino phosphorodiamidate (PMO) oligomers bearing polyA or polyT tails.
Delivery of short interfering RNA (siRNA) targeting genes such as enhanced green fluorescent protein (eGFP) in mammalian cells.
Delivery of plasmid DNA expressing proteins such as eGFP in various mammalian cell lines.
Facilitating splice correction of defective luciferase gene pre-mRNA in HeLa pLuc705 cells through intracellular delivery of nucleic acid oligomers.
Interested in licensing this patent?