Amphipathic DNA Elements for Nucleic Acid Delivery
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Solution Overview
Problem
Current methods for delivering nucleic acids, such as oligonucleotides and siRNAs, face challenges including inefficient cellular uptake, endosomal entrapment, and cytotoxicity, particularly for uncharged nucleic acid analogues like PNAs and PMOs, and the complexity of existing delivery systems like DPCs, which are costly and difficult to scale up.
Innovation Solution
Development of amphipathic trans-acting phosphorothioate DNA elements, like PS-dT8[3+], PS-dA8[3+], and PS-dC8[3+], which form complexes with nucleic acids and facilitate their internalization into cells using thiophosphate triester linkages, allowing for efficient delivery of both charged and uncharged nucleic acids with minimal cytotoxicity in various media conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If cationic lipids are used as carriers for cellular transfection, then cellular uptake of nucleic acids is improved, but cytotoxicity increases and stability in serum proteins decreases
Solution Approach 1:
The patent modifies the chemical parameters of the carrier by using phosphorodiamidate morpholino oligomers (PMOs) with morpholino rings instead of traditional lipid-based carriers. This structural parameter change eliminates the positive charge that causes cytotoxicity while maintaining cellular uptake capability through alternative mechanisms.
Solution Approach 2:
The invention creates composite delivery systems by conjugating PMO carriers with cell-penetrating peptides or other functional moieties. This composite approach combines the stability of PMOs with the cellular internalization capabilities of peptide conjugates, achieving effective delivery without the cytotoxicity of cationic lipids.
2Productivity
If cationic cell penetrating peptides are conjugated to PNA or PMO oligomers, then cellular internalization is improved, but covalent attachment challenges arise including insolubility and functional inhibition
Solution Approach 1:
The patent introduces linker molecules as intermediaries between the PMO/PNA oligomers and cell-penetrating peptides. These linkers facilitate covalent attachment while maintaining solubility and preserving the functional activity of both components, thus enabling successful conjugation without the problems of insolubility or functional inhibition.
Solution Approach 2:
The invention applies local modification by attaching cell-penetrating peptides only to specific ends or positions of the PMO/PNA oligomers rather than throughout the entire structure. This localized conjugation maintains the solubility and core functionality of the oligomer while providing cellular internalization capability at the interface.
3Reliability
If dynamic polyconjugates are used for siRNA delivery, then gene silencing activity is improved, but manufacturing complexity increases and scale-up becomes difficult
Solution Approach 1:
The patent segments the delivery system into separate functional components: the PMO carrier molecule, the siRNA payload, and optional peptide conjugates. These modular segments can be independently synthesized and purified, then combined through controlled conjugation reactions, greatly simplifying manufacturing and scale-up compared to synthesizing complex dynamic polyconjugates as single molecules.
Solution Approach 2:
The invention performs preliminary actions by pre-synthesizing and characterizing the PMO carrier and peptide conjugates separately before final assembly with siRNA. This pre-preparation of standardized building blocks enables consistent quality control and facilitates scale-up by allowing parallel synthesis of components that are later assembled under controlled conditions.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
These DNA elements enable efficient and cost-effective cellular uptake and nuclear delivery of nucleic acids, achieving significant splice correction and gene silencing activities with reduced cytotoxicity, outperforming traditional cationic lipids and CPPs in both serum-free and serum-containing media.
Implementation Method 1
amphipathic trans-acting phosphorothioate DNA elements, like PS-dT8[3+], PS-dA8[3+], and PS-dC8[3+], which form complexes with nucleic acids and facilitate their internalization into cells
Implementation Method 2
using thiophosphate triester linkages, allowing for efficient delivery of both charged and uncharged nucleic acids
Data Source
AI summary
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.