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Polynucleotide constructs having disulfide groups

A technology of polynucleotides and constructs, applied in phosphorus organic compounds, organic chemistry, compounds of group 5/15 elements of the periodic table, etc., can solve problems such as cytotoxicity and ineffective delivery of transfection reagents

Inactive Publication Date: 2016-10-26
SOLSTICE BIOLOGICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite widespread use, transfection reagents fail to achieve effective delivery into many cell types, especially primary cells and hematopoietic lineages (T and B cells, macrophages)
Furthermore, lipofection reagents often result in varying degrees of cytotoxicity ranging from mild in tumor cells to high in primary cells

Method used

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  • Polynucleotide constructs having disulfide groups
  • Polynucleotide constructs having disulfide groups
  • Polynucleotide constructs having disulfide groups

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0639] Example 1 Synthesis and purification of nucleotides and polynucleotides of the present invention

[0640] general synthesis program

[0641] The polynucleotide constructs of the invention can be prepared according to the general and specific methods described herein. For example, a thiol-containing starting material undergoes a reaction with a 2,2'-bipyridyl disulfide to give the corresponding dipyridyl disulfide compound (see, for example, Scheme 1), which is then Disulfide compounds are subjected to reaction with nucleoside phosphoramidites to produce nucleotide constructs of the invention (eg, see Scheme 1). These nucleotide constructs are then used in standard oligonucleotide synthesis protocols to form polynucleotide constructs. These polynucleotide constructs were then deprotected and purified using HPLC.

[0642] plan 1

[0643]

[0644] Specific synthesis of nucleotides of the present invention

[0645] Exemplary syntheses of nucleotides of the invention...

example 2

[1273] Example 2 in vitro activity assay

[1274] Polynucleotides targeting the luciferase gene (GL3) were synthesized and used to generate polynucleotides with attached internucleotide bridging groups (phosphotriesters) and / or terminal groups (phosphodiesters) or phosphotriesters) of one or more disulfide-linked polynucleotide constructs.

[1275] To assess the in vitro activity of these disulfide phosphotriesters, human ovarian SKOV-3 cells stably expressing luciferase (GL3) were used. Cells were grown in McCoy's 5A medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS), 100 μg / ml streptomycin, and 100 U / ml penicillin. Cells (1×10 4 / well) were seeded in 96-well microtiter plates and incubated at 37°C in 5% CO 2 Incubate overnight.

[1276] Controls: A control siRNA targeting the luciferase gene or a non-targeting control gene were transfected into cells at the indicated concentrations (typically 0.01-30 nM) using lipofectamine RNAiMax (Life Technologi...

example 3

[1298] Example 3 Cell Binding Experiment

[1299] Disulfide phosphotriester oligonucleotide-Cy3 cell binding general protocol: Incorporate a core of the invention containing a disulfide group attached to one or more internucleotide bridging groups and / or terminal groups The nucleotide construct anneals to G at a final concentration of 10 mM 2’Mod -Cy3 (guide strand).

[1300] Cell Treatment Setup: Seed 40,000 cells per well in a 48-well plate; allow cells to adhere overnight. Then, cells were washed once with 500 μl of PBS, and then 150 μl was added for treatment (note: for free folic acid samples, cells were treated with medium containing 2.3 mM folic acid for 1 hour prior to treatment). Cells were treated for 4 hours; after 4 hours, cells were washed once with PBS, trypsinized and analyzed by flow cytometry for siRNA-Cy3 cell association.

[1301] The results of these experiments are in Figure 9 A. Figure 9 B. Figure 10 A. Figure 10 B. Figure 11 A. and Figure ...

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Abstract

The invention features polynucleotide constructs containing one or more components (i) containing a disulfide linkage, where each of the one or more components is attached to an internudeotide bridging group or a terminal group of the polynucleotide construct, and each of the one or more components (i) contains one or more bulky groups proximal to the disulfide group. The invention also features polynucleotide constructs containing one or more components (i) containing a disulfide linkage, where each of the one or more components (i) is attached to an internudeotide bridging group or a terminal group of the polynucleotide construct, and each of the one or more components (i) contains at least 4 atoms in a chain between the disulfide linkage and the phosphorus atom of the internudeotide bridging group or the terminal group; and where the chain does not contain a phosphate, an amide, an ester, or an alkenylene. The invention also features methods of delivering a polynucleotide to a cell using the polynucleotide constructs of the invention.

Description

technical field [0001] The present invention relates to compositions and methods for transfecting cells. Background technique [0002] In vitro and in vivo nucleic acid delivery to cells has been performed using various recombinant viral vectors, lipid delivery systems, and electroporation. Such techniques attempt to treat various diseases and conditions by knocking down gene expression, provide genetic constructs for gene therapy, or attempt to study various biological systems. [0003] Polyanionic polymers such as polynucleotides do not readily diffuse across cell membranes. To overcome this problem for cultured cells, cationic lipids are typically combined with anionic polynucleotides to facilitate uptake. Unfortunately, this complex is often toxic to cells, meaning that both the exposure time and concentration of the cationic lipid must be carefully controlled in order to ensure transfection of viable cells. [0004] The discovery of RNA interference (RNAi) as a cellu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07F9/02C12P19/34
CPCC07F9/65586C07F9/65616C07H21/00A61P35/00A61P43/00C12N15/1137C12N2310/14C12N2310/311C12N2310/321C12N2310/351
Inventor C.W.布拉肖L.埃尔特普A.卡巴基比S.林B.刘D.刘B.R.米德S.萨卡穆里
Owner SOLSTICE BIOLOGICS LTD
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