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Compositions for Transfecting Resistant Cell Types

a technology of resistant cell types and compositions, applied in the direction of nervous system cells, biochemistry apparatus and processes, pharmaceutical non-active ingredients, etc., can solve the problems of transfection efficiency, stability and toxicity, and the notorious difficulty of cns tissue transfection, so as to increase and reduce the ratio of cationic lipid to structural lipid

Inactive Publication Date: 2021-09-16
PRECISION NANOSYST INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is about a way to make a special type of lipid nanoparticle that can target neurons. The method involves making the nanoparticle with a high ratio of a specific lipid called cationic lipid to a different lipid called structural lipid. This can improve the effectiveness of the nanoparticle in delivering its contents to specific types of cells in the brain.

Problems solved by technology

Delivering nucleic acids into cells or tissues presents challenges because nucleic acids are relatively large, negatively charged, hydrophilic compounds which are not capable of passively diffusing across the cell membrane and are also vulnerable to nucleases.
Cationic lipids and polymers have also been used in experiments, but each has issues of transfection efficiency, stability and toxicity.
CNS tissue is notoriously difficult to transfect (O'Mahony, Godinho et al.
The cells are sensitive to their conditions and are hard to maintain in vitro.

Method used

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  • Compositions for Transfecting Resistant Cell Types
  • Compositions for Transfecting Resistant Cell Types
  • Compositions for Transfecting Resistant Cell Types

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0167]LNP Characterization

[0168]Particle size (hydrodynamic diameter of the particles) was determined by Dynamic Light Scattering (DLS) using a ZetaSizer Nano ZS™, Malvern Instruments, UK). He / Ne laser of 633 nm wavelength was used as the light source. Data were measured from the scattered intensity data conducted in backscattering detection mode (measurement angle 173°). Measurements were an average of 10 runs of two cycles each per sample. Z-Average size was reported as the LNP size, and is defined as the harmonic intensity averaged particle diameter.

[0169]RNA encapsulated was measured using the Ribogreen® dye method. The RiboGreen® dye is a fluorescent nucleic acid stain for quantitating intact RNA. RiboGreen® assay provides RNA quantitation with minimal consumption of sample. A standard curve established using known concentrations of control RNA sample. Then the test solutions are run and their measurements translated using the standard curve.

TABLE 1Physico-chemical parameters o...

example 2

[0173]mRNA Transfection and Expression in Neurons

[0174]Messenger RNAs reagents were purchased from Trilink Biotechnologies, San Diego, Calif. The mRNA was used to study the transfection ability of the nanoparticles.

[0175]EGFP mRNA (5meC, ψ)) Enhanced Green Fluorescent Protein mRNA (5-methylcytidine, pseudouridine), 1.0 mg / mL in 10 mM Tris-HCl, pH 7.5, Length: 996 nucleotides (Seq Id. No 1). Then unknown bases are a portion of alpha globin which the vendor would not reveal.

[0176]Encapsulation efficiency was established for formulations as set out supra in Table 1. Size of the nanoparticles, as well as homogeneity of the size of the nanoparticles (PDI) was measured.

[0177]LNP Formulations Lipid Mix A, B, C, and D containing messenger RNA (GFP) were tested on enriched neurons in order to identify those that are most promising for GFP expression. A table of the observed intensity of signals in photographs, ELISAS, and flow cytometry as shown in the columns below was prepared to aid compa...

example 3

[0187]siRNA Transfection Procedures

[0188]mRNA silencing by RNA interference is induced by double stranded oligoribonucleotides with a length of about 22 base pairs with 2-nucleotide long 3′ overhangs. This type of small dsRNA is called “small interfering RNA” or “siRNA”. For experimental purposes, siRNA can be produced synthetically and then transfected into the target cells, or it can be expressed in vitro or in vivo using a specially designed expression plasmid.

[0189]Enriched neurons were treated with Lipid Nanoparticles containing an off-the-shelf dicer-substrate siRNA (DsiRNA) HPRT (SEQ ID NO 2, sense, SEQ ID NO 3, antisense)) or control siRNA DS NC1, a nonsilencing, negative control DsiRNA that does not recognize any sequences in human, mouse, or rat transcriptomes (SEQ ID NO 4, sense, SEQ ID NO 5, antisense). Both DsiRNA and DsNC1 were purchased from Integrated DNA Technologies, Inc., Coralville, Iowa, U.S.)

[0190]HPRT is an acronym for hypoxanthine phosphoribosyltransferase 1,...

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Abstract

A transfection reagent composition comprising: 30-60 MOL % of an cationic lipid, or pharmaceutical acceptable salt thereof; 10-60 MOL % structural lipid; a sterol and 0.1 to about 10 MOL % of a stabilizing agent is provided. The reagent is particularly adapted for neuron and related cell types. A method of manufacturing LNP including nucleic acid for selective uptake into either neurons or astrocytes or neural progenitor cells is also provided.

Description

[0001]This application claims the benefit of U.S. application No. 62 / 403,640 filed on Oct. 3, 2016.BACKGROUND OF THE INVENTIONField of Invention[0002]The field of the invention is the transfer of active nucleic acids into cells.Related Art[0003]Nucleic acids in the form of polynucleotides or oligonucleotides can be used to focus treatment on a particular genetic target, either by interfering with its expression, or by restoring or augmenting its expression, or by editing the gene.[0004]Delivering nucleic acids into cells or tissues presents challenges because nucleic acids are relatively large, negatively charged, hydrophilic compounds which are not capable of passively diffusing across the cell membrane and are also vulnerable to nucleases. (Akhtar, Basu S Fau-Wickstrom et al. 1991)[0005]Existing approaches for delivering these nucleic acids across the cell membrane include viruses such as adeno-associated viruses as vectors for gene restoration, but these can cause immune response...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/113
CPCC12N15/113C12N2310/11C12N2310/12C12N2310/16C12N2310/14C12N2310/531C12N2310/141C07J41/0055C07F9/106C12N15/111C12N2320/32A61K9/5123C12N15/88A61K9/14A61K47/14A61K47/18C12N5/0619C12N15/85C12N2500/35C12N2500/36C12N2500/50C12N15/87
Inventor THOMAS, ANITHADE SOUZA, REBECCA ANNE GRACEOUELLET, ERICTHARMARAJAH, GRACE THARMINISINGH, JAGBIRGARG, SHYAM MADHUSUDAN
Owner PRECISION NANOSYST INC