Compositions and methods for nucleic acid and/or protein payload delivery

a nucleic acid and/or protein technology, applied in the direction of powder delivery, dna/rna fragmentation, macromolecular non-active ingredients, etc., can solve the problems of low cellular transfection level of nanoparticle-based payload delivery technology and limited transfection effectiveness, so as to induce proliferation and/or bias differentiation of target cells

Pending Publication Date: 2020-03-26
LIGANDAL INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0003]Effective introduction of nucleic acid and / or protein payloads into cells, e.g., for genome editing and / or altering gene expression, is an important objective for therapeutic strategies and for research methodologies. To achieve effective introduction of a payload, it is important to appropriately package the payload to protect it from degradation prior to cellular entry, to permit entry into cells, to direct the payload away from the lysosomal degradation pathway, and to direct delivery to the appropriate subcellular compartment. In addition, the timing of release of a payload from the packaging following cellular entry can influence the effectiveness of the payload.

Problems solved by technology

Many nanoparticle-based technologies for payload delivery offer low levels of cellular transfection and limited effectiveness upon transfection.

Method used

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  • Compositions and methods for nucleic acid and/or protein payload delivery
  • Compositions and methods for nucleic acid and/or protein payload delivery
  • Compositions and methods for nucleic acid and/or protein payload delivery

Examples

Experimental program
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Effect test

example 1

[0376]To determine core formulation parameters, fluorescence spectroscopy was used to monitor nucleic acid condensation (for double stranded DNA payloads). The emission spectra of intercalating Ethidium bromide (EtBr) was measured after the addition of condensing agents at increasing charge ratios. De-intercalation of ethidium bromide caused by polymer induced nucleic acid condensation results in a drop in fluorescent signal. This is because ethidium bromide exhibits a much higher quantum fluorescent yield in the DNA-bound state, than unbound state. The results are depicted in FIG. 1.

[0377]FIG. 1 depicts results from a fluorimetric assay testing various parameters (e.g., cation: anion charge ratio) for condensation of nucleic acid payloads. The result showed, e.g., that a charge ratio of 2 works well for the condensation of plasmids encoding Cas9 and guide RNA molecules. 100 μl of Anionic solution was added to each well: 100 ng / μl DNA, 80 ng / μl poly(D-Glutamic Acid) (PDE), 0.5 ng / μl...

example 2

e Synthesis Method

[0378]Procedures were performed within a sterile, dust free environment (BSL-II hood). Gastight syringes were sterilized with 70% ethanol before rinsing 3 times with filtered nuclease free water, and were stored at 4° C. before use. Surfaces were treated with RNAse inhibitor prior to use.

Nanoparticle Core

[0379]A first solution (an anionic solution) was prepared by combining the appropriate amount of payload (in this case plasmid DNA (EGFP-N1 plasmid) with an aqueous mixture (an ‘anionic polymer composition’) of poly(D-glutamic Acid) and poly(L-glutamic acid). This solution was diluted to the proper volume with 10 mM Tris-HCl at pH 8.5. A second solution (a cationic solution), which was a combination of a ‘cationic polymer composition’ and a ‘cationic polypeptide composition’, was prepared by diluting a concentrated solution containing the appropriate amount of condensing agents to the proper volume with 60 mM HEPES at pH 5.5. In this case, the ‘cationic polymer com...

example 3

cle Uptake

[0386]In these studies (e.g., see FIG. 5), nanoparticles with various surface chemistries and charge ratios were tested. Formulations were (charge ratio refers to the nanoparticle core):

[0387]HTT018B: charge ratio (cations / anions) was 2; surface coat was poly(L-Arginine)

[0388]HTT019B: charge ratio was 5; surface coat was poly(L-Arginine)

[0389]HTT020B: charge ratio was 2, surface coat was N-acetyl Semax

[0390]HTT021B: charge ratio was 5, surface coat was N-acetyl Semax

[0391]HTT022B: charge ratio was 2, surface coat was N-acetyl Selank

[0392]HTT023B: charge ratio was 5, surface coat was N-acetyl Selank

[0393]L3000GFP: lipofectamine (non-nanoparticle) delivery of a nucleic acid encoding GFP (plasmid encoding GFP)

[0394]L3000CRISPR: lipofectamine (non-nanoparticle) delivery of CRISPR / Cas components with no GFP or fluorescent tag.

[0395]Nanoparticles were generated. For HTT018B-023B, the core components included a nucleic acid payload (CRISPR / Cas encoding nucleic acids: one plasmid ...

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Abstract

Provided are methods and compositions for nanoparticle delivery of payloads (e.g., nucleic acid and / or protein payloads) to cells. In some embodiments, a subject nanoparticle includes a core and a sheddable layer encapsulating the core, where the core includes (i) an anionic polymer composition; (ii) a cationic polymer composition; (iii) a cationic polypeptide composition; and (iv) a nucleic acid and / or protein payload; and where: (a) the anionic polymer composition includes polymers of D-isomers of an anionic amino acid and polymers of L-isomers of an anionic amino acid, and / or (b) the cationic polymer composition comprises polymers of D-isomers of a cationic amino acid and polymers of L-isomers of a cationic amino acid. In some cases, the polymers of D-isomers of an anionic and / or cationic amino acid are present at a ratio, relative to the polymers of L-isomers, in a range of from 10:1 to 1:10.

Description

CROSS-REFERENCE[0001]This application is a continuation of U.S. application Ser. No. 15 / 842,829, filed Dec. 14, 2017, which claims the benefit of U.S. Provisional Patent Application No. 62 / 517,346, filed Jun. 9, 2017, of U.S. Provisional Patent Application No. 62 / 443,567, filed Jan. 6, 2017, of U.S. Provisional Patent Application No. 62 / 443,522, filed Jan. 6, 2017, and of U.S. Provisional Patent Application No. 62 / 434,344, filed Dec. 14, 2016, all of which applications are incorporated herein by reference in their entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE[0002]A Sequence Listing was provided in U.S. application Ser. No. 15 / 842,829 on Dec. 14, 2017, as a text file, “LGDL-004_SeqList_ST25.txt” created on Dec. 14, 2017 and having a size of 128 KB. The contents of the text file are incorporated by reference herein in their entirety.INTRODUCTION[0003]Effective introduction of nucleic acid and / or protein payloads into cells, e.g., for genome editing a...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/85A61K47/68A61K48/00A61K47/54A61K31/7105A61K31/40A61K47/42A61K47/64A61P37/00A61P3/10A61P35/00A61K9/51A61K47/34C12N15/10B82Y5/00C12N15/87A61K9/00C12N15/11
CPCC12N15/102C12N2310/20A61K47/42A61K47/645A61P35/00A61P37/00A61K48/0075A61K47/6807A61K47/34A61K9/0085C12N15/87A61P3/10C12N2310/3517C12N15/85A61K47/549B82Y5/00A61K9/5146A61K48/0041C12N15/111A61K48/0016A61K47/64C12N2310/14A61K9/0019A61K31/7105A61K47/6455A61K31/40C12N2320/32C12N9/22A61K47/6937C12N15/113C07K14/605C07K14/50C07K14/70564C07K7/06C07K19/00
Inventor WATSON, ANDRE RONALDFOSTER, CHRISTIAN
Owner LIGANDAL INC
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