US-12622980-B2 - Nucleic acid loaded flowable hydrogels and compositions, systems and methods related thereto

US12622980B2US 12622980 B2US12622980 B2US 12622980B2US-12622980-B2

Abstract

The present disclosure provides, in part, nucleic acid loaded flowable hydrogels and compositions, systems and methods related thereto, to effectively deliver nucleic acids to cells that contact the flowable hydrogels.

Inventors

Tatiana Segura
Evan Kurt

Assignees

DUKE UNIVERSITY

Dates

Publication Date: 20260512
Application Date: 20200715

Claims (20)

1 . A scaffold comprising hydrogel particles, wherein the hydrogel particles comprise an irregular shape and comprise one or more polyplexes, and wherein the polyplexes comprise one or more copies of one or more nucleic acids and one or more nucleic acid complexing agents.
2 . The scaffold of claim 1 , wherein one or more hydrogel particles originate from a bulk hydrogel and one or more particles are discretely polymerized.
3 . The scaffold of claim 2 , wherein the polyplexes are substantially evenly distributed throughout the hydrogel particles that originate from a bulk hydrogel.
4 . The scaffold of claim 1 , wherein the irregularly shaped hydrogel particles comprise shred particles.
5 . The scaffold of claim 1 , wherein one or more hydrogel particles are devoid of nucleic acid and nucleic acid complexing agent (polyplexes), with the proviso that not all hydrogel particles are devoid of nucleic acid and nucleic acid complexing agent.
6 . The scaffold of claim 4 , wherein the hydrogel particles comprising an irregular shape comprises an average surface area of between about 100 μm 2 and 1000000 μm 2 , or between about 500 μm 2 and 500000 μm 2 , or between about 1000 μm 2 and 250000 μm 2 , or between about 2500 μm 2 and 100000 μm 2 , or between about 5000 μm 2 and 50000 μm 2 , or between about 7500 μm 2 and 40000 μm 2 , or between about 10000 μm 2 and 25000 μm 2 .
7 . The scaffold of claim 6 , wherein two or more hydrogel particles are annealed together, and wherein annealed together comprises covalent, electrostatic, hydrophobic, mechanical and transamination annealing.
8 . The scaffold of claim 1 , wherein the nucleic acid complexing agent comprises a cationic polymer, cationic peptide, cationic lipid, or mixture thereof.
9 . The scaffold of claim 1 , wherein the nucleic acid and the nucleic acid complexing agent are mixed at a N/P ratio, and wherein the N/P ratio comprises between about 1 and 100, or between about 1 and 90, or between about 2 and 75, or between about 3 and 65, or between about 4 and 50.
10 . The scaffold of claim 9 , wherein when the nucleic acid is DNA and the complexing agent is PEI, the N/P ratio comprises between about 5 and 35, or between about 15 and 25, or about 20.
11 . The scaffold of claim 1 , wherein the polyplexes further comprise a coating layer, wherein the coating layer comprises one or more coating layer agents, and wherein the coating layer agents comprise one or more biocompatible polymers, one or more mineral salts, and mixtures thereof.
12 . The scaffold of claim 1 , wherein the nucleic acid comprises between about 0.1 mg/ml nucleic acid and 20 mg/ml nucleic acid, or between about 0.2 mg/ml nucleic acid and 18 mg/ml nucleic acid, or between about 0.25 mg/ml nucleic acid and 16 mg/ml nucleic acid, or between about 0.3 mg/ml nucleic acid and 14 mg/ml nucleic acid, or between about 0.4 mg/ml nucleic acid and 12 mg/ml nucleic acid, or between about 0.5 mg/ml nucleic acid and 10 mg/ml nucleic acid, or between about 0.5 mg/ml nucleic acid and 8 mg/ml nucleic acid, or between about 0.5 mg/ml nucleic acid and 7 mg/ml nucleic acid, or between about 0.5 mg/ml nucleic acid and 6 mg/ml nucleic acid, or between about 0.5 mg/ml nucleic acid and 5 mg/ml nucleic acid, or between about 0.6 mg/ml nucleic acid and 6 mg/ml nucleic acid, or between about 0.7 mg/ml nucleic acid and 7 mg/ml nucleic acid, or between about 0.8 mg/ml nucleic acid and 8 mg/ml nucleic acid, or between about 0.9 mg/ml nucleic acid and 9 mg/ml nucleic acid, or between about 1 mg/ml nucleic acid and 10 mg/ml nucleic acid, or between about 1 mg/ml nucleic acid and 7.5 mg/ml nucleic acid, or between about 1 mg/ml nucleic acid and 5 mg/ml nucleic acid.
13 . A structure for localized and controlled release of nucleic acids, comprising: a scaffold comprising hydrogel particles, wherein the hydrogel particles comprise an irregular shape and comprise one or more polyplexes, and wherein the one or more polyplexes comprise one or more copies of one or more nucleic acids and one or more nucleic acid complexing agents; wherein the one or more nucleic acids is present at a total concentration of at least about 0.1 mg/ml, and further wherein the scaffold is characterized in that, when the structure is placed in contact with one or more cells so that the scaffold contacts the cells, the nucleic acid is released with a profile characterized by one or more of: a) a burst-free release; b) a sustained release; and c) exhibiting in vitro and/or in vivo biological effectiveness.
14 . The structure of claim 13 , wherein one or more hydrogel particles are devoid of nucleic acids and nucleic acid complexing agents, with the proviso that not all hydrogel particles are devoid of nucleic acid and nucleic acid complexing agent.
15 . The structure of claim 13 , wherein the concentration of the nucleic acid is at least about 0.1 mg/ml, about 0.2 mg/ml, about 0.3 mg/ml, about 0.4 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml, about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1 mg/ml, about 1.5 mg/ml, about 2 mg/ml, about 2.5 mg/ml, about 3 mg/ml, about 3.5 mg/ml, about 4 mg/ml, about 4.5 mg/ml, about 5 mg/ml, about 5.5 mg/ml, about 6 mg/ml, about 6.5 mg/ml, about 7 mg/ml, about 7.5 mg/ml, about 8 mg/ml, about 8.5 mg/ml, about 9 mg/ml, about 9.5 mg/ml, about 10 mg/ml, about 11 mg/ml, about 12 mg/ml, about 13 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 17 mg/ml, about 18 mg/ml, about 19 mg/ml, about 20 mg/ml.
16 . The structure of claim 13 , wherein the burst-free release is characterized by releasing less than about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50% of the nucleic acid agent in the first 24 hours after placement on the subject.
17 . The structure of claim 13 , wherein the burst-free release is characterized by the release of nucleic acids originating from only those particles in contact with the one or more cells.
18 . The structure of claim 13 , wherein the sustained release is characterized by the nucleic acid being released from the scaffold over an extended period of time of at least about 3 days, about 5 days, about 7 days, about 10 days, about 15 days, about 30 days, about 1 month, about 2 months, about 3 months, about 6 months, or about 1 year.
19 . The structure of claim 13 , wherein two or more hydrogel particles are annealed together, and wherein annealed together comprises covalent, electrostatic, hydrophobic, mechanical and transamination annealing.
20 . The structure of claim 13 , wherein the irregularly shaped hydrogel particles comprise shred particles.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a national phase application of International Application No. PCT/US2020/042131 filed Jul. 15, 2020, which claims the benefit under 35 U.S.C. § 119 of the U.S. Provisional Patent Application No. 62/874,074, filed Jul. 15, 2019, which applications are hereby incorporated by reference in their entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Federal Grant No. R01NS094599 awarded by the National Institutes of Health. The Federal Government has certain rights to this invention. INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY This application is being filed electronically via EFS-Web and includes an electronically submitted Sequence Listing in .txt format. The .txt file contains a sequence listing titled “20-1056-WO_SEQ-LISTING_ST25.txt” created on Jul. 19, 2022 and is 2 kilobytes in size. The Sequence Listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety. FIELD The present disclosure relates to nucleic acid loaded flowable hydrogels and compositions, systems and methods related thereto, to effectively deliver nucleic acids to cells that contact or infiltrate the flowable hydrogels. BACKGROUND Nucleic acid delivery has the potential to up-regulate and/or down-regulate any gene known to have bioactivity in humans to impact human health, introduce transgenes encoding recombinant proteins not otherwise present in a cell, or that encode the native bioactive signal, and more. Nucleic acid delivery has applications ranging from tissue engineering to vaccine development to infectious disease. This goal has driven the field of gene therapy for over two decades. A key goal of nucleic acid delivery is to achieve effective delivery into human cells. Most approaches to deliver nucleic material involve the systemic intravenous delivery of condensed nucleic acid (e.g., within a virus or a synthetic particle). However, this approach suffers from immune recognition, accumulation in first pass organs, and rapid clearance. This includes the FDA approved gene therapies onasemnogene abeparvovec (AAV injected intravenously) and patisiran (siRNA lipoplex delivered intravenously). To circumvent these limitations, local delivery approaches that inject and retain the nucleic acid cargo at desired locations are beneficial. For example, the FDA approved gene therapy voretigene neparvovec delivers AAV directly into the eye by subretinal injection, bypassing immune recognition, first pass organ accumulation, and rapid clearance. While the eye is an enclosed environment and has low risk for gene therapies diffusing to the rest of the body, this is not the case for most organs, which requires an alternative delivery method. SUMMARY The Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter. Accordingly, one aspect of the present disclosure provides a scaffold comprising hydrogel particles, wherein the particles comprise one or more polyplexes, which polyplexes comprise one or more copies of one or more nucleic acids and one or more nucleic acid complexing agents. In some embodiments, one or more particles originates from a bulk hydrogel, and in some embodiments, one or more particles can also be discretely polymerized, and mixtures thereof. In some embodiments, polyplexes are substantially evenly distributed throughout particles that originate from a bulk hydrogel, and in some embodiments, at least one particle comprises an irregular shape. In some embodiments, particles comprise regular and irregular shapes. In some embodiments, the scaffold comprises one or more particles devoid of nucleic acid, nucleic acid complexing agent and/or polyplexes, with the proviso that not all particles are devoid of nucleic acid, nucleic acid complexing agent and/or polyplexes. In some embodiments, the scaffold comprises particles of two or more types. In some embodiments, particles comprise an irregular shape with an average surface area that can range from about 100 μm2 and 1000000 μm2, as well as other size ranges. In some embodiments, the irregularly shaped particles comprise shred particles. In some embodiments, two or more particles are annealed together, wherein annealed together comprises covalent, electrostatic, hydrophobic, mechanical and transamination annealing. In some embodiments, nucleic acids comprise DNA and RNA, and in some embodiments, the nucleic acid complexing agent comprises a cationic polymer, cationic peptide, cationic lipid, or mixtures. In some embodiments, the nucleic acid and the nucleic acid complexing agent are mixed at a N/P ratio, which ratio comp