US-20260124245-A1 - METHODS AND COMPOSITIONS FOR USING LEUCINE ZIPPERS FOR CROSSLINKING OF CELLS AND DRUG CARRIERS

Abstract

Non-invasive, in situ forming depots for delivery of a therapeutic agents, containing heterodimerizing, synthetic leucine zippers for physical crosslinking mediated by competition-based dimerization. The heterodimerizing, synthetic leucine zippers form a self-assembling depot of the therapeutic agent at a target site in vivo. A library of such heterodimerizing, synthetic leucine zippers is provided, as well as methods of treating subjects using the same.

Inventors

• Juliane Nguyen
• Natalie Jasiewicz

Assignees

• THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL

Dates

Publication Date

20260507

Application Date

20230728

Claims (20)

1. 1 . A non-invasive, in situ forming depot for delivery of a therapeutic agent, comprising: one or more heterodimerizing, synthetic leucine zippers for physical crosslinking mediated by competition-based dimerization; and a therapeutic agent, wherein the one or more heterodimerizing, synthetic leucine zippers form a self-assembling depot of the therapeutic agent at a target site in vivo.
2. 2 . The non-invasive, in situ forming depot for delivery of a therapeutic agent of claim 1 , wherein the heterodimerizing, synthetic leucine zippers amplify an available binding area at a target site in vivo in a layer-by-layer fashion.
3. 3 . The non-invasive, in situ forming depot for delivery of a therapeutic agent of claim 1 , wherein the therapeutic agent comprises an affinity or attraction to a target site in vivo, optionally comprising a targeting ligand on a surface of the therapeutic agent, to enhance accumulation of the therapeutic agent at the target site.
4. 4 . The non-invasive, in situ forming depot for delivery of a therapeutic agent of claim 1 , wherein the non-invasive, in situ forming depots for delivery of a therapeutic agent achieve significantly enhanced retention and accumulation of the therapeutic agent at the target site, optionally wherein the non-invasive, in situ forming depots, including the therapeutic agent, are configured to persist at the target site for at least about 5 days, about 10 days, about 15 days, about 20 days, about 30 days, about 50 days, about 100 days, or more.
5. 5 . The non-invasive, in situ forming depot for delivery of a therapeutic agent of claim 1 , wherein the therapeutic agent comprises a cell, optionally a stem cell, a vesicle, an extracellular vesicle (EV), a nanoparticle, a microparticle, a small molecule, a biotherapeutic, a biologic, a pharmaceutical, and combinations thereof.
6. 6 . The non-invasive, in situ forming depot for delivery of a therapeutic agent of claim 1 , wherein the one or more heterodimerizing, synthetic leucine zippers are attached to a surface of the therapeutic agent, optionally attached via crosslinking, physical attachment or genetic fusion, optionally a heterobifunctional crosslinker.
7. 7 . (canceled)
8. 8 . (canceled)
9. 9 . The non-invasive, in situ forming depot for delivery of a therapeutic agent of claim 1 , wherein the therapeutic agent comprises a mesenchymal stem cell with a natural ability to migrate to sites of inflammation in vivo.
10. 10 . The non-invasive, in situ forming depot for delivery of a therapeutic agent of claim 1 , wherein the target site is an infarct site, wherein the therapeutic agent comprises a stem cell.
11. 11 . (canceled)
12. 12 . The non-invasive, in situ forming depot for delivery of a therapeutic agent of claim 1 , wherein the non-invasive, in situ forming depots are configured to be sequentially administered to a subject, whereby one or more layers of the in situ forming depots can be formed at a target site, optionally wherein the one or more heterodimerizing, synthetic leucine zippers cause physical multivalent crosslinking, wherein physical multivalent crosslinking causes retention and accumulation of the therapeutic agent at the target site and/or wherein each layer serves as an additional capturing surface for a subsequent dose of a non-invasive, in situ forming depot.
13. 13 . (canceled)
14. 14 . (canceled)
15. 15 . The non-invasive, in situ forming depot for delivery of a therapeutic agent of claim 1 , wherein the non-invasive, in situ forming depots are configured to be sequentially administered to a subject, whereby each sequential administration is configured to provide a different therapeutic agent and/or a different dosage of the therapeutic agent.
16. 16 . (canceled)
17. 17 . The non-invasive, in situ forming depot for delivery of a therapeutic agent of claim 1 , wherein the non-invasive, in situ forming depots are configured for intravenous administration subcutaneous, intramuscular, local, intracranial and/or intraarterial administration.
18. 18 . The non-invasive, in situ forming depot for delivery of a therapeutic agent of claim 1 , wherein multivalent crosslinking of the one or more leucine zippers increases resistance to venous washout, allowing the therapeutic agent to persist at the target site long term.
19. 19 . (canceled)
20. 20 . The non-invasive, in situ forming depot for delivery of a therapeutic agent of claim 1 , wherein the non-invasive, in situ forming depots, including the therapeutic agent, are configured to deposit at the target site at a concentration of at least about 2 times, about 5 times, about 10 times, about 20 times, about 50 times, or more, as compared to a therapeutic agent without the non-invasive, in situ forming depots.

Description

PRIORITY CLAIM The present application claims the benefit of U.S. Provisional Patent Application Ser. No. 63/393,417, filed Jul. 29, 2022, the disclosure of which is incorporated herein by reference in its entirety. GRANT STATEMENT This invention was made with government support under Grant Number EB023262 and HL161456 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD The subject matter disclosed herein relates generally to methods and compositions for using leucine zippers for crosslinking of cells and drug carriers. More particularly, the subject matter disclosed herein relates to compositions, formulations and methods for treating a condition using a leucine zipper therapeutic. REFERENCE TO SEQUENCE LISTING XML SUBMITTED ELECTRONICALLY The content of the Sequence Listing XML filed using Patent Center as an XML file (Name: 4210_0525WO 7-28-23-12.xml; Size: 16.992 bytes; and Date of Creation: Jul. 28, 2023) is incorporated herein by reference in its entirety. BACKGROUND Myocardial infarction remains a leading cause of death worldwide. Although the current standard of care for patients focuses on restoring perfusion as quickly as possible with percutaneous coronary interventions, patients are often left with dysregulated endogenous infarct repair and chronic severe morbidities, including ventricular arrhythmias and heart failure due to insufficient cardiac contractile function. The drug delivery and regenerative medicine fields have focused on cell delivery, particularly mesenchymal stem cell delivery, to combat these downstream effects due to their regenerative abilities. Despite these efficacious tools, delivery to the target site after intravenous injection remains highly ineffective, and traditional cell and other therapeutic interventions often require invasive intramyocardial injection or implantation. These invasive injections or implantations come with a variety of mechanical and biological risks such as arrhythmia, induction of fibrosis, and elevated serum myocardial biomarkers, indicative of cardiomyocyte death. Even with local delivery, promising in vitro MSC therapies, extracellular vesicle (EV) therapies, and other therapies are often plagued by poor therapeutic accumulation and retention at the infarct site of interest. Rapid cellular clearing diminishes effective doses and therefore potential therapeutic efficacy. Thus, there remains an urgent need for non-invasive approaches that enhance the accumulation and retention of cell-based therapeutics, drug carriers, delivery vehicles, nanocarriers, therapeutics, biologics, nucleic acids, etc. at the disease site. To address this gap, efforts were undertaken to develop a novel delivery strategy to enhance the accumulation and retention of cells in the injured myocardium. The goal is to create a modular platform of cells, drug carriers, delivery vehicles, nanocarriers, therapeutics, biologics, nucleic acids, etc. that crosslink to a scaffold at the infarct site or in the injured myocardium to serve as a drug depot. In some embodiments, the design criteria for the scaffold include, but are not limited to, (1) enhanced accumulation at the infarct site/injured myocardium, (2) prolonged retention, and/or (3) in case of drug complications or immunogenic reactions, the possibility to dissolve the scaffold into its individual subunits for elimination. SUMMARY This summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely an example of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise for purposes of example. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this summary does not list or suggest all possible combinations of such features. In one aspect, a non-invasive, in situ forming depot for delivery of a therapeutic agent, includes one or more heterodimerizing, synthetic leucine zippers for physical crosslinking mediated by competition-based dimerization, and a therapeutic agent, where the one or more heterodimerizing, synthetic leucine zippers form a self-assembling depot of the therapeutic agent at a target site in vivo. The non-invasive, in situ forming depot for delivery of a therapeutic agent may also include where the heterodimerizing, synthetic leucine zippers amplify an available binding area at a target site in vivo in a layer-by-layer fashion. In some aspects, the therapeutic agent includes an affinity or attraction to a target site in vivo, optionally includes a targeting ligand on a surface of the therapeutic agent, to enhance accumulation of the therapeutic agent at the target site. In some aspec