Patches for targeted delivery of carcinogenic cargo to tissues

Abstract

Disclosed is a patch configured to hold a carcinogenic cargo and deliver the cargo in a controlled manner to a predetermined location in a particular tissue. As an example, the patch can include a base layer configured to adhere to the predetermined location in the tissue and a hydrogel layer. The hydrogel layer can include a plurality of microchannels configured to store and release the genetically modified vector for a time and in an amount sufficient to generate a somatic tumor by manipulating the genome of the somatic cell at the predetermined location.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 306,449, filed February 3, 2022, entitled "Patch for Targeted Delivery of Carcinogenic Cargo to Tissue," which is incorporated herein by reference in its entirety.

[0002] Technical Field The present disclosure relates to devices, which may be in the form of patches, used for the in vivo delivery of physiologically active cargo to tissue.

[0003] Government support approval This invention was made with government support under CBET-0939511 awarded by the National Science Foundation. The government has certain rights in this invention. [Background technology]

[0004] In the development of drugs and medical devices for human use, a non-human animal or "preclinical" development stage must precede the clinical trial stage conducted in humans. Preclinical development therefore depends on the identification of suitable non-human animals that can model the potential effects of a drug or medical device before clinical trials can proceed. Identifying appropriate and predictive non-human animal models is critical for effectively and efficiently bringing new drugs and medical devices to market for the benefit of human health and well-being.

[0005] Appropriate animal models can be useful for evaluating the clinical feasibility, efficacy, safety, and comparative advantages and disadvantages of new drugs or medical devices prior to or as an adjunct to clinical trials on human subjects. The goal of preclinical testing of new drugs may be to determine the starting dose for first-in-human studies and / or to evaluate the product's potential toxicity. For medical devices, preclinical testing models can evaluate the device's practicality and potential limitations to its use. Furthermore, for medical diagnostics, preclinical models can provide a platform for testing or studying liquid biopsy assays or various imaging modalities.

[0006] As an example, oncolytic drugs and oncolytic medical devices can be studied using preclinical models (e.g., non-human animal models) of certain disease states, such as cancer. Well-established preclinical research typically relies on small experimental animal models (e.g., mice, rats, and rabbits), despite the many shortcomings of such animal models. These small experimental animal models can be flawed predictors of the performance and / or toxicity of medicines or medical devices in humans due to substantial differences from humans in anatomy, physiology, genetics, and metabolism.

[0007] Some recently developed preclinical models (e.g., particularly porcine models) are more homologous to humans and have similar tumor phenotypes. Such models have been shown to be better predictors of both the safety and efficacy of human drugs and medical devices. In the development of preclinical models for the treatment of human diseases, such as various human cancers, a significant challenge has been the lack of so-called autochthonous animal models that can recapitulate the clinical carcinogenesis process of tumor initiation at the site of tumor formation in specific tissues. Preclinical research on oncolytic drugs and oncolytic medical devices can rely on the use of transplantation models in which tumor-containing cells are implanted into test animals to study the effects of drugs or medical devices that may be useful in treating the tumor in question.

[0008] Thus, there is a need for more reliable and efficient methods of generating autostimulatory tumors in preclinical animal models so that preclinical research on drugs and medical devices can proceed with greater predictability of potential clinical outcomes from the use of such drugs and devices. Summary of the Invention

[0009] Disclosed herein are devices and methods for their use that enable targeted in vivo delivery of physiologically active cargoes (e.g., oncogenic gene-modifying vectors) to animal tissues. The devices disclosed herein include a carcinogenic cargo-containing matrix, which may be configured in the form of a patch that retains the carcinogenic cargo prior to its delivery to the tissue, e.g., for a period of time that may be predetermined. Accordingly, the devices disclosed herein include carcinogenic patches that can then be placed in vivo, e.g., in contact with a selected tissue at a predetermined location, to deliver the carcinogenic cargo. In this regard, the device may be surgically (or non-invasively, e.g., via an endoscope or needle) implanted to contact the selected tissue for delivery of the carcinogenic cargo at the predetermined site. More specifically, patches are disclosed that include a hydrogel having a plurality of microchannels configured to retain the carcinogenic cargo prior to delivery to the selected tissue. In particular, carcinogenic patches are disclosed that include an additional layer, e.g., a polydopamine adhesive layer, that can adhere the patch to the selected tissue. Hydrogel patches containing bioactive cargo in the form of oncogenic gene-modifying vectors can deliver carcinogens, viral vectors, gene-editing components, or combinations thereof, capable of inducing tumor growth in cells of selected tissues. In this manner, the patches and methods described herein can be utilized to initiate autologous tumor formation and subsequently induce tumor growth at selected sites in target tissues in intact animals. Construction and implantation of the cargo-carrying device thereby provides a preclinical animal model of tissue- and organ-specific tumor formation that can be used to predict the potential safety, efficacy, advantages, limitations, or other effects of pharmaceuticals and devices of potential clinical value in human subjects.

[0010] In one example, the patch may include a base layer configured to adhere to a predetermined location on tissue, and a hydrogel layer with a plurality of microchannels configured to store and release genetically modified vectors for a time and in an amount sufficient to generate a somatic tumor at the predetermined location via manipulation of the genome of somatic cells.

[0011] In another example, a method for generating a somatic tumor at a predetermined location in a tissue can include positioning at the predetermined location a patch comprising a base layer configured to adhere to the predetermined location and a hydrogel layer comprising a plurality of microchannels containing a releasable carcinogenic cargo stored within the plurality of microchannels, whereby the carcinogenic cargo is released from the hydrogel layer to generate a somatic tumor at the predetermined location via manipulation of the genome of a somatic cell.

[0012] In another example, a method of making a hydrogel patch containing microchannels can include forming a hydrogel by crosslinking gel-forming molecules in a crosslinking solution, adhering a base layer configured to adhere to tissue to a surface of the hydrogel, lyophilizing the hydrogel under conditions sufficient to produce a plurality of microchannels such that a cryogel having a plurality of microchannels is formed, and rehydrating the cryogel in a solution containing a selected cargo.

[0013] In another example, a method of making a hydrogel containing microchannels can include mixing a flexible cross-linked hydrogel with a solution containing a cargo, lyophilizing the flexible cross-linked hydrogel and the cargo under conditions sufficient to produce a plurality of microchannels in the hydrogel containing the cargo, and adhering a base layer configured to adhere to tissue to a surface of the hydrogel.

[0014] The above and other features of the present disclosure will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1A is a schematic diagram of an exemplary patch configured to hold and controllably release cargo.

[0016] FIG. 1B is a schematic diagram of the exemplary patch of FIG. 1A being positioned on tissue.

[0017] [Figure 2] FIG. 1 is a schematic diagram of an exemplary patch comprising a flexible cross-linked hydrogel and a polydopamine base layer.

[0018] [Figure 3] FIG. 3A shows an exemplary patch comprising alginate and polydopamine hydrogel with multiple microchannels or pores configured to be loaded with designated cargo.

[0019] FIG. 3B is a microscopic image of a portion of the patch of FIG. 3A, showing the pores in the patch.

[0020] [Figure 4] FIG. 4A shows the patch of FIG. 3A in a folded state.

[0021] FIG. 4B shows the patch of FIG. 3A in an unfolded state.

[0022] [Figure 5] FIG. 3B shows the patch of FIG. 3A adhered to tissue via its polydopamine base layer.

[0023] [Figure 6] FIG. 1 is a flowchart of a method for fabricating a patch comprising a flexible cross-linked hydrogel having a plurality of microchannels and a polydopamine-based layer, wherein the microchannels are configured to retain a selected cargo.

[0024] [Figure 7] FIG. 10 is a flowchart of another method for fabricating a patch comprising a flexible cross-linked hydrogel having a plurality of microchannels and a polydopamine-based layer, wherein the microchannels are configured to retain a selected cargo.

[0025] [Figure 8] FIG. 8A is a front view of an exemplary patch configured as a disk.

[0026] FIG. 8B is a side view of the patch of FIG. 8A.

[0027] [Figure 9] 8B shows the patch of FIG. 8A at various stages of deployment, thereby demonstrating its ability to be transformed into a tubular shape and then subjected to a deployment action.

[0028] [Figure 10] 10A-10C are schematic diagrams depicting an exemplary method for delivering a patch to a target tissue using an endoscope.

[0029] [Figure 11] 1 is a flowchart of an exemplary method for inducing tumor formation at a predetermined location in tissue using a patch.

[0030] [Figure 12] FIG. 1 presents microscopic images of three different sized patches and green fluorescent protein (GFP) expression in each of the patches after placement on the bladder epithelium.

[0031] [Figure 13] FIG. 1 presents brightfield images of tissues exposed to AdCre via patch and control patch, as well as images showing AdCre-GFP expression in tissues exposed to patch and control.

[0032] [Figure 14]FIG. 10 presents microscopic images showing tumor formation around the implanted patches compared to controls.

[0033] [Figure 15] FIG. 1 presents microscopic images of GFP expression in tissue exposed to patches containing AdCre and control patches containing only GFP.

[0034] [Figure 16] Figures 16A-16F are a series of panels illustrating the development of the Oncopig lung adenocarcinoma cell line. Figure 16A is a series of panels illustrating the development of the Oncopig lung adenocarcinoma cell line, showing images of primary Oncopig type II lung cells. Figure 16B is a series of panels illustrating the development of the Oncopig lung adenocarcinoma cell line, showing that treatment of Oncopig type II lung cells with Ad-Cre results in vacuolization and cell death. Figure 16C is an image showing positive TTF1 staining of the Oncopig lung adenocarcinoma cell line. Figure 16D is a gross image of an ex vivo lung nodule. Figure 16E is an image showing GFP expression in an ex vivo lung nodule. Figure 16F is a histology image showing structures consistent with lung adenocarcinoma.

[0035] [Figure 17] Figures 17A-17C are a series of panels showing the development of rabbit HCC cell lines. Figure 17A is an image showing rabbit primary hepatocytes. Figure 17B is a graph showing the editing rate of rabbit hepatocytes 3 weeks after CRISPR-Cas9 editing. Figure 17C is an image showing positive arginase-1 staining of rabbit TP53 and PTEN knockout cell lines, confirming their hepatocyte origin. DETAILED DESCRIPTION OF THE INVENTION

[0036] Disclosed herein are devices configured to deliver carcinogenic cargo to selected tissues, as well as methods of using such devices, such as preclinical in vivo models of predictive relevance for the potential clinical efficacy or outcome of pharmaceutical, diagnostic, and device use in diagnosing and treating human cancer. In some examples, the device is a carcinogenic patch comprising a flexible cross-linked hydrogel comprising a plurality of microchannels and a base layer attached to one side of the hydrogel and configured to adhere to tissue, e.g., at a predetermined location in the selected tissue. This configuration of the patch allows the patch to retain the cargo within the microchannels. Once adhered to the tissue, the cargo can be controllably released (e.g., over a period of time and at a predetermined rate of diffusion from the carcinogenic patch for delivery to cells of the tissue).

[0037] In some instances, the oncogenic patch may be flexible and may be at least partially folded or partially rolled up for insertion into a delivery device and relatively non-invasive delivery to the target tissue, hi some instances, the delivery device may be an endoscope or another delivery catheter.

[0038] In some examples, the cargo may be a cancer-causing cargo capable of inducing tumor formation, growth, or both, thereby resulting in a selected cancer appearing in the subject. The subject may, in some examples, include animals used as preclinical models (such as large animal models, e.g., pigs) for testing various anti-cancer drugs, medical devices, or diagnostic techniques. In certain examples, the patches and methods described herein may be tailored to effectively and efficiently generate self-generated tumors in preclinical animal models.

[0039] term Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Thus, "comprising A or B" means including A, or B, or A and B.

[0040] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0041] In order to facilitate review of the various examples of the present disclosure, the following explanations of specific terms are provided:

[0042] Cancer: A malignant tumor characterized by abnormal or uncontrolled cell growth. Other features often associated with cancer include metastasis, interference with the normal function of neighboring cells, release of abnormal levels of cytokines or other secretory products and suppressed or exacerbated inflammatory or immune responses, and infiltration of surrounding or distant tissues or organs, such as lymph nodes. "Metastatic disease" refers to cancer cells that have left the original tumor site and traveled to other parts of the body, for example, via the bloodstream or lymphatic system.

[0043] Cargo: One or more substances, such as viral vectors, genetically modified components, carcinogenic nanoparticles, carcinogens, or other substances that can be contained or incorporated into a patch (e.g., within the microchannels of the patch) and that can produce or treat disease at the site of implantation (such as a tissue). The cargo can be a carcinogenic cargo, i.e., a cargo that includes one or more bioactive substances that induce tumor formation or tumor growth, or both, in the tissue to which the cargo is delivered.

[0044] Carcinogenic: A molecule, material, or device that causes the development of one or more tumors.

[0045] OncoPig: An inducible pig cancer model called "OncoPig" or "OncoPig Cancer Model" (OCM). This pig cancer model uses the KRAS G12D and TP53 R167H Oncopigs are transgenic pig lines encoding a Cre recombinase-inducible porcine transgene encoding AdCre (see, e.g., Schook et al., PLOS One 10:e0128864, 2015). Cells derived from Oncopigs can be transformed in culture with an adenovirus encoding Cre (AdCre). Furthermore, injection of AdCre into transgenic pigs results in tumor formation.

[0046] Pharmaceutically acceptable carriers: Remington: The Science and Practice of Pharmacy, 22nd ed. London, UK: Pharmaceutical Press (2013) describes compositions and formulations suitable for pharmaceutical delivery of cargo or other compositions disclosed herein. Generally, the nature of the carrier will depend on the particular mode of administration being employed. For example, formulations for inclusion in the patches described herein can contain pharmaceutically and physiologically acceptable fluids as vehicles, such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, and the like. In addition to biologically neutral carriers, compositions administered or included in patches can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, e.g., sodium acetate or sorbitan monolaurate.

[0047] Subject: A category that includes living multi-cellular vertebrate organisms, humans, veterinary, and experimental subjects, including human and non-human mammals (mice, rats, rabbits, pigs, etc.).

[0048] Tumor: The product of neoplasia is a neoplasm (tumor), which is an abnormal growth of tissue resulting from excessive cell division. Tumors that do not metastasize are called "benign." Tumors that can invade surrounding tissues and / or metastasize are called "malignant" or "cancer." Neoplasia is an example of a proliferative disorder. "Autologous" tumors are tumors that arise in normal cells and in whole organisms (such as mice or pigs) and are thought to more closely mimic human tumors than models that utilize implanted or injected tumors or tumor cells (such as xenografts).

[0049] Patch: A hydrogel configured to adhere to tissue and carry a cargo within it. The patch is configured to be delivered to a target location in a selected tissue and then release its cargo over a predetermined period of time upon contact with the tissue. "Carcinogenic patch" refers to a patch containing a carcinogenic cargo.

[0050] Vector: A nucleic acid molecule that can be introduced into a host cell, thereby producing a transformed host cell. A recombinant DNA vector is a vector that contains recombinant DNA. A vector can contain a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. A vector can also contain one or more selectable marker genes and other genetic elements known in the art. A viral vector is a recombinant nucleic acid vector that contains at least some nucleic acid sequences derived from one or more viruses. A replication-deficient viral vector is a vector that, due to a deficiency in at least one replication-essential gene function, requires complementation of one or more regions of the viral genome required for replication.

[0051] Patch Device Description As outlined above, there is a need for preclinical models of various disease states, such as cancer, that are relatively inexpensive, amenable to high-throughput screening, and closely resemble the disease state presented in humans. As one example, it is desirable to recapitulate the complexity of specific human tumors within a preclinical in vivo (e.g., animal) model. While direct implantation or injection of cancer cells into selected preclinical animal models can result in tumor formation, such methods may not allow precise control of the site of tumor formation within the target tissue and / or may result in an uncontrollably rapid disease course. As a result, the applicability of such models for preclinical medicine, medical devices, and diagnostic testing may be limited.

[0052] The limited control of tumor location and size in preclinical models can be particularly problematic for preclinical models of luminal cancers such as bladder, esophageal, gastric, pancreatic, and colorectal cancer.

[0053] Thus, the present inventors have recognized that a minimally invasive delivery approach that allows for more controlled localization and release of tumor-inducing compositions may induce the formation of various cancers (such as those of the hollow organs described above) in more controlled and defined locations. Furthermore, the approaches provided herein may also be applied to non-hollow organs or other tissue types.

[0054] A patch configured to carry a cargo (which in some examples may include tumor-inducing materials or particles, as further described herein) can be configured to adhere to a target tissue and provide controlled, sustained delivery of the cargo at the target tissue. In some examples, the delivered cargo can induce tumor formation or growth in the target tissue (e.g., at or near the site of the carcinogenic patch). In this manner, the patch with its cargo can induce tumor formation or growth at a defined location and growth rate within the target tissue in vivo (e.g., in an animal model). The patch can be further configured to degrade after delivery of the cargo at the target tissue. In some examples, such a patch can be flexible so that it can be delivered endoscopically or via another minimally invasive targeted delivery method. As further described herein, this type of delivery approach can enable patch delivery procedures to be performed using existing veterinary, biomedical, and clinical facilities and technology.

[0055] 1A and 1B show an exemplary patch 100 configured to retain and controllably release cargo 102 ( FIG. 1A ) and adhere to tissue 104 ( FIG. 1B ). Patch 100 may also be referred to as a tumor-inducing patch (TIP) or tumor-modulating patch (TMP) (e.g., when cargo 102 includes an oncogenic viral vector, oncogenic nanoparticle, and / or carcinogen capable of inducing tumor formation or growth). In some examples, patch 100 may comprise a flexible cross-linked hydrogel configured to store cargo 102 (within microchannels or pores formed in the hydrogel, as further described below with reference to FIG. 2 ) and then controllably release cargo 102 ( FIG. 1A ) after implantation into tissue 104 for a predetermined period of time.

[0056] 1B, patch 100 can be delivered to a lumen 106 (e.g., an inner luminal surface) of tissue 104. For example, tissue 104 can be part of a hollow organ such as the bladder, esophagus, colon, or nerve. In other examples, tissue 104 can be part of a solid organ such as a lung, liver, heart, kidney, or bone. In some examples, tissue 104 can be the outer surface of tissue of an organ such as an ovary, neuron, or brain. Thus, patch 100 is flexible and can be configured to bend and conform to the curved lumen 106 of tissue 104 (or the curved, irregular, or convex outer surface of a solid organ).

[0057] FIG. 2 is a schematic diagram of an exemplary patch 200 that may be similar to patch 100 of FIGS. 1A and 1B and can be used in the same manner as described above with reference to FIGS. 1A and 1B. As introduced above, patch 200 can include a flexible cross-linked hydrogel 202. Hydrogel 202 can include gel-forming biocompatible molecules. In some examples, hydrogel 202 is an alginate and divalent hydrazide group cross-linked hydrogel. In other examples, hydrogel 202 can include alginate, hyaluronic acid, chitosan and derivatives thereof, poly(ethylene glycol) (PEG) diacrylate, PEG dimethacrylate, collagen, gelatin, gelatin methacrylate, poly(lactic-co-glycolic acid), or a combination thereof.

[0058] Hydrogel 202 can be configured to degrade after contact with tissue (e.g., tissue 104 in FIG. 1B) for a predetermined period of time. In some examples, patch 200 can degrade at a rate that supports tumor growth without interfering with the environment for tumor growth and while reducing or avoiding foreign body reactions (such as an undesirable immune response to the patch). In some examples, this predetermined period ranges from 3 to 5 weeks. For example, hydrogel 202 can include one or more biodegradable materials. The predetermined period for degradation of patch 200 can be tailored by adjusting the composition of hydrogel 202 (e.g., by selecting from one or more of the gel-forming molecules listed above to achieve the predetermined time for degradation).

[0059] The hydrogel 202 of the patch 200 can include a plurality of pores or microchannels 206 configured to receive a cargo 208 therein. For example, the patch 200 can be configured to house the cargo 208 within the lumina of the plurality of microchannels 206 (as shown in FIG. 2 ) and, upon contact with tissue, continuously release the cargo from the microchannels 206 over a predetermined period of time (which may be different from the predetermined period of time for degradation of the hydrogel 202). In this manner, the microchannels 206 can be configured to slowly diffuse the cargo 208 out of the microchannels 206 over a predetermined period of time and / or at a set diffusion rate.

[0060] In some examples, the multiple microchannels 206 can be anisotropically aligned with one another within the hydrogel 202. Furthermore, in some examples, the microchannels 206 (or pores) can have an opening width or diameter in the range of 50-1000 μm (e.g., 50-100 μm, 100-300 μm, 200-400 μm, 300-500 μm, 400-600 μm, 500-700 μm, 600-800 μm, 700-900 μm, or 800-1000 μm). In one example, the opening width or diameter is in the range of 200-300 μm. For example, while the microchannels 206 are shown in FIG. 2 as having rectangular cross-sections (for ease of illustration), in other examples, the microchannels 206 can have circular or rectangular cross-sections with diameters or major axes in the range of 50-1000 μm. The width or diameter of the microchannel 206 can be selected to achieve a desired diffusion rate or rate of release of the cargo 208 from the microchannel 206. For example, smaller pores or diameter of the microchannel 206 can result in a slower diffusion rate and a longer time for complete release of the cargo 208. In contrast, as the pore size or diameter of the microchannel 206 increases, the diffusion rate of the cargo can decrease. The formation of the microchannel 206 within the hydrogel 202 is further described below with reference to FIGS. 6 and 7.

[0061] Furthermore, in some examples, by adjusting the tortuosity, charge density, pore size, or a combination thereof, of the microchannels 206, different diffusion or release rates of the cargo 208 from the microchannels 206 can be achieved. In some examples, multiple patch layers of hydrogel 202 can be 3D printed with different pore sizes and compositions to enable release of different cargoes at different rates or times. Furthermore, in some examples, cargo-containing microparticles can be included within the patch 200, which allows for slower release of the cargo. Thus, using these approaches, multiple cargoes can be loaded into the patch 200 and delivered at different rates.

[0062] In some examples, the portion of the patch 200 that includes the microchannels 206 may be referred to herein as the cargo layer 214 of the patch 200 .

[0063] The patch 200 can further include a base layer 204 configured to adhere to tissue. In some examples, as shown in FIG. 2, the base layer 204 can be attached to one side of the hydrogel 202 and configured to adhere to tissue (e.g., tissue 104 in FIG. 1B). The base layer 204 can act as an adhesive, allowing the patch to adhere to tissue (e.g., the lumen lining of a tissue, as shown in FIG. 1B) until the patch 200 delivers its cargo and then dissolves or degrades. The base layer 204 can include fibrin or polydopamine (e.g., a fibrin or polydopamine-based tissue adhesive or adhesive layer).

[0064] In some examples, patch 200 can have a thickness 210 in the range of 0.05-5 mm, a length 212 in the range of 1-10 mm, and a width (perpendicular to length 212 and thickness 210 and in the direction shown on that page of FIG. 2) in the range of 1-10 mm. As further described below, the dimensions of some exemplary patches are shown in Table 1. The dimensions of patch 200 can be selected and varied based on a predetermined location in a target organ for implantation. In some examples, the dimensions of patch 200 can additionally or alternatively be selected based on a specified tumor size and growth rate.

[0065] 3A-5 show images of an exemplary patch 300 loaded with a selected cargo (e.g., a cancer-causing or tumor-inducing cargo, as described further below) and configured to release the cargo after adhering the patch 300 to tissue 302 (FIG. 5). In some examples, the patch 300 with its cargo can be configured to support the timely development (e.g., within a predetermined period of time) of site- and cell-specific solid tumors representing clinically relevant size and biological characteristics in a particular animal model (e.g., pig or other large animal).

[0066] Figure 3A shows an exemplary patch 300 comprising alginate and polydopamine hydrogel, and Figure 3B is a microscopic image of a portion of patch 300 showing pores 304 in patch 300. In an alternative example, patch 300 can include microchannels instead of pores 304, which are typically configured as open-ended channels that can be anisotropically aligned with one another within patch 300.

[0067] 4A shows patch 300 in a folded state or configuration, and FIG. 4B shows patch 300 in an unfolded state or configuration. Patch 300 can be repeatedly folded and unfolded as needed (e.g., during storage and / or delivery to tissue 302) due to its flexible hydrogel structure and other geometric properties described herein (e.g., its thickness).

[0068] As shown in FIG. 5, the patch 300 can include a base layer (e.g., base layer 204 of FIG. 2) that enables it to adhere (e.g., stick and remain attached) to the tissue 302. The patch 300 can be configured to remain adhered to the tissue 302 even under stretching or tension on the patch 300 (e.g., due to movement of the organ of which the tissue 302 is a part). Furthermore, the patch 300 can be configured to remain adhered to the tissue 302 until it has delivered its cargo and degraded. After implantation of the patch 300 and adhesion to the tissue 302, as shown in FIG. 5, the patch 300 can controllably and locally release the cargo disposed within the microchannels 304 via the microchannels 304 and degrade over a predetermined period of time without causing host inflammation.

[0069] The patches described herein can be constructed by forming a cross-linked hydrogel and lyophilizing the cross-linked hydrogel under conditions sufficient to generate a plurality of microchannels. Cargo to be delivered to tissue through the patch can be introduced into the hydrogel after formation of the microchannels (Figure 6), through a rehydration process, or by mixing the cargo with one or more components of the hydrogel before or after cross-linking (Figure 7).

[0070] As an example, Figure 6 presents a method 400 for making a patch, such as any one of the patches described herein. Method 400 begins at 402 by forming a hydrogel by crosslinking one or more gel-forming molecules in a crosslinking solution. As introduced above, the gel-forming molecules can include alginate, hyaluronic acid, chitosan and their derivatives, poly(ethylene glycol) (PEG) diacrylate, PEG dimethacrylate, collagen, gelatin, gelatin methacrylate, poly(lactic-co-glycolic acid), or combinations thereof. As one specific example, forming an alginate-based hydrogel at 402 can include crosslinking uronic acids of alginate and hydrolytically unstable molecules with divalent hydrazide groups.

[0071] Method 400 continues at 404 with adhering a base layer (e.g., base layer 204 of FIG. 2 , which may include fibrin or polydopamine in some examples) to the surface of the formed hydrogel. A mixture of fibrinogen and thrombin can form a fibrin layer on the hydrogel base layer. The fibrinogen and thrombin concentrations can be varied to adjust the adhesive strength of the fibrin base layer. In another example, a polydopamine adhesive base layer can be formed by polymerizing dopamine in an oxidizing environment. In another example, polydopamine and fibrin can be hybridized together in the base layer to increase the toughness and adhesive strength of the base layer. In some examples, the method at 406 can be followed by the method at 404, as described below.

[0072] At 406, the method includes freeze-drying the hydrogel under conditions sufficient to produce a plurality of microchannels. For example, freeze-drying at 406 can include placing the hydrogel on a metal (e.g., copper) substrate at sub-freezing temperatures to allow uniaxially oriented ice crystals to form throughout the hydrogel, followed by freeze-drying (e.g., lyophilization and / or dehydration). This uniaxial freezing process can induce anisotropic ice crystal growth in the hydrogel while excluding cross-linked polymer chains from the ice crystals. Subsequent freeze-drying removes the ice crystals, resulting in a cryogel with anisotropically aligned microchannels.

[0073] In some examples, uniaxial freezing and freeze-drying of the hydrogel at 406 can result in anisotropically aligned microchannels (e.g., microchannel 206 shown in FIG. 2) with diameters ranging from 50 to 1000 μm. Such microchannel structures enable rapid loading of cargo into the hydrogel (or cryogel) through a rehydration process. The method at 408 can include rehydrating the cryogel (e.g., dehydrated hydrogel) in a solution containing a selected cargo. In some examples, the solution can include deionized water and / or a biocompatible buffer or other pharmaceutically acceptable carrier along with the selected cargo. As described further below, in some examples, the cargo can include a gene-modifying vector or oncogenic molecule configured to induce tumor formation or growth, such as an oncogenic viral vector, a gene-editing component, a oncogenic nanoparticle, a carcinogen, or a combination thereof.

[0074] In some examples, method 400 can end at 406, and the dehydrated patch (which may be sterilized) can be stored without the loaded cargo for a period of time prior to use (e.g., up to about 1 year at room temperature, up to about 3 years frozen). When ready for use, the dehydrated hydrogel can be rehydrated with a cargo-containing solution as described above with reference to method 408.

[0075] In some examples, the dehydrated patch and the selected cargo-containing solution can be kept separate and included in a single kit. In this way, the dehydrated patch can be used for a variety of applications (e.g., different tumor or cancer models for therapeutic applications, etc.) by selecting a cargo-containing solution from multiple available cargo-containing solutions. In this way, a kit for treating disease or creating a preclinical cancer model can include a dehydrated patch (as described above) and a selected cargo-containing solution. In other examples, the dehydrated patch can be combined with a user-selected cargo-containing solution for customized use.

[0076] 7 presents a method 500 for making a patch, such as any one of the patches described herein. Method 500 begins by combining a flexible cross-linked hydrogel with a solution comprising a cargo, at 502. In some examples, combining the cross-linked hydrogel with the solution comprising the cargo, at 502, may include combining alginate or one or more other gel-forming molecules with the selected cargo (alone or in solution) and a cross-linking solution (such as those described above in 402 of method 400) to form a cross-linked hydrogel containing the selected cargo.

[0077] The method 500 continues at 504, which includes lyophilizing the flexible cross-linked hydrogel containing the selected cargo under conditions sufficient to generate a plurality of microchannels containing the cargo. The method at 504 can be similar to the method at 406, as described above with reference to FIG. 6.

[0078] At 506, the method includes adhering a base layer onto the surface of the hydrogel (similar to that described above for the method at 404 in FIG. 6).

[0079] The method 500 then continues at 508, where the cryogel formed by the methods of 504 and 506 can be rehydrated with a rehydration solution. In some examples, the rehydration solution can include deionized water and / or a biocompatible buffer or other pharmaceutically acceptable carrier.

[0080] In this manner, in some examples, methods 400 and 500 of Figures 6 and 7, respectively, can be used to form a patch configured to adhere to the surface of a selected tissue (such as the luminal surface of a hollow organ or the external surface of a solid organ) and provide controlled, sustained delivery of a selected cargo to a precise location within or on the tissue (e.g., a predetermined location such as a luminal structure), thereby resulting in the induction of spontaneous cancer with a defined location and growth rate in the target tissue in vivo. Thus, animal models of selected disease states (e.g., cancer) can be generated for preclinical research. In other examples, the patch is configured to provide controlled, sustained delivery of a selected therapeutic compound or candidate therapeutic compound.

[0081] In other examples, the patches disclosed herein can be formed by 3D printing a hydrogel (or cargo layer), as described above, and then loading the 3D-printed hydrogel with a cargo. As described herein, 3D printing can enable the formation of multiple patch layers with different pore sizes (or microchannel diameters) and compositions, thereby allowing multiple cargoes to be released from the multilayer patch at different rates or times.

[0082] cargo The cargo loaded into and contained within the patches described herein can be oncogenic (e.g., tumor- or cancer-causing) cargo, including one or more oncogenic viral vectors, gene-modifying components, recombinant proteins, carcinogens, or combinations thereof, capable of inducing the formation or growth of selected tumors. In some examples, the cargo is a gene-modifying vector or gene-modifying agent, and in some examples, is capable of manipulating the genome of somatic cells of the tissue in which the patch is positioned. In some examples, the cargo is contained in a nanoparticle (e.g., a carcinogenic nanoparticle or a nanoparticle containing a carcinogen) for delivery.

[0083] As an example, the cargo can be a viral vector containing one or more nucleic acids capable of inducing the formation or growth of a tumor (e.g., an oncogenic viral vector). In some examples, the subject to which the patch is administered or applied is a transgenic animal (such as a mouse or pig) containing one or more inducible genes that, when expressed in the animal, can result in the formation or growth of a tumor. For example, the animal can be transgenic for one or more Cre-inducible nucleic acids capable of inducing tumor formation or growth, such as one or more transgenes containing cancer driver mutations. Thus, in some examples, the cargo is a viral vector (e.g., an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector) containing a nucleic acid encoding Cre recombinase. Expression of Cre recombinase at or near a location containing cells containing the Cre-inducible nucleic acid, if the inducible nucleic acid is oncogenic, results in the expression of the inducible nucleic acid and the formation or growth of a tumor. One example is the "OncoPig" pig line, which is transgenic for nucleic acids encoding Cre recombinase-inducible porcine transgenes encoding KRASG12D and TP53R167H (see, e.g., Schook et al., PLOS One 10:e0128864, 2015). Expression of Cre, e.g., from an AdCre vector, results in the formation of tumors within tumors.

[0084] In other examples, the cargo is a vector (such as an expression vector) that encodes an oncogenic nucleic acid (such as a nucleic acid that encodes a driver mutation).

[0085] In other examples, the cargo comprises a genetic modification component that can induce tumor formation or growth upon release from the carcinogenic patch by editing the genome with specific nucleotide changes, additions or deletions ranging from single nucleotides to entire genes, etc., or by inducing specific gene expression. The genetic modification cargo may comprise a recombinant protein, a carcinogenic expression vector, a gene expression vector, a gene editing component, an epigenetic modifier, a senescence modifier, or a combination thereof.

[0086] Exemplary gene editing technologies include those based on genome editing proteins, such as zinc finger nucleases, TALENs, and CRISPR systems. In a particular example, the gene editing is a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated) system. The CRISPR system uses an RNA-guided nuclease called a CRISPR-associated or "Cas" endonuclease (e.g., Cas9 or Cpf1) to cleave DNA. In a typical CRISPR / Cas system, the Cas endonuclease is directed to a target nucleotide sequence (e.g., a site in the genome to be sequence-edited) by a sequence-specific non-coding guide RNA that targets single-stranded or double-stranded DNA sequences. One type of CRISPR system includes a type II Cas endonuclease, such as Cas9, a CRISPR RNA (crRNA), and a trans-activating crRNA (tracrRNA). The Cas9crRNA comprises a spacer sequence, typically an RNA sequence of about 20 nucleotides (in various examples, this is 20, 21, 22, 23, 24, 25, or up to about 30 nucleotides in length) that corresponds to (e.g., is identical to or nearly identical to, or complementary to or nearly complementary to) a target DNA sequence of approximately equal length. The Cas9crRNA also comprises a region that binds to the Cas9 tracrRNA to form a partially double-stranded structure that is cleaved by RNase III, resulting in a crRNA:tracrRNA hybrid or duplex. The crRNA:tracrRNA hybrid then allows the Cas9 endonuclease to recognize and cleave the target DNA sequence. In some examples, the tracrRNA and crRNA (e.g., a crRNA containing a spacer sequence) can be comprised in a chimeric nucleic acid called a single guide RNA (sgRNA).

[0087] Other CRISPR nucleases useful in the methods and compositions of the present disclosure include Cpf1, C2c1 (also known as Cas12b), and C2c3 (also known as Cas12c) (Shmakov et al. (2015) Mol. Cell, 60:385-397). For purposes of gene editing, CRISPR arrays can be designed to contain one or more guide RNAs (including spacer sequences) corresponding to the desired target DNA sequence; see, e.g., Cong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308. The design of effective guide RNAs for use in plant genome editing is disclosed in U.S. Patent Application Publication No. 2015 / 0082478 A1, the entire specification of which is incorporated herein by reference. More recently, efficient Cas9 gene editing has been achieved using chimeric "single guide RNAs" ("sgRNAs"), engineered (synthetic) single RNA molecules that mimic the naturally occurring crRNA-tracrRNA complex and contain both a tracrRNA sequence (for binding to Cas9 nuclease) and at least one crRNA sequence (a guide RNA sequence containing a spacer sequence to guide Cas9 nuclease to the targeted sequence for editing); see, e.g., Cong et al. (2013) Science 339:819-823; Xing et al. (2014) BMC Plant Biol 14:327-340. Chemically modified sgRNAs have been demonstrated to be effective for Cas9 genome editing; see, e.g., Hendel et al. (2015) Nature Biotechnol 33:985-991.

[0088] In one example, the cargo comprises CRISPR gene editing components, which may comprise a nucleic acid or a ribonucleoprotein complex. For example, the cargo may be a nucleic acid designed to introduce one or more mutations into a cancer driver gene or knock out the expression of a tumor suppressor, and a nucleic acid encoding a CRISPR endonuclease (such as an appropriate guide RNA and Cas9). In another example, the CRISPR gene editing components are provided in the form of a ribonucleoprotein complex comprising a gene editing nucleic acid and an endonuclease protein.

[0089] In other examples, cargo includes synthetic vectors or materials that electrostatically bind to DNA or RNA, thereby condensing the genetic material into nanometer-scale complexes (e.g., tens to hundreds of nanometers in diameter) that protect the genes and allow them to enter cells. Such materials can include cationic peptides, proteins, polymers, and liposomes. Exemplary synthetic vectors used for in vitro gene transfer include (diethylamino)ether (DEAE)-dextran and calcium phosphate.

[0090] In yet another example, the patch can include one or more auxiliary or additional cargoes that can further modulate tumor biology, tumor microenvironment, and tumor growth rate. Such additional cargoes can include cell division factors, angiogenesis factors, carcinogens, or combinations thereof. These factors can include stem cell and growth factors to increase cell division and tumor growth rate, angiogenic factors to increase neovascularization, immunomodulatory factors to alter the tumor immune microenvironment, and carcinogens to further promote tumorigenesis and heterogeneity.

[0091] In other examples, the cargo is a carcinogen. For example, a carcinogen is a compound that induces the local formation or growth of tumors at the site of administration. Exemplary carcinogens that can be used include azoxymethane (AOM) and dextran sulfate sodium (DSS). Additional carcinogens can be selected by those skilled in the art depending, for example, on the subject and the type of tumor to be induced.

[0092] In some examples, the cargo is formulated into a carcinogenic nanoparticle delivery system. In some examples, the nanoparticle structure spontaneously forms from a polymer in aqueous solution. Polymers used to fabricate nanoparticles include, for example, poly(acrylamide), poly(ester), poly(alkyl cyanoacrylate), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(D,L-lactic-co-glycolic acid) (PLGA). The nanoparticles may have an average diameter of 50 to 1000 nm and can be taken up by cells. The nanoparticles release their cargo as a function of their degradation. In some examples, the nanoparticles comprise a cationic polymer that can self-assemble with a negatively charged expression vector (e.g., a viral vector or an expression vector) to form nanoparticles with a diameter of 100 to 1000 nm. In one example, the nanoparticle is a liposome. Liposomes are typically lipid vesicles with one or more concentric phospholipid bilayers. In some cases, phospholipids are composed of a hydrophilic head group and two hydrophobic chains to allow for the encapsulation of both hydrophobic and hydrophilic compounds. Liposomes for use as delivery systems have been described (see, for example, Paszko and Senge, Currmed ​​Chem 19(31)5239-5277, 2012; Immordino et al., Int J Nanomedicine 1(3):297-315, 2006; U.S. Patent Application Publication Nos. 2011 / 0268655 and 2010 / 00329981).

[0093] How to use For example, methods are provided herein for using the disclosed patches to deliver one or more cargoes that promote or induce tumor formation or growth, or alternatively, to deliver therapeutic agents or candidate therapeutic agents. As discussed above, one use of the patches is to generate animal tumor models that more closely mimic human disease, for example, for use in preclinical models of cancer treatment. In other examples, the disclosed patches can be used to deliver therapeutic agents to either preclinical animal models or human or veterinary subjects. In one example, the method includes delivering a patch containing a cargo to a predetermined location in tissue (such as the lumen of a target tissue), adhering a base layer of the patch to the predetermined location in tissue, and releasing the cargo from the delivered patch to the predetermined location in tissue to which the cargo is adhered over a period of time to induce tumor formation or growth at the predetermined location in tissue.

[0094] A patch comprising a hydrogel having a base layer (e.g., a polydopamine or fibrin base layer), such as one of the hydrogel patches described above, can be flexible, thereby allowing for various manipulations of the patch, such as repeated folding and unfolding.

[0095] 8A-8B show an exemplary patch 600 including a hydrogel and base layer containing a plurality of microchannels, the patch configured as a disk having a diameter 602 of approximately 10 mm (shown in the front view of FIG. 8A) and a thickness 604 of approximately 1.5 mm (shown in the side view of FIG. 8B). It should be noted that the dimensions of patch 600 are exemplary, and other patch dimensions are possible, as described herein.

[0096] FIG. 9 shows patch 600 at various stages of deployment, demonstrating its ability to be transformed into a tubular shape (e.g., for transportation and / or delivery to a target tissue) and then undergo spontaneous (unassisted) or force-induced deployment (e.g., via an external device such as a biopsy forceps). At 610, the patch is rolled into a tube that, in some instances, can be loaded into a delivery device such as an endoscope (as described further below). After delivery at the implantation site (target tissue or predetermined location in tissue), patch 600 can begin to deploy, as shown at 612. Further stages of deployment of patch 600 are shown at 614, 616, and 618 until patch 600 is fully deployed at 620. FIG. 9 shows exemplary durations for various stages of deployment of patch 600 (over a period of approximately 30 minutes from 610 to 620). However, these durations are exemplary and may vary based on the size and composition of the patch.

[0097] Therefore, due to its flexible nature, it may be possible to deliver the patch to the target tissue via a delivery device such as an endoscope, catheter, or needle. Such a delivery device provides minimally invasive placement of the patch in the target tissue (compared to surgical implantation of tumor cells). In some examples, the patch may be delivered percutaneously via a needle (e.g., by placing the patch inside the needle and pushing the patch out of the needle with a stylet when the implantation site is reached) to the exterior surface of a solid organ or inside a solid organ.

[0098] An exemplary method for delivering a patch to a target tissue using an endoscope is shown in Figures 10A-10C. Additionally, an exemplary method 800 for using a patch to induce tumor growth in a target tissue (or generate a somatic tumor at a predetermined location in the tissue) is shown in Figure 11. The following description of method 800 refers to Figures 10A-10C as an example of delivering a patch to a target tissue. However, other delivery methods are possible, such as using a needle to deliver a patch to the exterior or interior of a solid organ.

[0099] Method 800 begins at 802 with loading a hydrogel-containing patch with cargo. The patch may be any one of the patches described herein, and is shown as patch 706 in the example presented in FIGS. 10A-10C, as further described below. For example, the patch may comprise a plurality of microchannels within a hydrogel body (or cargo layer of the patch) and a base layer disposed at the base of the body. Thus, loading at 802 may include loading any of the cargoes described above into a plurality of microchannels of the patch (e.g., via method 400 of FIG. 6 or method 500 of FIG. 7). In other examples, the method utilizes a patch pre-loaded with cargo (e.g., the method begins at 804 below).

[0100] Method 800 continues at 804, which includes delivering the patch to a predetermined location in the selected tissue. In some examples, the delivery at 804 can include delivering the patch to the lumen of the target tissue (e.g., as shown in FIG. 1B ). In other examples, the selected tissue can be tissue of a solid organ, and the delivery at 804 can include delivering the patch to the exterior of the selected tissue. In particular examples, the target tissue is the bladder, esophagus, stomach, pancreas, intestine (such as the colon), liver, lung, heart, kidney, bone, etc. The selected tissue is present in a subject, such as a laboratory, veterinary, or human subject. In some examples, the subject is a mouse, rabbit, or pig. The subject can be a transgenic animal, e.g., transgenic for one or more inducible genes. In one example, the transgenic animal is an oncopig. In other examples, the subject is a wild-type animal (e.g., an animal that is not transgenic or otherwise genetically modified).

[0101] In some examples, delivering at 804 may include folding or rolling the patch into a folded or rolled delivery state (such as the configuration shown at 610 in FIG. 9 ), inserting it into a lumen of a delivery device, and then delivering the patch to the target tissue using the delivery device. In some examples, the delivery device may be an endoscope or other type of delivery catheter. In other examples, the delivery device may be a syringe, needle, or other instrument that allows the patch to be delivered to a solid organ via percutaneous injection.

[0102] 10A , endoscope 700 can include a lumen 702 (or channel) defined by the interior wall of endoscope 700 that extends the entire length of endoscope 700. A catheter 704 can thus be disposed within lumen 702. In some examples, catheter 704 and endoscope 700 are coaxial with one another. Patch 706 (which can be any of the patches described herein) can be folded or rolled into a folded or rolled delivery state and then inserted into the lumen of catheter 704 in its folded or rolled state.

[0103] As the endoscope (or other delivery device) is moved (navigated) toward and approaches the implantation site of the patch 706, such as the lumen of the target tissue 708, the catheter 704 can be extended toward the target tissue 708, and the patch 706 can then be pushed out of the catheter 704 ( FIG. 10B ). As an example, as shown in FIG. 10B , the patch 706 can be pushed out of the catheter 704 using a pushing element 710, such as a shaft. In some examples, when the target tissue 708 is the lumen of a hollow organ, the patch 706 can be pushed into the lumen of the target tissue 708.

[0104] In some examples, once the patch 706 is pushed out of the catheter 704 ( FIG. 10B ), a grasping element 712, such as biopsy pinchers or forceps, that may extend through the endoscope 700 and / or the catheter 704 ( FIG. 10C ), may be used to further move or position the patch to a desired location in the target tissue 708 (e.g., relative to the lumen wall of the target tissue 708). The grasping element 712 may position the patch 706 at a desired location in the target tissue 708. The patch 706 may then spontaneously deploy or unfold relative to the target tissue 708 (e.g., relative to the lumen of the target tissue 708), or the grasping element 712 may be used to initiate or perform the deployment or unfolding action of the patch 706 relative to the target tissue 708. The endoscope 700 may then be removed from the implantation site and the subject.

[0105] Thus, the method of 804 can include positioning a patch at a predetermined location in tissue. Clinically relevant tumors are located in luminal or parenchymal regions of organs based on tissue anatomy. Also, in large organs, tumor location is important for delivery or removal of such tumors by either surgery, radiation, or directed energy. Thus, the patches and delivery methods of such patches described herein allow for precise positioning of the patch (e.g., at a predetermined location in tissue) to guide a given tumor to a given site at a defined time relative to other co-morbidities.

[0106] Returning to method 800 of FIG. 11 , at 806, the method can include adhering a polydopamine base layer of the patch to a predetermined location on tissue. For example, upon contact with the predetermined location on tissue, the base layer of the patch (e.g., base layer 204 shown in FIG. 2 ) can be activated to adhere to the tissue, thereby securing (effectively adhesively securing) the patch to the tissue in place. In some examples, a folded or rolled patch can be loaded onto a catheter (catheter 704 of FIG. 10A ) such that the base layer of the patch faces the tissue after being pushed out of the catheter. In some examples, activation of the base layer occurs upon exposure of the base layer to the tissue, for example, by unfolding or unrolling the patch.

[0107] After adhering to a predetermined location in the tissue (e.g., as shown in FIG. 5 ), method 800 continues at 808 with releasing cargo from the delivered patch to the predetermined location in the tissue to which it is adhered over a period of time to induce tumor formation or growth at the predetermined location in the tissue. For example, after contacting the tissue, the patch can begin to controllably (e.g., at a predetermined diffusion rate based on the size and tortuosity of the microchannels and the charge density of the hydrogel) and locally release its cargo through the microchannels. By controllably and substantially continuously releasing its cargo over a period of time (e.g., 1-3 weeks, or minutes to weeks), rather than all at once, the cargo can effectively trigger the cell division required to induce tumor growth.

[0108] In some examples, the method of 808 can further include automatically degrading the patch in the target tissue over a predetermined period of time. For example, within a predetermined period (e.g., 3-5 weeks), the patch can disappear due to degradation of the hydrogel and polydopamine layers, while causing little or no inflammation in the subject (e.g., in the target tissue). The patches described herein can be configured to minimize an immune response (e.g., inflammation) upon implantation into the target tissue. For example, by utilizing a biocompatible hydrogel in the patch and by containing cargo within the microchannels of the patch (increasing the surface area within the patch), the patches described herein induce little or no immune response at the implantation site.

[0109] [Example] The following examples are provided to illustrate particular features and / or configurations. These examples should not be construed as limiting the disclosure to the particular features or configurations described.

[0110] Example 1 - Ex vivo testing with patches Patches loaded with AdCre as their cargo, ranging in size from 1 x 1 mm, 5 x 5 mm, and 10 x 10 mm (Figure 12), were immobilized on bladder epithelium ex vivo. AdCre effectively translocated into epithelial cells, inducing Cre-GFP (green fluorescent protein) expression within 24 hours. Figure 12 shows microscopic images of the three sizes of patches after placement on the bladder epithelium, along with GFP expression in each patch. Furthermore, as shown in Figure 13, induced Cre-GFP expression was restricted to cells located under the patch and did not result in transduction of surrounding bladder epithelial cells by diffusion. For example, Figure 13 presents brightfield images of tissue exposed to AdCre via patch and control tissue, as well as Ad-GFP expression (indicated by green fluorescence) in tissue exposed to patch and control tissue.

[0111] Example 2 - In vivo tumor induction by patch Patches (hereafter referred to as TIPs) were injected (loaded) with either AdCre or an adenoviral vector encoding only GFP (AdGFP) and placed in the subcutaneous space in the neck region between the ears of several oncopigs (n = 2). This positioning allowed for visual observation of tumor induction while limiting the oncopigs' ability to contact or puncture the incision site. Each oncopig received a TIP containing AdCre on the left side and a TIP loaded with AdGFP on the right side. Thus, each animal served as its own control. After 7 days of TIP placement within the oncopigs, tumor mass formation was observed on the left side of both oncopigs (Figure 14). Figure 14 shows a microscopic image (indicated by 906) demonstrating tumor formation around the implanted TIPs. As shown in Figure 14, no tumor formation occurred around the control TIPs (indicated by 904) or in the subcutaneous tissue area without TIP exposure (indicated by 902).

[0112] The animals were euthanized one week after tumor induction, and the tumor mass was dissected similarly to the right incision site to allow for analysis of whether TIP survival and GFP activity could be observed. As shown in microscopy image 906 of Figure 14, AdCre-loaded TIPs induced tumor masses within 7 days in vivo. TIPs were identified within the tumor mass, indicating that the TIPs were still intact and had not biodegraded by 7 days, thus still providing tumorigenic signals. In the control TIP site (AdGFP-loaded TIP), the TIPs were still evident, but no tumor-like mass developed around the TIPs, as shown in microscopy image 904 of Figure 14. No foreign body inflammation or nodule formation was observed.

[0113] The resulting tumor and control TIP sites were subjected to fluorescence microscopy within 3 hours of tissue capture to assess GFP expression. As shown in Figure 15, GFP activity was observed in tumors induced by TIP with AdCre (indicated by 910) and in the subcutaneous tissue surrounding control TIP with AdGFP (indicated by 912). This provides evidence that TIP features support the sustained release of functional adenoviral vectors in vivo, suggesting that these vectors do not immediately diffuse to the tissue site but are instead contained within and protected from tissue enzyme-induced degradation processes. Thus, TIP may support longer-term tumor induction in vivo. When such experiments were performed in vitro, cells exposed to AdCre or AdGFP began to express GFP within 24 hours, with fluorescence returning to background levels within 3–14 days.

[0114] Thus, the TIP allows for the defined release of adenoviral vectors and delays the immediate cellular uptake of AdCre. Although AdCre is capable of transducing all cells, it only triggers tumor formation in dividing cells (removal of the STOP signal), and is therefore limited to activating transgene expression in actively dividing cells at the time of AdCre injection. Therefore, local tissue growth and AdCre clearance rates affect the magnitude and efficacy of tumor induction when AdCre is directly injected into oncopig tissue. However, the engineered TIP provides continuous release of AdCre and AdGFP to extend the exposure time, thus extending the time course of induction.

[0115] Additionally, patches (TIP or TMP) of various sizes, shapes, and microstructures have been developed that allow for up to 1,000-fold differences in viral vector loading (e.g., AdCre and AdGFP vector loading), as shown in Table 1. The ability to manipulate patch geometry and adenoviral vector load further demonstrates that the patches described herein can provide a defined signal for a defined period of time. Table 1. Exemplary patch dimensions, coverage and vector delivery [Table 1]

[0116] Example 3 - Development of OncoPig lung adenocarcinoma cell lines To generate Oncopig lung adenocarcinoma cell lines, type II pneumocytes were isolated from Oncopig lung samples and exposed to AdCre (Figure 16A). Consistent with findings in humans, expression of the KRASG12D mutant protein induced vacuolization and cell death (Figure 16B). To circumvent this induced cell death, Oncopig type II pneumocytes were transduced with a lentiviral vector encoding Cre recombinase and a CDK4R24L expression vector under the control of a type II pneumocyte-specific promoter, resulting in cell proliferation and positive staining for clinically relevant lung adenocarcinoma diagnostic markers (Figure 16C). Ex vivo delivery of this lentiviral vector into Oncopig lung segments resulted in lung masses containing GFP-expressing cells and a histological structure consistent with lung adenocarcinoma (Figures 16D-F). These tumor masses also stained positive for Ki-67 (indicating increased proliferation) and the KRASG12D transgene. The results of this study demonstrate the ability to induce tumorigenesis in oncopig cells through a combination of gene editing and transgene activation.

[0117] Example 4 - Development of rabbit HCC cell lines To demonstrate the ability to induce tumorigenesis in wild-type animals through gene editing alone, isolated rabbit hepatocytes were exposed to CRISPR-Cas9 components targeting TP53 and PTEN. Transfection of isolated hepatocytes resulted in TP53 and PTEN KO, as confirmed by sequencing (Figures 17A and 17B). While primary hepatocytes rapidly died after 2 weeks of culture, TP53 and PTEN KO cell lines proliferated and persisted in culture for over 4 months. These cells stained positive for the hepatocyte-specific marker arginase-1, thus confirming their hepatocyte origin (Figure 17C). Together, these results support the ability to utilize gene editing techniques to induce tumorigenesis in primary wild-type cells.

[0118] Example 5 - Method for making hydrogel patches Alginate (MW ≈ 250,000 gmol-1, FMC Biopolymer) sterilized by filtration was dissolved in 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer at a concentration of 2% (w / v). This alginate solution was sequentially mixed with microparticles (if needed), sulfonated N-hydroxysuccinimide (sulfo-NHS, Thermo Scientific), adipic acid dihydrazide (AAD, Sigma-Aldrich), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, Thermo Scientific). The pregelled mixture was allowed to harden in the space between two glass plates separated by a 1 mm spacer. The hydrogel sheet was then incubated in DI water at room temperature for 12 hours. To prepare the microchanneled hydrogel, the gel was then placed on a copper plate at a controlled temperature. All gels were surrounded by styrene foam for insulation. The frozen samples were freeze-dried to introduce microchannels through the alginate gel disc. Finally, the dehydrated microchanneled matrix was rehydrated by dropping aqueous medium or a cancer gene suspension onto the top of the sample. In contrast, microporous gels were prepared by sequential freezing, freeze-drying, and rehydration of alginate gels in copper containers.

[0119] [Additional Examples of the Disclosed Technology] In consideration of the above implementations of the disclosed subject matter, the present application discloses the following additional embodiments: It should be noted that one feature of an embodiment alone, or two or more features of an embodiment taken in combination, and optionally in combination with one or more features of one or more additional embodiments, are further embodiments included in the disclosure of the present application.

[0120] [Example 1] A patch comprising a base layer configured to adhere to a predetermined location on tissue, and a hydrogel layer having a plurality of microchannels configured to store and release a genetically modified vector for a time and in an amount sufficient to generate a somatic tumor at the predetermined location through manipulation of the genome of somatic cells.

[0121] [Example 2] Any example herein, particularly the patch of Example 1, wherein the patch is configured to continuously release genetically modified vectors from multiple microchannels over a period of time while positioned at a predetermined location on tissue.

[0122] Example 3. The patch of any example herein, particularly either Example 1 or Example 2, configured to degrade after 3 to 5 weeks of contact with tissue.

[0123] [Example 4] The patch of any example herein, particularly any one of Examples 1-3, wherein the hydrogel layer further comprises a genetically modified vector, the genetically modified vector comprising an oncogenic viral vector disposed within the lumen of the microchannel.

[0124] [Example 5] The patch of any example herein, particularly any one of Examples 1 to 3, wherein the hydrogel layer further comprises a genetically modified vector, the genetically modified vector comprising an adenoviral vector expressing Cre recombinase (AdCre) disposed within the lumen of the microchannel.

[0125] [Example 6] The patch of any example herein, particularly either Example 4 or Example 5, wherein the genetically modified vector is carried within a carcinogenic nanoparticle disposed within the lumen of the microchannel.

[0126] [Example 7] The patch of any example herein, particularly any one of Examples 1-6, wherein the microchannels of the plurality of microchannels are anisotropically aligned with one another within the hydrogel of the hydrogel layer.

[0127] [Example 8] The patch of any example herein, particularly any one of Examples 1-7, wherein the cargo layer of the patch comprises alginate and a divalent hydrazide group cross-linked hydrogel.

[0128] [Example 9] The patch of any example herein, particularly any one of Examples 1-8, wherein the diameter of the plurality of microchannels is in the range of 50-1000 μm.

[0129] [Example 10] Any example herein, particularly a patch of any one of Examples 1 to 9, wherein the thickness of the patch is in the range of 0.05 to 1.5 mm, the length of the patch is in the range of 1 to 10 mm, and the width of the patch is in the range of 1 to 10 mm.

[0130] [Example 11] A method for generating a somatic tumor at a predetermined location in a tissue, the method comprising positioning at the predetermined location a patch comprising a base layer configured to adhere to the predetermined location and a hydrogel layer comprising a plurality of microchannels containing a releasable carcinogenic cargo stored within the plurality of microchannels, whereby the carcinogenic cargo is released from the hydrogel layer to generate a somatic tumor at the predetermined location via manipulation of the genome of a somatic cell.

[0131] [Example 12] The method of any example herein, especially Example 11, wherein the predetermined location is the luminal surface of the tissue.

[0132] [Example 13] The method of any example herein, especially Example 12, wherein the tissue is tissue of one of the pancreas, bladder, colon, rectum, esophagus, stomach, or throat.

[0133] [Example 14] The method of any example herein, especially Example 11, wherein the predetermined location is an outer surface of the tissue.

[0134] [Example 15] The method of any example herein, especially Example 14, wherein the tissue is one of neuron, brain, or ovary.

[0135] [Example 16] The method of any example herein, particularly any one of Examples 11-15, wherein the patch is configured to be folded and positioned against one of the luminal or exterior surface of the tissue.

[0136] [Example 17] The method of any of the examples herein, particularly any one of Examples 11-16, wherein positioning the patch at a predetermined location on the tissue includes adhering a base layer of the patch to the luminal or outer surface of the tissue.

[0137] Example 18 The method of any example herein, especially Example 17, wherein the base layer comprises one of fibrin or polydopamine adhesive.

[0138] [Example 19] The method of any example herein, particularly any one of Examples 11-18, further comprising, prior to positioning the patch at a predetermined location on the tissue, folding the patch into a folded delivery state and inserting it into a lumen of a delivery device, and moving the delivery device toward the predetermined location on the tissue.

[0139] [Example 20] The method of any example herein, particularly Example 19, wherein the delivery device is an endoscope.

[0140] [Example 21] The method of any example herein, especially Example 19, wherein the delivery device is a needle.

[0141] [Example 22] The method of any of the examples herein, particularly any one of Examples 19-21, wherein positioning the patch at a predetermined location on the tissue includes pushing a folded patch out of the distal end portion of the delivery device at the predetermined location on the tissue, and unfolding the patch from the folded delivery state to an unfolded state such that the base layer is positioned against the surface of the predetermined location on the tissue.

[0142] [Example 23] The method of any of the examples herein, particularly any one of Examples 11-22, further comprising automatically disintegrating the patch at the predetermined location on the tissue over a predetermined length of time after positioning the patch at the predetermined location on the tissue.

[0143] Example 24 The method of any example herein, particularly Example 23, wherein the predetermined length of time is in the range of 3 to 5 weeks.

[0144] [Example 25] The method of any example herein, particularly any one of Examples 11-24, wherein the oncogenic cargo comprises a genetically modified vector.

[0145] Example 26. The method of any example herein, particularly any one of Examples 11-24, wherein the oncogenic cargo comprises a nucleic acid comprising CRISPR components for introducing one or more tumor driver mutations into somatic cells of the tissue.

[0146] Example 27. The method of any example herein, particularly any one of Examples 11-24, wherein the carcinogenic cargo is carried within nanoparticles stored within a plurality of microchannels of the hydrogel layer of the patch.

[0147] [Example 28] The method of any example herein, particularly any one of Examples 11-24, wherein the carcinogenic cargo comprises one or more of a carcinogen, a mitogenic factor, or an angiogenic factor.

[0148] [Example 29] The method of any example herein, particularly any one of Examples 11-27, wherein the hydrogel layer of the patch further comprises an auxiliary cargo stored within a plurality of microchannels, and further comprising releasing the auxiliary cargo from the hydrogel layer to modulate one or more of the biological characteristics, microenvironment, or growth rate of a somatic tumor generated at a predetermined location in the tissue.

[0149] [Example 30] The method of any example herein, especially Example 29, wherein the auxiliary cargo comprises a carcinogen.

[0150] [Example 31] The method of any example herein, particularly either Example 29 or Example 30, wherein the auxiliary cargo comprises one or more of a mitogenic factor and an angiogenic factor.

[0151] [Example 32] The method of any example herein, particularly any one of Examples 11-31, wherein the tissue is in a subject, and the subject is a transgenic animal or a wild-type animal.

[0152] [Example 33] A method for making a hydrogel patch containing microchannels, the method comprising: forming a hydrogel by crosslinking gel-forming molecules in a crosslinking solution; adhering a base layer configured to adhere to tissue to the surface of the hydrogel; freeze-drying the hydrogel under conditions sufficient to produce a plurality of microchannels so as to form a cryogel having a plurality of microchannels; and rehydrating the cryogel in a solution containing a selected cargo.

[0153] [Example 34] The method of any example herein, especially Example 33, wherein the selected cargo is an oncogenic viral vector.

[0154] [Example 35] The method of any Example herein, especially Example 33, wherein the selected cargo is an adenoviral vector expressing Cre recombinase (AdCre).

[0155] [Example 36] The method of any example herein, especially Example 33, wherein the selected cargo is a carcinogenic nanoparticle carrying a cancer-causing agent.

[0156] [Example 37] The method of any example herein, especially Example 33, wherein the selected cargo is a carcinogen.

[0157] Example 38. The method of any example herein, particularly any one of Examples 33-37, wherein the generated microchannels of the plurality of microchannels are anisotropically aligned with one another in the hydrogel.

[0158] Example 39. The method of any example herein, particularly any one of Examples 33-38, wherein the hydrogel is an alginate and divalent hydrazide group crosslinked hydrogel.

[0159] [Example 40] The method of any example herein, particularly any one of Examples 33-39, wherein the microchannel diameter of the plurality of microchannels is in the range of 50-1000 μm.

[0160] [Example 41] The method of any example herein, particularly any one of Examples 33-40, wherein the patch thickness is in the range of 0.05-1.5 mm, the patch length is in the range of 1-10 mm, and the patch width is in the range of 1-10 mm.

[0161] Example 42: A method for producing a hydrogel containing microchannels includes mixing a flexible cross-linked hydrogel with a solution containing a cargo, freeze-drying the flexible cross-linked hydrogel and cargo under conditions sufficient to produce a plurality of microchannels containing the cargo in the hydrogel, and adhering a base layer configured to adhere to tissue to a surface of the hydrogel.

[0162] [Example 43] The method of any example herein, especially Example 42, wherein the cargo is an oncogenic viral vector.

[0163] [Example 44] The method of any Example herein, especially Example 42, wherein the cargo is an adenoviral vector expressing Cre recombinase (AdCre).

[0164] [Example 45] The method of any example herein, particularly Example 42, wherein the cargo is a carcinogenic nanoparticle carrying a cancer-causing substance.

[0165] [Example 46] The method of any example herein, especially Example 42, wherein the cargo is a carcinogen.

[0166] [Example 47] The method of any example herein, particularly any one of Examples 42-46, wherein the generated microchannels of the plurality of microchannels are anisotropically aligned with one another in the hydrogel.

[0167] [Example 48] The method of any example herein, particularly any one of Examples 42-47, wherein the hydrogel is an alginate and divalent hydrazide group crosslinked hydrogel.

[0168] Example 49 The method of any example herein, particularly any one of Examples 42-48, wherein the microchannel diameter of the plurality of microchannels is in the range of 50-1000 μm.

[0169] [Example 50] The method of any of the examples herein, particularly any one of Examples 42-49, wherein the patch thickness is in the range of 0.05-1.5 mm, the patch length is in the range of 1-10 mm, and the patch width is in the range of 1-10 mm.

[0170] In view of the many possible examples to which the principles of the present disclosure may be applied, it should be recognized that the illustrated configurations represent examples of the disclosed technology and should not be construed as limiting the scope of the disclosure or the claims. Rather, the scope of claimed subject matter is defined by the following claims and their equivalents.

Claims

1. A patch, a base layer configured to adhere in place to tissue; a hydrogel layer comprising a plurality of microchannels configured to store and release genetically modified vectors for a time and in an amount sufficient to generate a somatic tumor through manipulation of the genome of a somatic cell at said predetermined location; patch.

2. The patch is configured to continuously release the genetically modified vector from the plurality of microchannels over a period of time that the patch is positioned at the predetermined location on the tissue. The patch of claim 1 .

3. the patch is configured to degrade after 3 to 5 weeks of contact with the tissue; The patch of claim 1 .

4. the hydrogel layer further comprises the genetically modified vector; The genetically modified vector comprises an oncogenic viral vector disposed within the lumen of the microchannel; The patch of claim 1 .

5. the hydrogel layer further comprises the genetically modified vector; The genetic modification vector comprises an adenovirus vector (AdCre) expressing Cre recombinase, which is disposed within the lumina of the microchannel. The patch of claim 1 .

6. The gene-modified vector is carried within a carcinogenic nanoparticle disposed within the lumina of the microchannel. The patch according to claim 4 or claim 5.

7. 1. A method for the generation of a somatic tumor in a predetermined location in a tissue, comprising: positioning at the predetermined location a patch comprising a base layer configured to adhere to the predetermined location and a hydrogel layer comprising a plurality of microchannels containing a releasable carcinogenic cargo stored within the plurality of microchannels, whereby the carcinogenic cargo is released from the hydrogel layer to generate a somatic tumor at the predetermined location via manipulation of the genome of a somatic cell. provided, however, that the tissue does not exist in a human. method.

8. and automatically disintegrating the patch at the predetermined location on the tissue over a predetermined length of time after positioning the patch at the predetermined location on the tissue. The method of claim 7.

9. the predetermined length of time is in the range of 3 to 5 weeks; The method of claim 8.

10. the oncogenic cargo comprises a genetically modified vector; The method of claim 7.

11. the oncogenic cargo comprises a nucleic acid comprising a CRISPR component for introducing one or more tumor driver mutations into the somatic cells of the tissue; The method of claim 7.

12. the carcinogenic cargo is carried within nanoparticles stored within the plurality of microchannels of the hydrogel layer of the patch; The method of claim 7.

13. The oncogenic cargo comprises one or more of a carcinogen, a mitogenic factor, or an angiogenic factor; The method of claim 7.

14. the hydrogel layer of the patch further comprises an auxiliary cargo stored within the plurality of microchannels, and the method further comprises releasing the auxiliary cargo from the hydrogel layer to modulate one or more of a biological characteristic, a microenvironment, or a growth rate of the somatic tumor generated at the predetermined location in the tissue. The method of claim 7.

15. the auxiliary cargo comprises a carcinogen; 15. The method of claim 14.

16. The auxiliary cargo comprises one or more of a cell division factor and an angiogenesis factor.

16. The method of claim 14 or claim 15.

17. The tissue is in a subject, and the subject is a transgenic or wild-type animal. The method of claim 7.

18. 1. A method for making a hydrogel patch containing microchannels, comprising: forming a hydrogel by crosslinking gel-forming molecules in a crosslinking solution; adhering a base layer to a surface of the hydrogel, the base layer being configured to adhere to tissue; freeze-drying the hydrogel under conditions sufficient to create a plurality of microchannels, such that a cryogel having the plurality of microchannels is formed; and rehydrating the cryogel in a solution containing a selected cargo. method.

19. The thickness of the patch is within the range of 0.05 to 1.5 mm, the length of the patch is within the range of 1 to 10 mm, and the width of the patch is within the range of 1 to 10 mm.

20. The method of claim 18.

20. 1. A method for making a hydrogel patch containing microchannels, comprising: mixing the flexible cross-linked hydrogel with a solution containing a cargo; freeze-drying the flexible cross-linked hydrogel and the cargo under conditions sufficient to produce a plurality of microchannels containing the cargo in the hydrogel; and adhering a base layer to a surface of the hydrogel, the base layer being configured to adhere to tissue. method.

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