EP4665300A1

EP4665300A1 - Methods and compositions for formulating recombinant viral vectors

Info

Publication number: EP4665300A1
Application number: EP24757596.2A
Authority: EP
Inventors: George M. Church; Denitsa M. MILANOVA; David B. Thompson; Madison Ski KRIEGER
Original assignee: Harvard University
Current assignee: Harvard University
Priority date: 2023-02-16
Filing date: 2024-02-14
Publication date: 2025-12-24
Also published as: WO2024173497A1

Abstract

Devices for delivering an adeno-associated viral vectors into patients, methods for making the same, and associated articles and methods are generally provided.

Description

METHODS AND COMPOSITIONS FOR FORMULATING RECOMBINANT VIRAL VECTORS

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63/485,377, filed February 16, 2023, and entitled “Methods and Compositions for Formulating Recombinant Viral Vectors,” which is incorporated herein by reference in its entirety for all purposes.

FIELD

Devices for delivering adeno-associated viral vectors into patients and methods for making or using the same are generally described.

BACKGROUND

Adeno-associated viral vectors can advantageously be employed as gene therapy vectors. However, they are commonly delivered by methods that cause them to spread uncontrollably to the lymphatic system instead of to the tissue of interest.

Accordingly, improved device and methods for delivering adeno-associated viral vectors into patients are needed.

SUMMARY

Devices for delivering adeno-associated viral vectors into patients, related components, and related methods are generally described.

In some embodiments, a device for delivering an adeno-associated viral vector into a patient is provided. The device comprises a matrix and an adeno-associated viral vector. The matrix comprises a hydrophilic polymer and a sugar. The adeno-associated viral vector is crystalline.

In some embodiments, a method is provided. The method comprises drying a solution to form a solid device for delivering an adeno-associated viral vector into a patient. The solution comprises a hydrophilic polymer in an amount of at least 10% w/v, a sugar, the adeno-associated viral vector, and water.

Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

FIG. 1 shows one non-limiting example of a device including a matrix and an adeno- associated viral vector, in accordance with some embodiments;

FIG. 2 shows one example of a device that comprises a component comprising a matrix and adeno-associated viral vector and further comprises a component lacking the matrix and the adeno-associated viral vector, in accordance with some embodiments;

FIG. 3 shows a method that comprises drying a solution comprising an adeno- associated viral vector, a hydrophilic polymer, and a sugar to form a solid device for delivering the adeno-associated viral vector to a patient, in accordance with some embodiments;

FIG. 4 shows one non-limiting example of a method of delivering an adeno- associated viral vector to a patient, in accordance with some embodiments;

FIG. 5 shows the percent transduction for two formulations comprising AAVs, in accordance with some embodiments;

FIG. 6 shows micrographs of mice to which AAVs have been delivered and negative controls, in accordance with some embodiments;

FIG. 7 shows data from AAV redosing, in accordance with some embodiments;

FIG. 8 shows data associated with three different crystallization conditions, in accordance with some embodiments;

FIG. 9 shows infectivity as a function of PVA concentration, in accordance with some embodiments; and FIG. 10 shows a micrograph of a mouse dosed with AAVs, in accordance with some embodiments.

DETAILED DESCRIPTION

In some embodiments, a device for delivering an adeno-associated viral vector into a patient comprises the adeno-associated viral vector in crystalline form. Advantageously, such devices in some embodiments may be capable of including a relatively large amount of the adeno-associated viral vector in stable form, e.g., in comparison to devices including adeno-associated viral vectors in other forms.

In some embodiments, a device for delivering an adeno-associated viral vector into a patient comprises a hydrophilic polymer and/or a sugar. Such components may advantageously assist, in certain embodiments, with crystallizing the adeno-associated viral vector during device fabrication and/or stabilizing the adeno-associated viral vector in crystalline form after device fabrication. In some embodiments, such components may also enhance the suitability of the device for delivery of the adeno-associated viral vector into the patient. As one example, a device may comprise a hydrophilic polymer that that both assists with crystallizing the adeno-associated viral vector and has mechanical properties that facilitate the penetration of the skin of a patient. As another example, a device may comprise a hydrophilic polymer that undergoes erosion, solubilization, and/or biodegradation in the patient at a rate that promotes the delivery of the adeno-associated viral vector into the patient over a period of time that is desirable. It is of course also possible for a device to comprise hydrophilic polymers and/or sugars that have some, but not all, of these attributes and/or that have none of the above attributes.

Some embodiments relate to methods of fabricating devices for delivering adeno- associated viral vectors into patients. Some such methods may comprise drying a solution comprising the adeno-associated viral vector. The solution may comprise a relatively high amount (e.g., at least 10% w/v, or other amounts such as discussed herein) of a hydrophilic polymer. This may desirably render the solution relatively viscous in certain embodiments, which may enhance the tendency of the adeno-associated viral vector to crystallize.

FIG. 1 shows one non-limiting example of a device 100 including a matrix 102 and an adeno-associated viral vector 104A and 104B. As can be seen in FIG. 1, the adeno- associated viral vector may take the form of a plurality of adeno-associated viral vector domains that are dispersed within the matrix, such as crystallites comprising the adeno- associated viral vector that are dispersed within the matrix. It is also possible for the adeno- associated viral vector to be uniformly distributed through the device (not shown). The adeno-associated viral vector (and/or adeno-associated viral vector domains) may be positioned on a surface of the device (e.g., as shown in FIG. 1, the adeno-associated viral vector 104A is positioned at a surface 106 of the device 100) and/or distributed through the bulk of the device (e.g., as shown in FIG. 1, the adeno-associated viral vector 104B is positioned in the bulk of the device 100). The matrix, too, may be positioned at a surface of the device (e.g., at the surface 106 of device 100 as shown in FIG. 1) and/or dispersed throughout the bulk of the device (e.g., distributed throughout the bulk of the device 100, as also shown in FIG. 1).

In some embodiments, a device comprises a component that comprises the matrix and the adeno-associated viral vector and further comprises an additional component that lacks the adeno-associated viral vector or both the matrix and the adeno-associated viral vector. As an example, a device may further comprise a support, a handle, a delivery device, packaging, and/or another component lacking the adeno-associated viral vector. The further component may comprise the matrix, or may be formed from components other than the matrix. FIG. 2 shows one example of a device 200 that comprises a component comprising the matrix 202 and adeno-associated viral vector 204A and 204B and further comprises a component 208 lacking the matrix and the adeno-associated viral vector. When the device comprises both the component comprising the matrix and the adeno-associated viral vector, the component comprising the matrix and the adeno-associated viral vector may be present at a surface of the device (e.g., as shown in FIG. 2) or may be positioned internal to the device (not shown).

A variety of types of devices may be suitable for delivering adeno-associated viral vectors into patients. In some embodiments, the device is a solid and/or comprises a solid. For instance, non-limiting examples of suitable devices include devices comprising microneedles, patches, microparticles, pills, sutures, and/or surgical threads. It is also possible for a device for delivering adeno-associated viral vectors into patients to comprise a non-solid material, such as a gel (e.g., a hydrogel, a thermo-responsive gel), an emulsion, and/or liquid crystals. In some embodiments, a device comprises a solid material that may be hydrated to form a non-solid material, such as one or more precursors that may be hydrated to form one or more of the foregoing non-solid materials. It is also possible for the non-solid material to be provided in non-solid form and/or not require further hydration. When the components described above are present in a device, the adeno-associated viral vector and/or the matrix may be positioned in the component and/or may be positioned in a different component that is also present in the device.

Without wishing to be bound by any particular theory, it is believed that microneedles may be particularly suitable for delivering adeno-associated viral vectors into patients. Microneedles may be capable of performing delivery that is relatively minimally invasive and/or relatively rapid. Microneedles may also be capable of delivering adeno-associated viral vectors into patients in manners that do not compromise the skin barrier, result in the presence of open skin punctures, and/or lead to scarring. When a device comprises microneedles that comprise adeno-associated viral vectors positioned at a surface thereof, the adeno-associated viral vectors may be positioned at the tips of the microneedles and/or on some or all of the sidewalls of the microneedles. Adeno-associated viral vectors may also be positioned in the bulk of the microneedles.

In some embodiments, a device for delivering adeno-associated viral vectors into patients comprises one of the above-described components that itself comprises another of the above-described components. As one example, in some embodiments, a device comprises a microparticle positioned in a microneedle. As noted above, the adeno-associated viral vector, and, possibly the matrix, may be positioned in either or both components (e.g., the adeno-associated viral vector and, possibly, the matrix may be encapsulated in a microparticle positioned in a microneedle). In embodiments in which an adeno-associated viral vector is present in a microparticle positioned in a microneedle, the microparticle may reduce direct contact between the adeno-associated viral vector and the skin. This may advantageously reduce the immune response of the skin to the adeno-associated viral vector. In some embodiments, a microneedle comprises a microparticle comprising an adeno- associated viral vector that has a higher degree of swelling upon exposure to water (e.g., in a patient’s tissue) than the microneedle. In such embodiments, upon hydration, the microparticle may rapidly break away from the microneedle, thereby releasing the adeno- associated viral vector. It is also possible for a microparticle to swell to a degree that is similar to or lower than the swelling of the microneedle upon exposure to water (e.g., in a patient’s tissue).

Some embodiments relate to methods. As one example, an embodiment may relate to a method of fabricating a device for delivering an adeno-associated viral vector into a patient, such as one or more of the devices described herein. One example of such a method is shown in FIG. 3. As shown in FIG. 3, a method may comprise drying a solution comprising an adeno-associated viral vector, a hydrophilic polymer, and a sugar to form a solid device for delivering the adeno-associated viral vector to a patient (shown by reference sign 310).

It is also possible for one or more steps to be performed prior to this step. For instance, one or more steps that enhance the suitability of the solution for undergoing the drying process may be performed. As shown in FIG. 3, one non-limiting example of such a step is a step in which the solution is centrifuged and/or ultracentrifuged (step 312 in FIG. 3). Without wishing to be bound by any particular theory, these steps may concentrate the solution that it can more readily undergo the drying process and/or separate solid contaminants from the solution so that it can more readily undergo the drying process. Centrifuging and/or ultracentrifuging a solution may also concentrate, sediment, and/or otherwise change the distribution of one or more components present therein (e.g., an adeno- associated viral vector). The resultant distribution may be more advantageous than the initial distribution for delivery of the component to a tissue. As one example, the resultant distribution may concentrate the component into the tip of a needle being formed within a mold, such as a microneedle.

In some embodiments, a method comprises one or more steps subsequent to performing the drying process. As one example, a method may comprise rehydrating the device (step 314 in FIG. 3). This may occur directly prior to use of the device (e.g., as described in further detail below). Without wishing to be bound by any particular theory, it is believed that the device may be more stable during storage when in solid form, but may be easier to introduce into a patient once rehydrated.

Some embodiments relate to methods of delivering adeno-associated viral vectors to patients by employing a device described herein. FIG. 4 shows one non-limiting example of a method of delivering an adeno-associated viral vector to a patient. As shown in FIG. 4, the method may comprise contacting a device comprising a matrix and an adeno-associated viral vector with a patient’s cells (step 416). Contacting the device with the patient’s cells may deliver the adeno-associated viral vector from the device to the patient. If there is adeno- associated viral vector at the surface of the device, this may occur immediately upon contact and/or upon solubilization, erosion, and/or biodegradation of the adeno-associated viral vector in the patient’s tissue. Adeno-associated viral vector present in the bulk of the device may delivered subsequent to solubilization, erosion, and/or biodegradation of any covering material (e.g., any covering component of the device, any covering matrix) in the patient’s tissue. The method may also comprise one or more steps prior to contacting the device with the patient’s cells. As an example, and as shown in FIG. 4, the method may comprise pre- permeabilizing the tissue of the patient (step 418). Without wishing to be bound by any particular theory, it is believed that such pre-permeabilization may enhance the ability of the adeno-associated viral vectors present in the device to be introduced into the patient’s tissue upon contact of the device therewith. Another example shown in FIG. 4 is step 420, in which the tissue of a patient is immunosuppressed prior to the contact of the device with the patient’s cells. This may be accomplished by treating the tissue with an immunosuppressant drug. Without wishing to be bound by any particular theory, it is believed that such immunosuppression may reduce the immune response of the patient to the device and/or to one or more components present in the device (e.g., an adeno-associated viral vector positioned therein).

It is also possible for the method to comprise one or more steps after contact of the device with the patient’s cells. One example of such a step is performing immunosuppression on a patient’s tissue (e.g., as described above). FIG. 4 also shows another example of such a step. In FIG. 4, step 422 comprises contacting the device with the patient’s cells a second time. The second contact between the device and the patient’s cells may deliver a further amount of adeno-associated viral vector to the patient. Without wishing to be bound by any particular theory, it is believed that this may allow for redosing. For instance, a patient may be dosed with an initial dose of the adeno-associated viral vector and then redosed with a second dose of the adeno-associated viral vector from the same device. Advantageously, this may allow for a single device to be employed multiple times, and possibly in multiple locations, in a single patient. It is also possible for a single device to be employed multiple times in the same location in a single patient (e.g., for the device to be contacted with a patient’s cells a third time, a fourth time, or even more times).

In some embodiments, one or more steps are performed concurrently with contact of the device with the patient’s cells. For instance, immunosuppression performed on a patient’s tissue (e.g., as described above) may be performed concurrently with contact of the device with the patient’s cells.

As described above, in some embodiments, a device described herein comprises an adeno-associated viral vector and/or a solution from which a device is formed comprises an adeno-associated viral vector. It is also possible for a device and/or a solution to comprise two or more different adeno-associated viral vectors. The adeno-associated viral vector may take the form of a small (e.g., approximately 5 kb long) virus that can serve as a gene-transfer vehicle. The adeno-associated viral vector itself may be non-enveloped. In some embodiments, the adeno-associated viral vector packages a single- stranded linear DNA genome, which may be a positive strand or a negative strand. Adeno-associated viral vectors may comprise coding regions that are flanked by inverted terminal repeats. The inverted terminal repeats may act as the origins for DNA replication and/or serve as the primary packaging signal. In some embodiments, one of the two inverted terminal repeats includes a small deletion. Adeno-associated viral vectors having this property may be capable of being packaged as self-complementary vectors in which the genome self-anneals after viral uncoating.

A variety of suitable adeno-associated viral vectors may be included in the devices herein. In some embodiments, the adeno-associated viral vector is a recombinant adeno- associated viral vector. Non-limiting examples of suitable adeno-associated viral vectors include those derived from one or more of the following AAV serotypes: AAV1, AAV2 (e.g., rAAV2/2), AAV3, AAV4, AAV5 (e.g., rAAV2/5), AAV6 (e.g., rAAV2/6), AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and variants of the foregoing. The adeno- associated viral vectors may comprise one or more transgenes. The transgenes may be suitable for treating a variety of diseases, such as monogenic skin diseases and/or multigenic skin diseases (e.g., UV-induced aging, intrinsic skin aging). In some embodiments, a transgene may code for a gene that produces proteins that provide protection against pathogens and/or other infectious agents. Adeno-associated viral vectors may be employed to deliver a donor DNA sequence for targeted DNA repair. In some embodiments, an adeno- associated viral vector comprises DNA encoding the LAMB3 gene and/or the COL3A1 gene. Some adeno-associated viral vectors may encode an RNA guided nuclease and/or a guide RNA. Such adeno-associated viral vectors may be capable of performing targeted knockout and/or dual-guided targeted exon skipping.

In some embodiments, an adeno-associated viral vector comprises one or more capsid mutations. The capsid mutation(s) may affect the solubility of the adeno-associated viral vector (e.g., in a patient, in a solution from which the device is formed, in one or more device components), its interaction with extracellular matrix proteins and/or other molecules within the skin, its interactions with cell surface components, its interaction with other adeno- associated viral vector capsids, its interaction with cargo molecules, its hydrophilicity, its pH tolerance, and/or its charge. As one example, in some embodiments, an adeno-associated viral vector comprises a mutation that adds or removes one or more of the following from the capsid: lysine, arginine, aspartic acid, glutamic acid, and histidine.

As another example, in some embodiments, an adeno-associated viral vector comprises a mutation that substitutes a hydrophilic amino acid (e.g., serine, threonine) for a hydrophobic amino acid (or vice versa). Such mutations may enhance the solubility of the adeno-associated viral vector in solutions including other solutes (e.g., polymers, sugars), especially when the other solutes are present at relatively high concentrations.

As a third example, in some embodiments, an adeno-associated viral vector comprises a mutation that substitutes an acidic amino acid (e.g., glutamate, aspartate) for a basic amino acid (e.g., lysine, arginine, histidine). Such mutations may alter the protonation state of the adeno-associated viral vector at one or more values of pH and/or affect the stability of the adeno-associated viral vector at one or more values of pH. Additionally, such mutations may affect the interaction of the adeno-associated viral vector with one or more charged molecules (e.g., salts, metals, ionic polymers) also present in a solution in which the adeno-associated viral vector is dissolved. It is also possible that such mutations may affect interaction of the adeno-associated viral vector with one or more tissue components (e.g., one or more components of skin, extra-cellular matrix, cell surface components). This may, in turn, affect diffusion of the adeno-associated viral vector within the tissue and/or transduction of cells in the tissue.

In some embodiments, a device comprises an adeno-associated viral vector that is crystalline. The crystalline adeno-associated viral vector may be present in the form of crystallites that are dispersed in the matrix.

Adeno-associated viral vectors may be present in the solutions described herein at a variety of suitable concentrations. In some embodiments, a solution may be supersaturated with the adeno-associated viral vector and/or the adeno-associated viral vector may be present in solution in a substantially unaltered state. Supersaturation may be determined by analyzing the solution to assess whether the adeno-associated viral vector crystallizes and/or precipitates from the solution. Such crystallites and precipitates may be detected by turbidity. In some embodiments, a solution comprises the adeno-associated viral vector at a concentration of greater than or equal to 1 • IO¹⁰ GC/pL, greater than or equal to 2 • IO¹⁰ GC/pL, greater than or equal to 5 • IO¹⁰ GC/pL, greater than or equal to 7.5 • IO¹⁰ GC/pL, greater than or equal to 1 • 10¹¹ GC/pL, greater than or equal to 2 • 10¹¹ GC/pL, greater than or equal to 5 • 10¹¹ GC/pL, or greater than or equal to 7.5 • 10¹¹ GC/pL. In some embodiments, a solution comprises the adeno-associated viral vector at a concentration of less than or equal to 1 • 10¹² GC/pL, less than or equal to 7.5 • 10¹¹ GC/pL, less than or equal to 5 • 10¹¹ GC/pL, less than or equal to 2 • 10¹¹ GC/pL, less than or equal to 1 • 10¹¹ GC/pL, less than or equal to 7.5 • IO¹⁰ GC/pL, less than or equal to 5 • IO¹⁰ GC/pL, or less than or equal to 2 • IO¹⁰ GC/pL. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 1 • IO¹⁰ GC/pL and less than or equal to 1 • 10¹² GC/pL). Other ranges are also possible. When a solution comprises two or more adeno-associated viral vectors, each may independently make up an amount of the solution in one or more of the above-referenced ranges and/or all of the adeno-associated viral vectors together may make up an amount of the solution in one or more of the above-referenced ranges.

In some embodiments, adeno-associated viral vectors may be present in the devices described herein at a variety of suitable concentrations. In some embodiments, a device surface comprises adeno-associated viral vectors at a concentration of greater than or equal to 10⁸ GC/cm² (GC stands for genome copies), greater than or equal to 10⁹ GC/cm², greater than or equal to IO¹⁰ GC/cm², or greater than or equal to 10¹¹ GC/cm². In some embodiments, a device surface comprises adeno-associated viral vectors at a concentration of less than or equal to 10¹² GC/cm², less than or equal to 10¹¹ GC/cm², less than or equal to IO¹⁰ GC/cm², or less than or equal to 10⁹ GC/cm². Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 10⁸ GC/cm² and less than or equal to less than or equal to 10¹² GC/cm²). Other ranges are also possible.

As described above, in some embodiments, a device described herein comprises a matrix. In some embodiments, a solution from which a device is formed comprises one or more components that become the matrix upon device fabrication and/or that have a composition. It is also possible for a device described herein to comprise one or more components suitable for inclusion in a matrix in a component of the device that lacks the matrix. Similarly, it is possible for a solution from which a device is formed to comprise one or more matrix components that do not become incorporated into the matrix. As an example, a solution may comprise one or more components described below with respect to the matrix that are incorporated into a crystal comprising an adeno-associated viral vector, are incorporated into another device component, and/or are removed from the solution during device fabrication. Further examples of materials that may be incorporated into the matrix, a device component other than a matrix, and/or a solution for forming a device are described in further detail below. In some embodiments, a matrix, a solution for forming a device, and/or a component of a device comprises a hydrophilic polymer. The hydrophilic polymer may be water- soluble, water-erodible, and/or biodegradable. In some embodiments, the hydrophilic polymer is soluble, erodible, and/or biodegradable when present in a patient’s tissue (e.g., in the tissue with which the device is contacted). The erosion that occurs may comprise surface erosion, bulk erosion, or both. The biodegradation that occurs may comprise biodegradation that is performed by cells and/or molecules (e.g., enzymes, non-enzymatic biomolecules, metabolites, small molecules) in the tissue of the patient with which the device is contacted. The solubility, erosion, and/or degradation of the hydrophilic polymer may occur over a time scale that promotes delivery of the adeno-associated viral vector from the matrix into the patient. As an example, in scenarios where rapid delivery is desirable, the hydrophilic polymer may dissolve, erode, and/or undergo biodegradation in water over a period of time of seconds to minutes. As another example, in scenarios where sustained delivery is desirable, the hydrophilic polymer may dissolve, erode, and/or undergo biodegradation in water over a period of time of days to weeks.

In some embodiments, a hydrophilic polymer present in a solution has one or more properties that facilitate the formation of a device from the solution in a manner that preserves the bioactivity of any adeno-associated viral vector also present in the solution. As one example, the hydrophilic polymer may be capable of being polymerized and/or crosslinked at room temperature, without the use of chemicals that would destabilize the adeno- associated viral vector, without exposure to UV light, and/or without exposure to freeze-thaw cycling. In some embodiments, the hydrophilic polymer may be capable of being crosslinked at a relatively low level.

Cross-linking may comprise forming covalent bonds and/or non-covalent bonds (e.g., the cross-links may comprise covalent bonds and/or non-covalent bonds). Non-limiting examples of suitable non-covalent bonds include hydrophobic interactions, charge -based interactions, polyelectrolyte complexes, hydrogen bonds, stereocomplexes, supramolecular chemical interactions. Cross-linking may be reversible or irreversible. In some embodiments, cross-linking may be reversible upon contact with tissue and/or after implantation in tissue. It is also possible for the cross-linking to be stable upon contact with tissue and/or after implantation in tissue.

Suitable hydrophilic polymers include hydrophilic polymers that are charged and hydrophilic polymers that are uncharged. Without wishing to be bound by any particular theory, it is believed that charged polymers may undergo charge-charge interactions with adeno-associated viral vector when both are present in a device, which may reduce the rate at which the adeno-associated viral vector is released from the device. Non-limiting examples of suitable hydrophilic polymers include polyethylene glycol and its copolymers, polyvinylpyrrolidone, gelatin, poly-gamma-glutamic acid, poly(methylvinylether/maleic anhydride), polyvinylpyrrolidone-polyvinyl alcohol copolymers, poly(vinylpyrrolidone-co- methacrylic acid), poly(vinylpyrrolidone-co-cyclodextrin), polylactic acid, poly glycolic acid, poly(lactic-co-glycolic acid), polycaprolactone and polysaccharides. Non-limiting examples of suitable polysaccharides include dextran, sodium chondroitin sulfate, hydroxypropyl cellulose, carboxymethyl cellulose, hydroxypropyl methylcellulose, sodium alginate, hyaluronic acid, and amylopectin.

The hydrophilic polymer may make up a variety of suitable amounts of the matrix, solution for forming the device (e.g., prior to the removal of any liquid therefrom and/or the performance of any drying steps), and/or the device as a whole. In some embodiments, the hydrophilic polymer makes up greater than or equal to 10% w/v, greater than or equal to 12.5% w/v, greater than or equal to 15% w/v, greater than or equal to 17.5% w/v, greater than or equal to 20% w/v, greater than or equal to 25% w/v, greater than or equal to 30% w/v, or greater than or equal to 35% w/v of the solution for forming the device. In some embodiments, the hydrophilic polymer makes up less than or equal to 40% w/v, less than or equal to 35% w/v, less than or equal to 30% w/v, less than or equal to 25% w/v, less than or equal to 20% w/v, less than or equal to 17.5% w/v, less than or equal to 15% w/v, or less than or equal to 12.5% w/v of the solution for forming the device. Combinations of the abovereferenced ranges are also possible (e.g., greater than or equal to 10% w/v and less than or equal to 40% w/v). Other ranges are also possible. When a solution comprises two or more hydrophilic polymers, each may independently make up an amount of the solution in one or more of the above-referenced ranges and/or all of the hydrophilic polymers together may make up an amount of the solution in one or more of the above-referenced ranges.

In some embodiments, a matrix, a solution for forming a device, and/or a component of a device comprises a polymer is thermoresponsive. The thermoresponsive polymer may have some or all of the properties described above with respect to hydrophilic polymers and the preservation of adeno-associated viral vector function during device formation. The matrix, the solution for forming a device, and/or the component of a device may comprise the thermoresponsive polymer in addition to the hydrophilic polymer and/or the hydrophilic polymer may itself be thermoresponsive. A thermoresponsive polymer may exhibit thermoresponsivity with respect to solubility in water and/or tissue, erodibility in water and/or tissue, and/or gelation in water and/or tissue. For instance, a thermoresponsive polymer may become less soluble in water and/or tissue, less erodible in water and/or tissue, and/or gel in water and/or tissue upon heating. In some embodiments, a thermoresponsive polymer undergoes a transition in one or more properties when the temperature is raised above a certain value. The value may be a value in between the temperature at which it is stored and the temperature of the tissue with which it is contacted (e.g., between room temperature and 37 °C).

In some embodiments, a thermoresponsive polymer comprises at least one repeat group that has a temperature-dependent solubility in water. In some embodiments, a thermoresponsive polymer that is a copolymer is provided. The copolymer may comprise one repeat group that is relatively hydrophilic and another that is relatively hydrophobic. One or both such repeat groups may exhibit a temperature-dependent solubility in water. One non-limiting example of a thermoresponsive copolymer is a polyethylene oxidepolypropylene oxide copolymer. In some embodiments, the polyethylene oxidepolypropylene oxide copolymer is a polyethylene oxide-polypropylene oxide-polyethylene oxide triblock polymer. For instance, the thermoresponsive copolymer may be a pluronic (e.g., Pluronic F-127, Pluronic F-68).

In some embodiments, a solution for forming a device and/or a component of a device (e.g., a support, backing, and/or applicator) comprises a polymer that is not soluble in water, not erodible in water, and/or not biodegradable. In such embodiments, the polymer may remain relatively stable when the device is positioned in a patient. Advantageously, such polymers may support other species and/or components in the device that are soluble in water, erodible in water, and/or biodegradable when the device is positioned in a patient. The soluble, erodible, and/or biodegradable species may dissolve, erode, and/or biodegrade away while the non-soluble and/or non-erodible polymer remains substantially unchanged. The non-soluble and/or non-erodible polymer may retain its mechanical integrity, and may facilitate removal of the device from the patient upon conclusion of the delivery of the adeno- associated viral vector into the patient.

It is also possible for a matrix, a solution for forming a device, and/or a component of a device to comprise a polymer that is not soluble in water, not erodible in water, and/or not biodegradable (e.g., a first polymer) but is in some manner coupled to a component (e.g., a second polymer and/or second component) that is soluble in water, erodible in water, and/or biodegradable. The coupling may take the form of covalent bonding (e.g., when the first polymer and/or repeat units thereof are present in a copolymer that also comprises a second polymer and/or at least some repeat units thereof) and/or encapsulation (e.g., when the first polymer is encapsulated in the second component). Polymers that are not soluble in water, erodible in water, and/or biodegradable but are coupled to a component that has one or more of these properties may have some or all of the properties described above with respect to hydrophilic polymers and the preservation of adeno-associated viral vector function during device formation. Non-limiting examples of polymers that are neither soluble in water nor erodible in water nor biodegradable include polystyrene and polycarbonate.

In some embodiments, a matrix, a solution for forming a device, and/or a component of a device comprises a sugar. The sugar may be water-soluble and/or water-erodible. Nonlimiting examples of suitable sugars include sucrose, trehalose, and/or raffinose, etc. Other sugars that may be used, e.g., as a cryoprotectant or a lyoprotectant, will be known to those of ordinary skill in the art.

The sugar may make up a variety of suitable amounts of the matrix, solution for forming the device, and/or the device as a whole. In some embodiments, the sugar makes up greater than or equal to 10% w/v, greater than or equal to 12.5% w/v, greater than or equal to 15% w/v, greater than or equal to 17.5% w/v, greater than or equal to 20% w/v, greater than or equal to 25% w/v, greater than or equal to 30% w/v, greater than or equal to 35% w/v, greater than or equal to 40% w/v, or greater than or equal to 45% w/v of the solution for forming the device. In some embodiments, the sugar makes up less than or equal to 50% w/v, less than or equal to 45% w/v, less than or equal to 40% w/v, less than or equal to 35% w/v, less than or equal to 30% w/v, less than or equal to 25% w/v, less than or equal to 20% w/v, less than or equal to 17.5% w/v, less than or equal to 15% w/v, or less than or equal to 12.5% w/v of the solution for forming the device. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 10% w/v and less than or equal to 50% w/v, or greater than or equal tol0% w/v and less than or equal to 20% w/v). Other ranges are also possible. When a solution comprises two or more sugars, each may independently make up an amount of the solution in one or more of the above-referenced ranges and/or all of the sugars together may make up an amount of the solution in one or more of the abovereferenced ranges.

In some embodiments, a matrix, a solution for forming a device, and/or a component of a device comprises a salt. The salt may be water-soluble and/or water-erodible. When present in the solution, the salt may be a buffering agent. It is also possible for the salt to facilitate crystallization of the adeno-associated viral vector and/or to assist with maintaining the stability of the adeno-associated viral vector. Non-limiting examples of suitable salts include phosphate salts, carbonate salts, borate salts, l-ethyl-3-methylimidazolium trifluoromethanesulfonate and l-ethyl-3-methylimidazolium acetate.

The salt may make up a variety of suitable amounts of the matrix, solution for forming the device, and/or the device as a whole. In some embodiments, the salt makes up greater than or equal to 20 mM of the matrix, solution for forming the device, and/or the device as a whole.

In some embodiments, a matrix, a solution for forming a device, and/or a component of a device comprises a small molecule. The small molecule may be water-soluble and/or water-erodible. In some embodiments, the small molecule may serve as a solvent, a surfactant (e.g., a non-ionic surfactant), a buffering agent, a drug (e.g., an immunosuppressant drug, a pain medication, a drug that targets skin aging), and/or a prodrug (e.g., a prodrug for any of the drugs described herein). It is also possible for the small molecule to facilitate crystallization of the adeno-associated viral vector and/or to assist with maintaining the stability of the adeno-associated viral vector.

Non-limiting examples of suitable small molecules include bis-tris propane, HEPES, citric acid, 2-propanol, succinic acid, acetic acid, creatinine, MES, bis-tris methane, ADA, ACES, MOPSO, PIPES, imidazole, BES, MOPS, TES, TAPSO, HEPSO, tris, tricine, bicine, TAPS, ammediol, CHES, ethanolamine, CAPSO, glycerol, CAPS, cholamine chloride, DIPSO, acetamidoglycine, POPSO, HEPPSO, HEPPS, glycinamide, glycylglycine, mannitol, sorbitol, Tween-80, polysorbate 80, nucleic acids, and amino acids. Non-limiting examples of suitable amino acids include serine, arginine, aspartic acid, glutamic acid, glycine, histidine, lysine, and proline.

Non-limiting examples of suitable small molecules that are immunosuppressant drugs include dexamethasone, prednisone, cyclosporin, rapamycin, tacrolimus, and mycophenolate mofetil. The immunosuppressant drugs may reduce immune cell infiltration and/or blunt cytotoxic T-cell and/or B-cell response to the adeno-associated viral vector.

Non-limiting examples of suitable small molecules that are drugs that target skin aging include senolytics that upregulates pro-apoptotic proteins to induce cell death (e.g., inhibitors to the proteins of the BCL-2 family), drugs that target specific pathways (e.g., P13K, AKT, mTOR, MDM2, HSP90, HDAC, OXR1, TAK1, JAK, p38 MAPK, IKK/NF- kB), drugs that target autophagy, drugs that target metabolism, drugs that target lysosomal activity, nucleic acids that target telomeric sequences that drive cellular DNA damage response (e.g., antisense oligonucleotides having this property), drugs that act upon melanogenesis (e.g., along the p53, p21, and/or Wnt pathways), drugs that modulate tyrosinate levels, drugs that modulate the cell cycle, drugs that affect production and/or translocation of melanosomes in the epidermis, drugs that regulate autophagy, drugs that inhibit cytokines (e.g., cytokines involved in paracrine effects and/or factors that induce angiogenesis, collagen degradation and/or other external stimuli affecting melanin synthesis, photoaging and/or intrinsic aging of skin), drugs that treat hyperpigmentation (e.g., age spots), drugs that lighten skin, drugs that treat fine lines, drugs that treat wrinkles, drugs that inhibit factors locally secreted from aged and/or senescent fibroblasts (e.g., GDF15, SCF, SDF1), and/or drugs that are inhibitors to pro-aging blood factors.

In some embodiments, a matrix, a solution for forming a device, and/or a component of a device comprises an antibody-cleaving enzyme. Without wishing to be bound by any particular theory, it is believed that antibody-cleaving enzymes may inhibit pre-existing neutralizing antibodies by reducing their binding to adeno-associated viral vectors. This may allow for enhanced delivery of the adeno-associated viral vector, reduced phagocytosis thereof, and/or reduced transport of adeno-associated viral vector-derived antigens to lymph nodes by antigen-presenting cells. One non-limiting example of an antibody-cleaving enzyme is a cysteine protease that cleaves immunoglobulin G (e.g., specifically).

In some embodiments, a matrix, a solution for forming a device, and/or a component of a device comprises one or more agents for performing gene editing. Non-limiting examples of such agents include charged polymers (e.g., cationic polymers, such as supercharged cationic polymers), charged lipids (e.g., cationic lipids), and/or functional effector proteins (e.g., RNA-guided genome editing enzymes, such as Cas9 and/or Casl2a nucleases, ZFN, and/or TALEN). Functional effector proteins may be capable of performing a single dsDNA break (e.g., to deactivate a gene), may be capable of performing a double dsDNA break (e.g., to perform exon skipping), may be a base editor, and/or may be capable of being employed to knock-in a therapeutic sequence into a genome. In some embodiments, an agent for performing gene editing comprises a genome editing enzyme that is delivered as a ribonuclear protein that is complexed with a cationic and/or ionizable polymer.

In some embodiments, a solution comprises a combination of components that promote protein-protein intermolecular interactions between the adeno-associated virus present in the solution. Without wishing to be bound by any particular theory, such interactions may promote supersaturation and/or crystallization. Also without wishing to be bound by any particular theory, such interactions may depend on the charge of the adeno- associated viral vector, its surface zeta potential, and its concentration in the solution. In some embodiments, a solution described herein comprises water. In other words, it may be an aqueous solution, such as a phosphate-buffered saline solution. Non-limiting examples aqueous solutions that may be particularly suitable for forming devices include: an aqueous solution comprising 16% w/v polyvinyl alcohol 31000, 27% w/v polyethylene glycol 3350, 16% w/v sucrose, 5% w/v l-3thyl-3-methylimidazolium trifluoromethanesulfonate, and 0.09 M bis-tris propane; and an aqueous solution comprising 16% w/v polyvinyl alcohol 31000, 27% w/v polyethylene glycol 3350, 16% w/v sucrose, 5% w/v l-ethyl-3- methylimidazolium acetate, and 0.09 M HEPES.

In some embodiments, a solution described herein has a relatively high viscosity. Advantageously, the relatively high viscosity may facilitate the formation of devices that have desirable structural properties (e.g., in which the adeno-associated viral vector is crystalline.

The devices described herein, and/or components thereof that comprise the matrix and/or the adeno-associated viral vector, may have one or more properties that facilitate the delivery of an adeno-associated viral vector into a patient. As described above with respect to the matrix, the device and/or one or more components thereof (e.g., one or more components comprising the adeno-associated viral vector and/or the matrix, one or more other components) is water-soluble, water-erodible, and/or biodegradable.

In some embodiments, the device and/or one or more components thereof (e.g., one or more components comprising the adeno-associated viral vector and/or the matrix, one or more other components) assists with stabilizing the adeno-associated viral vector. For instance, it may protect the adeno-associated viral vector from undergoing agglomeration, unfolding, mechanical disruption, and/or loss of infectivity during storage.

It is also possible for the device and/or one or more components thereof (e.g., one or more components comprising the adeno-associated viral vector and/or the matrix, one or more other components) to be non-toxic and/or non-immunogenic. Such devices and/or components may allow the device to be contacted with a patient’s tissue more than once without causing the patient to exhibit an adverse event. It is also possible for such devices and/or components to allow the device to be contacted with a patient’s tissue more than once without causing additional transgene expression (e.g., by a mechanism involving neutralization of adeno-associated viral vector particles positioned therein and/or clearance of transduced cells).

It is also possible for the device and/or one or more components thereof (e.g., one or more components comprising the adeno-associated viral vector and/or the matrix, one or more other components) to be capable of inducing an immune response specific to an antigen present in the a component of the adeno-associated viral vector (e.g., a transgene therein). In some embodiments, the device and/or one or more components thereof (e.g., one or more components comprising the adeno-associated viral vector and/or the matrix, one or more other components) provides protection to the patient against pathogens and/or other infectious agents. This may be accomplished by the inclusion of adeno-associated viral vectors that include genes for proteins that provide such protection.

In some embodiments, a device comprises one or more components that are relatively tough and/or rigid. Such components may facilitate the penetration of the device into a patient’s tissue and/or extraction of the device from a patient’s tissue (e.g., without undergoing breakage). In other words, they may have a suitable toughness and/or rigidity to be capable of penetrating the patient’s tissue and/or being extracted therefrom. The tough and/or rigid component(s) of the device may include component(s) of the device that comprise the adeno-associated viral vector and/or the matrix, and/or may comprise component(s) of the device that provide mechanical support to such components. As one example, in some embodiments, a device comprises microneedles that are relatively tough and/or rigid. The microneedles may comprise the adeno-associated viral vector and the matrix.

In some embodiments, a device may be shelf-stable without refrigeration. For instance, some devices may be shelf-stable for up to two weeks at room temperature. Advantageously, devices that are shelf-stable without refrigeration may be capable of being manufactured in larger and/or less frequent production runs than other types of devices, which may reduce production cost. Such devices may also be capable of being transported without the need for refrigerated or cryogenic shipping.

As described above, some devices may comprise microneedles. When present, the microneedles may be attached to be attached to an additional device component, such as a device component lacking the adeno-associated viral vector and-or the matrix. In some embodiments, the additional component may support the microneedles and/or facilitate the penetration of the microneedles into a patient’s tissue.

One non-limiting example of a suitable additional component is a backing (e.g., a solid plastic backing). The backing may be relatively rigid or may be flexible. Without wishing to be bound by any particular theory, it is believed that flexible backings may facilitate the application of the microneedles to a tissue having a relatively uneven topography. In some embodiments, a backing further comprises an adhesive (e.g., an adhesive that adheres the backing to the skin upon penetration of microneedles thereinto).

Another non-limiting example of a suitable additional component is an applicator, such as a spring-loaded applicator. Some suitable spring-loaded applicators may be capable of being rotated and/or offset after insertion of the microneedles into a patient’s tissue. This may facilitate detachment of the microneedles from the applicator (and retention thereof in the patient).

When present, microneedles may have any of a variety of suitable shapes. Nonlimiting examples of suitable shapes that microneedles may have include inverted square pyramids, tetrahedrons, hexagonal pyramids, and cones.

Microneedles may have a variety of suitable widths at their bases. In some embodiments, a device comprises a plurality of microneedles having an average width at the base of greater than or equal to 150 microns, greater than or equal to 200 microns, greater than or equal to 250 microns, greater than or equal to 300 microns, greater than or equal to 350 microns, greater than or equal to 400 microns, greater than or equal to 450 microns, greater than or equal to 500 microns, greater than or equal to 550 microns, greater than or equal to 600 microns, greater than or equal to 650 microns, greater than or equal to 700 microns, or greater than or equal to 750 microns. In some embodiments, a device comprises a plurality of microneedles having an average width at the base of less than or equal to 800 microns, less than or equal to 750 microns, less than or equal to 700 microns, less than or equal to 650 microns, less than or equal to 600 microns, less than or equal to 550 microns, less than or equal to 500 microns, less than or equal to 450 microns, less than or equal to 400 microns, less than or equal to 350 microns, less than or equal to 300 microns, less than or equal to 250 microns, or less than or equal to 200 microns. Combinations of the abovereferenced ranges are also possible (e.g., greater than or equal to 150 microns and less than or equal to 800 microns). Other ranges are also possible. The width of a microneedle at its base may be determined by measuring the longest dimension of the microneedle base perpendicular to the direction of microneedle projection. The average width of a plurality of microneedles at the base may be determined by averaging the widths of the microneedles in the plurality of microneedles at their bases.

Microneedles may have a variety of suitable heights (i.e., lengths in the direction of projection from their bases). In some embodiments, a device comprises a plurality of microneedles having an average height of greater than or equal to 500 microns, greater than or equal to 800 microns, greater than or equal to 1 mm, greater than or equal to 1.5 mm, greater than or equal to 2 mm, greater than or equal to 2.5 mm, greater than or equal to 3 mm, or greater than or equal to 3.5 mm. In some embodiments, a device comprises a plurality of microneedles having an average height of less than or equal to 4 mm, less than or equal to 3.5 mm, less than or equal to 3 mm, less than or equal to 2.5 mm, less than or equal to 2 mm, less than or equal to 1.5 mm, less than or equal to 1 mm, or less than or equal to 800 microns. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 500 microns and less than or equal to 4 mm). Other ranges are also possible. The average height of a plurality of microneedles may be determined by averaging the heights of the microneedles in the plurality of microneedles.

Without wishing to be bound by any particular theory, it is believed that microneedles having different heights may be suitable for delivering adeno-associated viral vectors to different depths in a patient’s skin. For instance, microneedles having heights between 500 microns and 800 microns may be particularly suitable for delivering adeno-associated viral vectors to the epidermal-dermal junction, microneedles having heights between 800 microns and 2 mm may be suitable for delivering adeno-associated viral vectors to the dermis, and/or microneedles having heights between 2 mm and 4 mm may be suitable for delivering adeno- associated viral vectors to muscle.

Microneedles may extend at a variety of suitable angles from a backing. In some embodiments, microneedles extend from the backing at angle relatively close to perpendicular. It is also possible for microneedles to extend from the backing at an oblique angle. In some embodiments, microneedles extend from a backing at an angle of greater than 0°, greater than or equal to 5°, greater than or equal to 10°, greater than or equal to 15°, greater than or equal to 20°, greater than or equal to 25°, greater than or equal to 30°, greater than or equal to 35°, or greater than or equal to 40°. In some embodiments, microneedles extend from a backing at an angle of less than or equal to 45°, less than or equal to 40°, less than or equal to 35°, less than or equal to 30°, less than or equal to 25°, less than or equal to 20°, less than or equal to 15°, less than or equal to 10°, or less than or equal to 5°. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0° and less than or equal to 45°). Other angles are also possible. The ranges described above may independently characterize the angle of any particular microneedle with respect to a backing and/or the average angle of all of the microneedles present with respect to a backing. Microneedles may extend from backings at relatively constant angles or over a range of angles. Microneedles may be positioned over a variety of suitable areas. In some embodiments, a plurality of microneedles extends over an area (e.g., an area on a device component supporting the microneedles) of greater than or equal to 1 cm², greater than or equal to 2 cm², greater than or equal to 5 cm², greater than or equal to 7.5 cm², or greater than or equal to 10 cm².

A device may comprise a variety of suitable numbers of microneedles. In some embodiments, a device comprises greater than or equal to 2 microneedles, greater than or equal to 5 microneedles, greater than or equal to 7 microneedles, greater than or equal to 10 microneedles, greater than or equal to 15 microneedles, greater than or equal to 20 microneedles, greater than or equal to 30 microneedles, greater than or equal to 50 microneedles, greater than or equal to 75 microneedles, greater than or equal to 100 microneedles, greater than or equal to 150 microneedles, greater than or equal to 200 microneedles, greater than or equal to 300 microneedles, greater than or equal to 500 microneedles, or greater than or equal to 750 microneedles. In some embodiments, a device comprises less than or equal to 1000 microneedles, less than or equal to 750 microneedles, less than or equal to 500 microneedles, less than or equal to 300 microneedles, less than or equal to 200 microneedles, less than or equal to 150 microneedles, less than or equal to 100 microneedles, less than or equal to 75 microneedles, less than or equal to 50 microneedles, less than or equal to 30 microneedles, less than or equal to 20 microneedles, less than or equal to 15 microneedles, less than or equal to 10 microneedles, less than or equal to 7 microneedles, or less than or equal to 5 microneedles. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 2 microneedles and less than or equal to 1000 microneedles). Other ranges are also possible.

Microneedles may be spaced from each other at a variety of suitable spacings. In some embodiments, the spacing may be sufficient to prevent skin infection upon contact of the microneedles with a patient’s skin.

As described above, some embodiments relate to methods in which a solution is dried to form a solid device and/or a component of a solid device. The solution may be dried in a variety of suitable manners, non-limiting examples of which include lyophilization and extrusion. It is also for the drying to comprise precipitation and/or centrifugation. In some embodiments, the solution is dried in contact with a gas, such as air. The fluid may be undergoing laminar flow (e.g., a gas may contact the solution and dissolves fluid evaporating from the solution during drying). Without wishing to be bound by any particular theory, it is believed that drying under laminar flow may result in the removal of liquid from the solution at a relatively uniform and steady rate. This is believed to facilitate the formation of devices and/or components thereof in the form of uniform solid articles that have relatively low levels of undesirable imperfections, such as voids, cracks, and the like. It is also possible for a solution to be dried in the presence of a desiccant (e.g., in a desiccation chamber). In some embodiments, a solution is dried by performing two or more of the above-described processes sequentially (e.g., drying under laminar flow and then in a desiccation chamber).

In some embodiments, a solution from which a device and/or a device component is formed may be dried in a manner such that the adeno-associated viral vector crystallizes during drying. Crystallization may be facilitated by the inclusion of components in the solution that nucleate crystallites and/or reduce the solubility of the adeno-associated viral vector in the solution or matrix (as described above with respect to the types of components that may be included in solutions and matrices) and drying at a rate that is sufficiently slow to allow crystals to form.

In some embodiments, a solution from which a device and/or a device component is formed may be dried in a mold. As one example, in some embodiments, a solution is dried in a mold comprising indentations having the shape of microneedles. Subsequent to drying, the resultant solid device may be removed from the mold and have a surface topography that is the inverse of the mold. When employed, molds may be formed from a variety of suitable materials, one non-limiting example of which is PDMS.

As also described above, some methods comprise centrifuging and/or ultracentrifuging a solution to be dried. The centrifuging and/or ultracentrifuging may be performed at a temperature of less than or equal to 4 °C.

The solution may be stored for a period of time prior to drying (e.g., prior to undergoing centrifugation and/or ultracentrifugation, after undergoing centrifugation and/or ultracentrifugation). The solution may be stable throughout the storage period (e.g., the adeno-associated viral vector disposed therein may not lose an appreciable degree of functionality). In some embodiments, the solution may be stored for greater than or equal to 2 weeks, or greater than or equal to 1 month.

As described above, some embodiments relate to methods in which a device is contacted with a patient’s cells to deliver adeno-associated viral vector to the patient. A variety of suitable patients may be treated in this manner. The patient may be a human or a non-human mammal (e.g., a non-human primate, a canine, an equine, a feline, a porcine, a bovine, an ungulate, a lagomorph. In various embodiments, the subject can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, as an outpatient, or other clinical context. In certain embodiments, the subject may not be under the care or prescription of a physician or other health worker.

Contacting a patient’ s cells with the devices described herein may be performed in a variety of ways. As one example, contacting the device with the patient’s cells may comprise injecting the device and/or a portion of the device into the patient. As further examples, contacting the device with the patient’s cells may comprise disposing the device on a patient’s tissue (e.g., skin, cornea, heart, kidney), implanting the device in the patient, and/or having the patient ingest the device.

Pre-permeabilization of a patient’s tissue may comprise sonication, heating, microneedling, and/or micro-coring the patient’s tissue and/or any tissue of the patient covering the tissue to which the adeno-associated viral vector is to be delivered (e.g., the patient’s skin). One or more of these techniques may enhance the permeability of the patient’s tissue to the device, which may reduce the amount of force needed to be applied to the device to cause it to contact the patient’s tissue.

In some embodiments, a method comprises employing a device described herein to deliver the adeno-associated viral vector to a target site. The target site may comprise one or more portions of a patient’s tissue (e.g., skin, epidermal tissue, dermal tissue, hair follicles, sebaceous glands). In some embodiments, a target site comprises skin cells, immune cells, resident cells (e.g., resident immune cells), and/or transient cells (e.g., transient immune cells). For example, the target site may comprise epidermal skin cells (e.g., epidermal cycling progenitor cells, epidermal stem cells), fibroblasts, dermal fibroblasts (e.g., senescent dermal fibroblasts), keratinocytes, melanocytes, Langerhans cells, Merkel cells, and/or hair follicle stem cells. Non-limiting examples of target sites include the dermal-epidermal junction, the dermis, follicles, glands, muscle, and corneas. In some embodiments, delivery to a target site comprises delivery through one or more layers of skin (e.g., across the epidermal layers, to the skin’s basal layer, into the upper and/or middle layers of the dermis). The skin may be healthy skin, diseased skin, and/or hair-bearing skin. The skin may be intact, and, in some embodiments, may surround open skin (e.g., skin comprising wounds, such as chronic wounds, and/or skin comprising a compromised barrier).

Delivery to the target site may be performed in vivo or ex vivo. In some embodiments, spread of the adeno-associated viral vector beyond the target site may be relatively limited. For instance, the spread of the adeno-associated viral vector may be limited to the tissue cells bound by vasculature and/or prevented from entering into the blood stream and/or throughout the patient’s circulatory system. This may be accomplished by employing a device in which the adeno-associated viral vector is provided in a form that has a viscosity too high to allow for appreciable transport beyond the target site.

Delivery of an adeno-associated viral vector to a patient may be performed to treat a variety of conditions. In some embodiments, a skin condition is treated. Non-limiting examples of skin conditions that may be treated include genetic skin conditions like Junctional Epidermolysis Bullosa (e.g., that manifests with chronic and/or acute localized skin wounds), intrinsic skin aging dysfunctions (e.g., wrinkles), and extrinsic skin aging dysfunctions (e.g., UV-damaged skin cells, which may be resistant to apoptosis, have entered senescence, and/or exhibit ROS and/or DNA damage).

U.S. Pat. Apl. Ser. No. 63/485,377, filed February 16, 2023, is incorporated herein by reference in its entirety for all purposes.

EXAMPLE 1

To assay the effect of polyvinyl alcohol (PVA)/sucrose solutions in stabilizing the function adeno-associated viral vector (AAV) particles during desiccation-based concentration, rAAV2-EGFP particles were diluted in into either IX PBS or IX PBS with PVA/sucrose 16%/16% w/v. These dilutions were treated as “dried” following drying in a laminar flow hood overnight and then drying for an additional 48 hours in a vacuum desiccator, after which they were rehydrated at 37 °C for 1 hour. The viral preparations so- treated were then applied to 293T cells in culture. Function of the AAV vector particles was measured by gene expression of the reporter gene by flow cytometry of the treated 293T cells, assessing the % of cells transduced as a function of vector preparation, and shown in FIG. 5. As can be seen from FIG. 5, the devices prepared from solutions comprising the PVA and sucrose exhibited a much higher efficiency of transduction than those prepared from the solutions lacking the sucrose and PVA.

EXAMPLE 2

To test the ability of microneedle-based AAV formulations to enable redo sable delivery to the skin, mice were treated with l*10ⁿ GC particles of AAV-luciferase through either our microneedle-based delivery method (MN) or a standard intradermal (ID) injection. One day prior and on the day of administration, mice were subjected to local, immune suppression via topical application of a solution of dexamethasone in ethanol. 39-days post injection, expression of the AAV-delivered luciferase transgene was measured by IVIS following IP injection of D-luciferin. 39 days after the first injection, mice were subjected to AAV vector challenge to assess the effect of MN vs ID dosing on the ability to re-dose. This vector redosing challenge proceeded through another course of topical immune suppression, and all mice were ID injected with l*10ⁿ GC vector doses. To assess local versus systemic immune effects, two such doses were delivered per mouse, at both the original first-dose skin site or at a distal site.

Re-dosing in immunocompetent, hairless SKH1 mice was assessed at day 39. Animals were pretreated with 0.25% dexamethasone applied topically. Re-dosing was tested at a previously treated site (lower back) and at a distal site (upper back), shown in FIGs. 6 and 7. The intensity of the luminescence provided by the luciferase is shown by heat maps in FIG. 6 and is the parameter measured by the y-axis of FIG. 7.

EXAMPLE 3

1536 crystallization screening experiments were performed to determine conditions that would facilitate AAV crystallization. The types of solutions tested included those comprising: (1) salt and buffer (36 salts at three concentrations combined with eight buffers); (2) PEG, salt, and buffer (eight PEGs at two concentrations, combined with 36 salts and eight buffers); and (3) commercially available screening solutions. Some of these solutions were further modified to include PVA and/or sucrose. As shown in FIG. 8, three solutions produced crystallization conditions for rAAV2.

EXAMPLE 4

Polymer microneedle formulations of AAV in hypertonic solutions in the presence of sugars were tested. The effects of osmolarity on AAV activity at various salt concentrations were also explored. Additionally, the in vitro infectivity of AAV embedded in polymeric matrices which are hydrophilic and uncharged are described. Various mixtures of hydrophilic synthetic polymers and sugar-based materials were explored. More specifically, selected electrochemical properties (ionic strength and tonicity) and their influence on AAV mono-dispersity and infectivity were assessed.

Table 1 lists three exemplary formulations. Briefly, they were prepared by: (1) Concentrating AAV particles to an amount of 2.7* IO¹⁰ GC in 1 microliter of solution; (2) Performing overnight dehydration of the solution at room temperature; (3) Rehydrating the dehydrated solution in cell culture media; and (4) Performing infectivity testing in human embryonic kidney (HEK293T) cells. The gene expression of fluorescent reporter gene mKate driven by rAAV2.CMV.mKate2.WPRE3 vector pre-mixed as described in the chemical formulations shown in Table 1 was assessed.

Table 1.

As can be seen from Table 1, formulations including sucrose, PVA, and sodium phosphate exhibited higher levels of infectivity than the positive control. FIG. 9, which depicts infectivity as a function of PVA concentration, also supports this conclusion. The ability of rAAV2.CASI.Luc2.WPRE in Formulation 1 (described in Table 1) to deliver transgenes uniformly over a contiguous area of intact skin in vivo was assessed using an immunocompromised mouse model. AAV2 particles carrying a luciferase transgene cargo were formulated into a 1 cm² microneedle array patches using either side-wall loaded (method F:A in Table 2) or uniformly loaded (method F:B in Table 2). Patches were applied topically to the skin on the left side and right side of the backs, respectively of hairless mice through a spring-loaded applicator. Seven days post-administration, the mice were injected IP (intraperitoneally) with D-luciferin and subjected to whole-body luminescence imaging to detect luciferase transgene expression. Both tested formulations resulted in transgene expression in the skin, while Method F:B was 4-fold more intense, with uniform expression covering 36% of the treated skin area.

Table 2.

FIG. 10 depicts the delivery of AAVs to mouse skin from polymer patches including a 1 cm by 1 cm microneedle area (including 100 microneedles total). Polymer Microneedle Fabrication

Water-soluble microneedle arrays encapsulating AAV-particles were fabricated using custom casting processes. Two distinct fabrication processes were tested - sidewall-loaded and uniformly-loaded. Sidewall-loaded: An AAV suspension at a concentration of 2.7* IO¹⁰ GC/microliter was eluted in 5% w/v sorbitol, 0.001% v/v Pluronic F68 and IX PBS to form the AAV mix referenced in Tables 1 and 2 above. It was then pipetted onto the top of the PDMS mold (20 microliters per array), and centrifuged at 3000 RCF for 10 min. Excess solution was cleared from the top of the mold, the array was inspected for air bubbles, and the solution was allowed to dry leaving behind particles at the tips and walls of the mold. This process was repeated when loading was lower than desired, unevenly distributed, or air bubbles formed. About 200 microliters of a 16% (w/v) sucrose and 16% (w/v) PVA solution in water was then dispensed on top of the mold and centrifuged at 3000 RCF for 10 min.

Uniformly -loaded: An AAV suspension at a concentration of 2.7* 10¹⁰ GC/pl was directly eluted in a 16% (w/v) sucrose and 16% (w/v) PVA solution at a dilution of 1:10 resulting in 14.4% (w/v) sucrose and 14.4% (w/v) PVA solution. It was then pipetted onto the top of the PDMS mold (20 microliters per array), and centrifuged at 3000 RCF for 10 min. Excess solution was cleared from the top of the mold, the array was inspected for air bubbles, and the solution was allowed to dry leaving particles uniformly dispersed throughout the structures of the mold. This process was repeated when loading was lower than desired, unevenly distributed, or air bubbles formed. About 200 microliters of a 14.4% (w/v) sucrose and 14.4% (w/v) PVA solution in water was then dispensed on top of the mold and centrifuged at 3000 RCF for 10 min.

In both cases of sidewall-loaded and uniformly-loaded arrays, the PDMS molds were left overnight in a laminar flow hood at room temperature. Acrylic discs were then fixed to double-sided tape and attached to the back side of the solidified AAV-loaded polymer microneedle arrays. The arrays were then separated from the mold and stored under vacuum desiccation to complete drying for 48 hours prior to use.

Plasmid

All ITR plasmid vectors were transformed into NEB Stable or Agilent SURE2 Supercompetent cells. 2 liters of LB with 100 microliters/mL Carbenicillin were inoculated with bacterial stock and grown at 30 °C for 24 hours in a shaker incubator. Plasmid was then isolated using Qiagen Plasmid Plus Gigaprep kits following the manufacturers protocol. All other plasmids were grown for 14-16 hours at 37 °C. All plasmids were checked for recombination via linearization using a restriction digest and running on an agarose gel. Non-linearized plasmid was also run on an agarose gel to ensure high quality >95% supercoiled plasmid was used during transfections. The AAV vector backbone contained AAV2-ITR1 in flip and ITR2 in flop orientations each 145bp in length annealed to a plasmid vector encoding either reporter or therapeutic genes. Other transgenes which were used in this study included Luciferase2 and mKate2 whose expression was driven from a CASI and CMV promoters. In all cases, the vectors contained signal elements for WPRE or WPRE3 (the short version of WPRE).

Tissue Culture

HEK293T (ATCC) cell cultures were maintained in T150 tissue culture flasks (Coming) in DMEM high glucose with GlutaMAX (Life Technologies) supplemented with 10% (v/v) FBS (Genclone) and 1% Pen/Strep (Life Technologies). Cells were passaged 3-4 times a week during maintenance and kept for 25 passages. Approximately 8*10⁷ HEK293T cells were seeded onto each T875 5-layer flask (Corning) 48 hours before transfection. During large-scale passaging 100 micromolar cell strainers were used to filter dissociated cells prior to addition of media to remove clumps. Cells were counted using either a Muse (EMD Millipore) cell counter or Countess II FL (Life Technologies) immediately prior to seeding.

Vector Particle Production

HEK293T cells were transfected 48 hours following seeding into 5-layer flasks as described above and at 70-80% confluency on day of transfection. 2x 5-layer flasks were prepared for each ITR plasmid. Plasmid ratios of 2:2: 1 (200 micrograms of adenovirus helper plasmid, 200 micrograms of AAV capsid plasmid, and 100 micrograms of the ITR- transgene-ITR plasmid) were added to 7.5 mL of serum-free DMEM after which 2.0 mL of PEI MAX 50K (at a concentration of 1 microgram/microliter) was added. Plasmid and PEI solution were mixed gently by agitation and allowed to incubate at room temperature for 15 min. During this time media in the 5-layer flasks were removed via pouring and new volumes of fresh complete media were prepared (125 mL per 5-layer flask). After incubation, the plasmid and PEI solution were mixed into the freshly prepared media and added onto the tissue culture flasks. Flasks were then moved back into the tissue culture incubator (37 °C 5% CO2).

On day 3 following the transfection, additional complete media was added to each of the flasks (62.5 mL per flask) to allow further virus production. Cultures were untouched until day 6 when they were harvested and prepared for downstream processing via NaCl dissociation and lysis. A 5 M solution of NaCl was prepared and sterile filtered. 20 mL of this was added to each 5-layer flask for a final concentration of 0.5 M NaCl in each flask and mixed to ensure equal distribution across layers. Flasks were then put back into the incubator for 3 hours, collected by gentle agitation to completely dissociate cells, and poured into polypropylene collection bottles. These bottles were placed at 4 °C overnight to allow the majority of the cellular protein and nucleic acids to settle to the bottom.

Due to the amount of cellular proteins and nucleic acid aggregates collected with viral particles, initial clarification of the harvest was required. After an overnight incubation at 4 °C, the bottles included a mostly transparent supernatant and a viscous and sticky sediment on the bottom. The supernatant was poured into 0.22 micromolar bottle top vacuum filtration systems while the solids were retained in the bottles and discarded. Supernatant was then filtered. Replacement of the filter due to fouling was necessary and was performed when approximate flow rate fell below 1 mL/min. This process took several hours and was performed on ice.

Concentration of viral capsid proteins from this large volume of clarified supernatant was then performed via PEG precipitation. A solution of 40% (w/v) PEG8000 in ddlLO was added to the bottles of clarified supernatant for a final concentration of 8% (1:4 dilution). The bottles were agitated to ensure even mixing before placing back at 4 °C overnight to allow precipitation. The supernatant went from a clear red solution to a cloudier appearance during this process. This step was not allowed to go longer than 1 overnight incubation.

The following day the PEG/supernatant mixture was poured into 500 mL conical centrifuge tubes and centrifuged at 3500G for 15 min. Supernatant was removed via aspiration, without disturbing the pellet. Pellet was then resuspended in 8 mL of PBS. 0.8 pL Benzonase was then added to this resuspended viral pellet and incubated at 37 °C for 45 min to remove residual DNA. After incubation, the solution (~8 mL in volume) was ready for purification via ultracentrifugation.

Purification

Purification of AAV capsids were performed via ultracentrifugation through an iodixanol density gradient. Solutions of 15%, 25%, 40%, and 54.5% iodixanol (all v/v) were prepared from a stock solution of 60% iodixanol. The 15% solution was diluted using 1 M NaCl, 1 mM MgCh, 2.5 mM KC1 in PBS. The 25% and 40% solutions were diluted using 1 mM MgCh, 2.5 mM KC1 in PBS. 20X PBS was added to the stock 60% solution for the final 54.5% dilution. Phenol red was also added to 25% and 54.5% solutions (675 pL into 180 mL for the 25% and 675 pL into 155 mL for the 54.5%) to allow visual identification of each layer.

Density gradients were then constructed in the ultracentrifuge tubes. First, 8 mL samples that have completed the downstream processing described above were then added to the tubes. Volumes of iodixanol solution were then added to the bottom of the tubes below the processed sample using a needle. The volumes of iodixanol for each tube were 5 mL, 9 mL, 8 mL, and 5 mL respectively for the 15%, 25%, 40%, and 54.5% layers. Each layer was added very carefully from the bottom and a fresh needle used every time to prevent crosscontamination.

Completed density gradients were then balanced to within 10 mg via addition of PBS prior to sealing with plugs and placing into the VTi50 rotor. The tubes were tightly secured into the rotor before placing into the ultracentrifuge and spun for 1 hr at 50,000RPM (242,000G) with temperature set at 18 °C.

During this time, 161.5 mL microcentrifuge tubes were prepared and labelled sequentially for each prep and arranged in a straight line on a tube rack. After ultracentrifugation and extraction from rotor, the plug seal was removed before carefully poking a hole into the bottom of the tube using a 19.5G needle. 5 mL of the centrifuged density gradient was first discarded into a waste container drop-wise from this hole before beginning to collect fractions in prepared tubes. The ultracentrifuge tube with flowing gradient was moved over each microcentrifuge tube allowing approximately 0.5mL fractions to collect drop-wise in each one before moving onto the next. The rest of the iodixanol density gradient was discarded.

Fractions were then assayed via SDS PAGE for purity of AAV capsid proteins. 10 pL samples from the latter half of the fractions (typically, 9 through 16) were denatured in 4XLDS + 2.5% B-mercaptoethanol (v/v) at 70°C for 10 min. Samples were then run on a 4- 12% Tris-Glycine gel for 40 min at 225V. Gels were then stained with 10 p L SYPRO Red in 50 mL of 7.5% (v/v) acetic acid for 1 hr on a shaker at room temperature. Gels were then destained for 5 min in fresh 7.5% acetic acid before imaging on a GelDoc. All exposures were done for 5 sec to make comparison of protein gels consistent. Clean fractions showed only three bands corresponding to VP1, VP2, and VP3 of the AAV capsid. The fractions above a certain point were dirty with additional bands of varying sizes and streaking throughout the entire lane. All these fractions were discarded. Clean fractions were then pooled together and polished using 100 kD centrifugal retention filters and cryogenic storage solution. Storage solution was prepared with 5% w/v sorbitol and 0.001% v/v Pluronic F68 in IX PBS. Pooled fractions were then mixed thoroughly 1:1 with storage solution. 15 mL of this was placed into 100 kD retention filters at a time and centrifuged in a swinging bucket rotor at 3500G for 15 min at 4°C. This was repeated until all samples were concentrated to under 500 pL total volume. 14 mL of storage solution was then added to each concentrated sample, mixed thoroughly, and then centrifugation was repeated with speed increased to 4000G. This was repeated once more to completely replace solution with storage buffer before continuing to centrifuge until volume is brought down below 200 pL.

Viral Titration

Purified and polished samples were then quantified using qPCR to obtain a genome copy titer. First, standard was prepared by use of a PCR purification kit on linearized ITR- transgene-ITR plasmid obtained via restriction digest. Plasmid containing IxlO⁵ copies was used for the first data point to build a standard curve. 2 pL was then used for serial 1:10 dilutions (2 pL into 18 pL) 7 times to obtain a standard curve. 2 pL of each sample were also diluted in series 6 times. 2 pL of each dilution or sample was added to a reaction consisting of 12.5 pL TaqMan Fast Advanced MasterMix, 1.25 pL 5mM forward primer (5’ GCT TTA ATG CCT TTG TAT CAT GCT AT 3’), 1.25 pL 5mM reverse primer (5’ CTG ATG AAG AGA CAG CAA CCA GG 3’), 2.5 pL 5 mM Probe (5’ / 56-FAM / CTT CCC GTA / ZEN / TGG CTT TCA TTT TCT CCT CC / 3IABkFQ / 3’), 5.5 pL H₂O. Primers for all VRC01 vectors were designed against WPRE. All reactions were performed with replicates. Ct values from the samples were then compared against the prepared standard to calculate final genome copy (GC) titer.

Analysis by FACS

After 3 days in culture, HEK293T cells were processed into single cells, and analyzed for native mKate fluorescence by flow cytometry using a BD CANTO II flow cytometer (BD Bioscience). Analyses were performed using Diva software (BD Bioscience) and Flowjo (Tree Star; Ashland, OR).

SCID Mouse

Human skin xenografting was performed on immunodeficient Fox Chase SCID (Prkdcscid) beige (Lystbg) mice. In these animals, the SCID mutation results in severe combined immunodeficiency affecting both the B and T lymphocytes, and the beige mutation - in defective natural killer (NK) cells.

Animals with successful grafts were selected and allowed to reach full dermal vascularization within a month. After this period, regenerated human skin tissues were used to assess the feasibility of AAV-mediated gene transfer via polymer microneedle delivery. Bioluminescent imaging

To image the intensity and cross-sectional distribution of luciferase driven by a CASI promoter (rAAV2.CASI.Luc2.WPRE), animals were injected IP (intraperitoneal injection) with D-luciferin and subjected to whole-body luminescence imaging.

While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

CLAIMS What is claimed is:

1. A device for delivering an adeno-associated viral vector into a patient, comprising: a matrix; and the adeno-associated viral vector, wherein: the matrix comprises a hydrophilic polymer and a sugar; and the adeno-associated viral vector is crystalline.

2. A method, comprising: drying a solution to form a solid device for delivering an adeno-associated viral vector into a patient, wherein the solution comprises a hydrophilic polymer in an amount of at least 10% w/v, a sugar, the adeno-associated viral vector, and water.

3. A device or method as in any preceding claim, wherein the device comprises a microneedle, a patch, a microparticle, a pill, a suture, and/or a surgical thread.

4. A device or method as in any preceding claim, wherein the device comprises a gel, an emulsion, and/or liquid crystals.

5 A device or method as in any preceding claim, wherein the device comprises a precursor that may be hydrated to form a gel, an emulsion, and/or liquid crystals.

6. A device or method as in any preceding claim, wherein the gel is a hydrogel.

7. A device or method as in any preceding claim, wherein the gel is thermo-responsive.

8. A device or method as in any preceding claim, wherein the adeno-associated viral vector is encapsulated in a microparticle positioned in a microneedle.

9. A device or method as in any preceding claim, wherein the matrix and the adeno- associated viral vector on positioned on a surface of the device.

10. A device or method as in any preceding claim, wherein the matrix and the adeno- associated viral vector are dispersed throughout the bulk of the device.

11. A device or method as in any preceding claim, wherein the device is water-soluble and/or biodegradable.

12. A device or method as in any preceding claim, wherein, the solution comprises at least 10% w/v of the sugar.

13. A device or method as in any preceding claim, wherein the sugar comprises sucrose, trehalose, and/or raffinose.

14. A device or method as in any preceding claim, wherein the solution and/or device comprises a thermoresponsive polymer.

15. A device or method as in any preceding claim, wherein the hydrophilic polymer is charged.

16. A device or method as in any preceding claim, wherein the hydrophilic polymer(s) comprises polyethylene glycol and/or one or more of its copolymers, polyvinylpyrrolidone, gelatin, poly-y-glutamic acid, poly(methylvinylether/maleic anhydride), a polyvinylpyrrolidone-polyvinyl alcohol copolymer, poly(vinylpyrrolidone-co-methacrylic acid), poly(vinylpyrrolidone-co-cyclodextrin), polylactic acid, polyglycolic acid, poly(lactic- co-glycolic acid), polycaprolactone and/or a polysaccharide.

17. A device or method as in any preceding claim, wherein the polysaccharide comprises dextran, sodium chondroitin sulfate, hydroxypropyl cellulose, carboxymethyl cellulose, hydroxypropyl methylcellulose, sodium alginate, hyaluronic acid, and/or amylopectin.

18. A device or method as in any preceding claim, wherein the thermoresponsive polymer comprises a polyethylene oxide-polypropylene oxide copolymer.

19. A device or method as in any preceding claim, wherein the polyethylene oxidepolypropylene oxide copolymer is a polyethylene oxide-polypropylene oxide-polyethylene oxide triblock polymer.

20. A device or method as in any preceding claim, wherein the solution and/or device further comprises polystyrene and/or polycarbonate.

21. A device or method as in any preceding claim, wherein the solution and/or device comprises a salt.

22. A device or method as in any preceding claim, wherein the salt comprises a phosphate salt, a carbonate salt, a borate salt, l-ethyl-3-methylimidazolium trifluoromethanesulfonate and/or l-ethyl-3-methylimidazolium acetate.

23. A device or method as in any preceding claim, wherein the solution and/or device comprises a small molecule.

24. A device or method as in any preceding claim, wherein the small molecule comprises a solvent.

25. A device or method as in any preceding claim, wherein the small molecule comprises a surfactant.

26. A device or method as in any preceding claim, wherein the small molecule and/or the salt comprises a buffering agent.

27. A device or method as in any preceding claim, wherein the small molecule comprises bis-tris propane, HEPES, citric acid, 2-propanol, succinic acid, acetic acid, creatinine, MES, bis-tris methane, ADA, ACES, MOPSO, PIPES, imidazole, BES, MOPS, TES, TAPSO, HEPSO, tris, tricine, bicine, TAPS, ammediol, CHES, ethanolamine, CAPSO, glycerol, CAPS, cholamine chloride, DIPSO, acetamidoglycine, POPSO, HEPPSO, HEPPS, glycinamide, glycylglycine, mannitol, sorbitol, Tween-80, polysorbate 80, one or more nucleic acids, and/or one or more amino acids.

28. A device or method as in any preceding claim, wherein the one or more amino acids comprise serine, arginine, aspartic acid, glutamic acid, glycine, histidine, lysine, and/or proline.

29. A device or method as in any preceding claim, wherein the adeno-associated viral vector is derived from one or more of the following AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and variants of the foregoing.

30. A method as in any preceding claim, wherein the drying comprises lyophilization.

31. A method as in any preceding claim, wherein the drying comprises extrusion, precipitation, and/or centrifugation.

32. A method as in any preceding claim, wherein the drying is performed under laminar flow.

33. A method as in any preceding claim, wherein the adeno-associated viral vector crystallizes during drying.

34. A method as in any preceding claim, further comprising centrifuging and/or ultracentrifuging the solution prior to drying.

35. A method as in any preceding claim, wherein the centrifuging and/or ultracentrifuging is performed at a temperature of less than 4 °C.

36. A method comprising contacting the device of any preceding claim with the patient’s cells, thereby delivering the adeno-associated viral vector to the patient.

37. A method as in any preceding claim, further comprising contacting the device of claim 1 with the patient’s cells a second time, thereby delivering a further amount of the adeno-associated viral vector to the patient.

38. A method as in any preceding claim, wherein the patient is a human.

39. A method as in any preceding claim, wherein the contacting comprises injecting the device and/or a portion of the device into the patient.

40. A method as in any preceding claim, further comprising treating the tissue of the patient with an immunosuppressant drug prior to the injection.

41. A method as in any preceding claim, further comprising pre-permeabilizing the tissue of the patient prior to the injection.

42. A method as in any preceding claim, wherein the pre-permeabilization comprises sonication, heating, microneedling, and/or micro-coring.

43. A method as in any preceding claim, wherein the adeno-associated viral vector is delivered to a target site.

44. A method as in any preceding claim, wherein the target site comprises resident cells, transient cells, immune cells, and/or skin cells.

45. A method as in any preceding claim, wherein the skin cells comprise epidermal skin cells, epidermal stem cells, fibroblasts, dermal fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, and/or hair follicle stem cells.

46. A method as in any preceding claim, wherein the target site comprises epidermal tissue, dermal tissue, a hair follicle, and/or sebaceous glands.

47. A method as in any preceding claim, wherein the adeno-associated viral vector is delivered ex vivo.

48. A method as in any preceding claim, wherein the adeno-associated viral vector is delivered in vivo.