Compositions for adipose tissue targeting and uses thereof

Abstract

The present disclosure pertains to compositions for targeting local delivery of a AAV particle comprising a payload to adipose tissue for local and / or systemic delivery of a payload. Also disclosed herein are methods of making and using the same.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application 63 / 625,893 filed on January 26, 2024, the entire contents of which is hereby incorporated by reference. BACKGROUND

[0002] Adipose tissue is distributed throughout the body in distinct depots and adipocytes make up a minority of the cells within the tissue. Given the diversity and distribution of adipose tissue, delivery of payloads to adipose tissue, including localized delivery, remains a challenge. SUMMARY

[0003] Adipose tissue is one of the largest endocrine organs, playing important roles in physiology and pathologies of multiple diseases. The present disclosure identifies that prior strategies to deliver vectors (e.g., rAAV particles) to animal (e.g., mice) adipose tissue resulted in unwanted distribution of said vectors (and concomitant payload expression) in sites that are distant from the site of administration, e.g., non-adipose tissue e.g., liver (see, e.g., Jimenez V., et. al., In vivo adeno-associated viral vector-mediated genetic engineering of white and brown adipose tissue in adult mice. Diabetes. 2013 Dec;62(12):4012-22, which is incorporated herein by reference in its entirety). Such unwanted vector distribution and payload expression can result in off-target effects that can be toxic and / or reduce the intended therapeutic effect(s) of the payload.

[0004] Among other things, the present disclosure provides technologies that can address certain limitations identified in existing methods for delivery of nucleic acid payloads to adipose tissue. The technologies provided herein are particularly useful for delivering vectors (e.g., recombinant AAV particles) comprising payloads to adipose tissue, with minimal (or substantially no) distribution of said vectors to tissue other than those at or near a site of administration. In some embodiments, technologies disclosed herein result in minimal (e.g., undetectable or none) distribution of a vector comprising a pay load to non-adipose tissue (e.g., other organs, non-adipose cell types). In some embodiments, technologies disclosed herein result in local transduction of adipose tissue (e.g., transduction of adipose tissue at or near a site of administration) and local expression and / or distribution of a payload in adipose tissue (e.g., adipose tissue at or near a site of administration). In some embodiments, technologies disclosed herein result in local transduction of adipose tissue (e.g., transduction of adipose tissue at or near a site of administration) and systemic distribution of a pay load (e.g., to one or more tissues other than adipose tissue at or near a site of administration, e.g., including other adipose tissue and / or organs and / or compartments such as blood). In some embodiments, technologies disclosed herein result in (1) local expression and / or distribution of a payload in adipose tissue, and (2) systemic distribution (e.g., subsequent systemic distribution) of a payload to non-adipose tissue. In some embodiments, a payload that is expressed in adipose tissue at or near a site of administration can be distributed locally, e.g., to adipose tissue at or near a site of administration. In some embodiments, a payload that is expressed in adipose tissue at or near a site of administration can be distributed systemically, e.g., to one or more tissues other than adipose tissue at or near a site of administration, e.g., including other adipose tissue and / or organs and / or compartments such as blood.

[0005] Among other things, provided herein is a method of delivering an adeno-associated (AAV) particle or a composition comprising the same to adipose tissue in a subject, wherein an AAV particle comprises an AAV capsid protein and a polynucleotide construct, and wherein a subject is a mammal, e.g., a non-rodent mammal.

[0006] This disclosure also provides a method for controlling and / or reversing a gene therapy, the method comprising: (1) delivering a first dose of a gene therapy comprising an adeno-associated (AAV) particle or a composition comprising the same to adipose tissue in a mammal (e.g., a non-rodent mammal), wherein the AAV particle comprises an AAV capsid and a polynucleotide construct encoding a payload; and (2) reducing or eliminating expression of the pay load in the adipose tissue by removing at least a portion of the adipose tissue transduced by the AAV particle.

[0007] In some embodiments of the methods disclosed herein, delivery comprises administration of the AAV particle or a composition comprising the same subcutaneously.

[0008] In some embodiments of the methods disclosed herein, an AAV particle or a composition comprising the same is administered once.

[0009] In some embodiments of the methods disclosed herein, an AAV particle or a composition comprising the same is administered repeatedly.

[0010] In some embodiments of the methods disclosed herein, an AAV particle or a composition comprising the same is administered at a pre-determined tissue depth in a subject. In some embodiments, an AAV particle or a composition comprising the same is administered between the dermis and Scapa’s fascia. In some embodiments, an AAV particle or a composition comprising the same is administered to superficial subcutaneous adipose tissue. In some embodiments, an AAV particle or a composition comprising the same is administered between Scapa's fascia and Camper's fascia. In some embodiments, an AAV particle or a composition comprising the same is administered to deep subcutaneous adipose tissue. In some embodiments, an AAV particle or a composition comprising the same is administered to visceral adipose tissue.

[0011] In some embodiments of the methods disclosed herein, an AAV particle or a composition comprising the same is administered at a pre-determined depth from the exterior surface of the skin. In some embodiments, a pre-determined depth is between at least about 2mm to about 6mm.

[0012] In some embodiments of the methods disclosed herein, adipose tissue is or comprises brown adipose tissue, beige adipose tissue, white adipose tissue, pink adipose tissue, or any combination thereof.

[0013] In some embodiments of the methods disclosed herein, adipose tissue is or comprises brown adipose tissue. In some embodiments, brown adipose tissue is located near or at the following locations in the subject: neck, kidney, adrenal glands, heart (e.g., aorta), mediastinum, shoulder, neck, and / or back. In some embodiments, brown adipose tissue is characterized as metabolizing fat to produce heat and / or has a role in energy metabolism.

[0014] In some embodiments of the methods disclosed herein, adipose tissue is or comprises beige adipose tissue. In some embodiments, beige adipose tissue is located near or at the following locations in the subject: abdomen, face, glute, and / or femur.

[0015] In some embodiments of the methods disclosed herein, adipose tissue is or comprises white adipose tissue. In some embodiments, white adipose tissue is located near or at the following locations in the subject: bone marrow, abdomen, and / or organs.

[0016] In some embodiments, white adipose tissue is characterized by its location. In some embodiments, white adipose tissue is present in a subject near or next to an organ in the form of a visceral depot.

[0017] In some embodiments, white adipose tissue comprises subcutaneous white adipose and / or visceral white adipose tissue. In some embodiments, white adipose tissue is characterized as storing fat.

[0018] In some embodiments of the methods disclosed herein, adipose tissue is or comprises pink adipose tissue. In some embodiments, pink adipose tissue is located near or at the following locations in the subject: abdomen, breast, bone marrow, and / or dermis.

[0019] In some embodiments of the methods disclosed herein, adipose tissue comprises adipocytes.

[0020] In some embodiments of the methods disclosed herein, an AAV particle does not substantially target (e.g., does not target) non-adipose tissue, non-adipocytes, and / or adipose tissue (e.g., adipose tissue at a different location) which was not administered, e.g., directly administered, the AAV particle or a composition comprising the same.

[0021] In some embodiments of the methods disclosed herein, an AAV particle does not substantially transduce (e.g., does not transduce) non-adipose tissue, non-adipocytes, and / or adipose tissue (e.g., adipose tissue at a different location) which was not administered, e.g., directly administered, the AAV particle or a composition comprising the same.

[0022] In some embodiments of the methods disclosed herein, an AAV particle is substantially not detected (e.g., not detectable) in non-adipose tissue (e.g., liver, skin, muscle), non-adipocytes, and / or adipose tissue (e.g., adipose tissue at a different location) which was not administered, e.g., directly administered, the AAV particle or a composition comprising the same.

[0023] In some embodiments of the methods disclosed herein, an AAV particle is substantially not detectable (e.g., not detectable) in a non-adipose tissue sample from the subject or adipose tissue sample (e.g., adipose tissue sample at a different location) which was not administered, e.g., directly administered, the AAV particle or a composition comprising the same.

[0024] In some embodiments of the methods disclosed herein, expression of a payload encoded by a polynucleotide construct is substantially not detectable (e.g., not detectable) in nonadipose tissue (e.g., liver, skin, muscle) non-adipocytes, and / or adipose tissue (e.g., adipose tissue at a different location) which was not administered, e.g., directly administered, the AAV particle or a composition comprising the same.

[0025] In some embodiments of the methods disclosed herein, administration of an AAV particle or a composition comprising the same to adipose tissue results in local expression and / or systemic distribution of the payload. In some embodiments of the methods disclosed herein, administration of an AAV particle or a composition comprising the same to adipose tissue results in local expression of the payload. In some embodiments of the methods disclosed herein, administration of an AAV particle or a composition comprising the same to adipose tissue results in systemic distribution of the payload. In some embodiments of the methods disclosed herein, administration of an AAV particle or a composition comprising the same to adipose tissue results in local expression and systemic distribution of the payload.

[0026] In some embodiments of the methods disclosed herein, a payload is expressed and / or distributed at a therapeutically effective level. In some embodiments, a therapeutically effective level is a level that can treat and / or prevent one or more symptoms of a disease, disorder or condition.

[0027] In some embodiments of the methods disclosed herein, systemic distribution is detected at least about 24 hours after administration. In some embodiments, systemic distribution persists for at least about 1 week after administration. In some embodiments, systemic distribution of the payload is dose dependent.

[0028] In some embodiments, a payload can have an effect, e.g., can modulate one or more activities, expression and / or localization of a cell or a biological molecule expressed by a cell (e.g., a polypeptide, a nucleic acid, a lipid, etc). In some embodiments, a payload.

[0029] In some embodiments of the methods disclosed herein, a payload is a secreted polypeptide.

[0030] In some embodiments, a payload acts systemically.

[0031] In some embodiments of any of the methods disclosed herein, a pay load acts locally, e.g., at or near the site of administration.

[0032] In some embodiments of the methods disclosed herein, detection of expression of a payload occurs in a biological sample from a subject. In some embodiments, a biological sample is or comprises cells, tissue, and / or bodily fluid.

[0033] In some embodiments of the methods disclosed herein, an AAV particle or a composition comprising the same is administered at a plurality of sites in the subject. In some embodiments, an AAV particle or a composition comprising the same is administered at a plurality of sites in a first section of adipose tissue. In some embodiments, a first section of adipose tissue comprises adipose tissue in a pre-selected location in a subject’s body. In some embodiments, an AAV particle or a composition comprising the same is administered at between 1 and 100 different sites in the first section of adipose tissue.

[0034] In some embodiments, a plurality of sites are non-overlapping.

[0035] In some embodiments, a plurality of sites are at a pre-specified distance from one another. In some embodiments, a pre-specified distance is about 0.1 cm.

[0036] In some embodiments of the methods disclosed herein, an AAV particle or a composition comprising the same is administered at another (e.g., a second) section of adipose tissue in the subject’s body. In some embodiments, another (e.g., a second) section of adipose tissue is at a different location from the first section of adipose tissue.

[0037] In some embodiments, an AAV particle or a composition comprising the same is administered at a plurality of sites in the another (e.g., a second) section of adipose tissue.

[0038] In some embodiments, a first or subsequent (e.g., a second) section of adipose tissue is selected based on one or more characteristics of adipocytes in the first or subsequent section.

[0039] In some embodiments of the methods disclosed herein, an AAV particle or a composition comprising the same is administered at a volume of at least lOuL.

[0040] In some embodiments of the methods disclosed herein, a method further comprises administering one or more doses of the AAV particle or a composition comprising the same.

[0041] In some embodiments of the methods disclosed herein, an AAV particle or a composition comprising the same is administered at a dose of at least 1 x 10(11) vector genomes (vg).

[0042] In some embodiments of the methods disclosed herein, an AAV particle or a composition comprising the same is administered at a dose, e.g., concentration, of at least 1 x 10(12) vg / mL.

[0043] In some embodiments of the methods disclosed herein, an AAV particle comprises a polynucleotide construct comprises one or more coding sequences. In some embodiments, one or more coding sequences encodes a payload.

[0044] In some embodiments, a payload is or comprises a polypeptide.

[0045] In some embodiments, a payload is effective in treating a disease and / or disorder. In some embodiments, a payload is effective in treating and / or preventing one or more symptoms of a disease and / or disorder.

[0046] In some embodiments, a payload is or comprises a therapeutic payload.

[0047] In some embodiments, a therapeutic payload comprises: GLA, LPL, FVIII, FIX, GLP-1, GIP, ADIPOQ, FGF19, FGF21, PPY, BMP7, LEP, Adalimumab, Etanercept, Pembrolizumab, Ustekinumab, Dupilumab, Nivolumab, Daratumumab, Risankizumab, Secukinumab, Pertuzumab, Emicizumab, Denosumab, Abatocept, Atezolizumab, Durvalumab, Guselkumab, Tocilizumab, Ixekizumab, Infliximab, Eculizumab, Ravulizumab, Insulin, or any combination thereof.

[0048] In some embodiments, a payload is or comprises a polyribonucleotide.

[0049] In some embodiments, a polyribonucleotide is or comprises a messenger RNA, an inhibitory RNA or a non-coding RNA.

[0050] In some embodiments, a polynucleotide construct comprises a transgene.

[0051] In some embodiments of the methods disclosed herein, a method further comprises removal of transduced adipose tissue and / or adipocytes from the subject. In some embodiments, all or substantially all of the transduced adipose tissue and / or adipocytes is removed. In some embodiments, removal comprises physical excision, biopsy, cryolypolysis, surgical removal, liposuction, laser mediated removal, radioablation, imaging-based ablation, or any combination thereof.

[0052] In some embodiments, removal results in a reduction in expression and / or activity of a pay load compared to a subject that has not undergone removal or compared to the same subject prior to removal. In some embodiments, removal results in ablation, e.g., complete reduction, in expression and / or activity of a payload.

[0053] In some embodiments of the methods disclosed herein, a mammal is a pig, cow, dog, cat, non-human primate, or human.

[0054] In some embodiments of the methods disclosed herein, a mammal is a human.

[0055] In some embodiments of the methods disclosed herein, a mammal is not a rodent, e.g., a mouse or a rat.

[0056] In some embodiments of the methods disclosed herein, an AAV particle comprises a regulatory element. In some embodiments, a regulatory element is one or more promoters or a fragment or variant thereof, one or more enhancers or a fragment or variant thereof, one or more silencers or a fragment or variant thereof, one or more insulators or a fragment or variant thereof, or any combination thereof.

[0057] In some embodiments, a regulatory element comprises one or more promoters or a fragment or variant thereof; and one or more enhancers or a fragment or variant thereof.

[0058] In some embodiments, a regulatory element is or is derived from a gene expressed in adipocytes.

[0059] In some embodiments, a regulatory element comprises one or more sequences provided in Table 2 or fragments or variants thereof.

[0060] In some embodiments, a regulatory element comprises: a mFabp4 regulatory element, a mAdipoq regulatory element, a hADIPOQ regulatory element, or combinations thereof.

[0061] In some embodiments, a regulatory element comprises a ubiquitous promoter and / or a ubiquitous enhancer. In some embodiments, a regulatory element reduces (e.g., prevents) expression of a payload in non-adipocytes.

[0062] In some embodiments, a regulatory element reduces (e.g., prevents) expression of a payload in adipocytes which are not present at a site of administration when the AAV particle is administered.

[0063] In some embodiments of the methods disclosed herein, an AAV capsid protein is or comprises: an AAV1 capsid protein, an AAV2 capsid protein, an AAV3B capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 (AAVrhlO) capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV-PHP.B capsid protein, an AAV8-PHP.eB capsid protein, an AAV-PHP.S capsid protein, an AAV-7m8 capsid protein, an AAV-DJ capsid protein, an AAV-OligOOl capsid protein or a Rec2 capsid protein, or a variant or fragment of any of the foregoing.

[0064] In some embodiments of the methods disclosed herein, an AAV capsid protein is or comprises an AAV1 capsid protein, or a variant or fragment thereof.

[0065] In some embodiments of the methods disclosed herein, an AAV capsid protein is or comprises a PHP.B capsid protein, or a variant or fragment thereof.

[0066] In some embodiments of the methods disclosed herein, an AAV capsid protein is or comprises a Rec2 capsid protein, or a variant or fragment thereof.

[0067] In some embodiments of the methods disclosed herein, an AAV capsid protein is or comprises a AAV2 capsid protein, or a variant or fragment thereof.

[0068] In some embodiments of the methods disclosed herein, an AAV capsid protein is or comprises a AAV3B capsid protein, or a variant or fragment thereof.

[0069] In some embodiments of the methods disclosed herein, an AAV capsid protein is or comprises a AAV4 capsid protein, or a variant or fragment thereof.

[0070] In some embodiments of the methods disclosed herein, an AAV capsid protein is or comprises a AAV5 capsid protein, or a variant or fragment thereof.

[0071] In some embodiments of the methods disclosed herein, an AAV capsid protein is or comprises a AAV6 capsid protein, or a variant or fragment thereof.

[0072] In some embodiments of the methods disclosed herein, an AAV capsid protein is or comprises a AAV7 capsid protein, or a variant or fragment thereof.

[0073] In some embodiments of the methods disclosed herein, an AAV capsid protein is or comprises a AAV8-PHP.eB capsid protein, or a variant or fragment thereof.

[0074] In some embodiments of the methods disclosed herein, an AAV capsid protein is or comprises an AAV-PHP.S capsid protein, or a variant or fragment thereof.

[0075] In some embodiments of the methods disclosed herein, an AAV capsid protein is or comprises an AAV-7m8 capsid protein, or a variant or fragment thereof.

[0076] In some embodiments of the methods disclosed herein, an AAV capsid protein is or comprises an AAV-DJ capsid protein, or a variant or fragment thereof.

[0077] In some embodiments of the methods disclosed herein, an AAV capsid protein is or comprises an AAV-OligOOl capsid protein, or a variant or fragment thereof.

[0078] In some embodiments of the methods disclosed herein, at least 10% of the transduced adipose tissue is removed. In some embodiments, all or substantially all of the transduced adipose tissue is removed. In some embodiments, a reduction in a level and / or expression (e.g., local and / or systemic) of a pay load correlates with a level of adipose tissue that is removed. Solely as an example, in some embodiments, as more transduced adipose tissue is removed, a larger decrease in a level and / or expression (e.g., local and / or systemic) of a payload is observed.

[0079] In some embodiments of the methods disclosed herein, removing the transduced adipose tissue reduces local expression of the payload and / or reduces systemic distribution of the payload.

[0080] In some embodiments of the methods disclosed herein, removing all or substantially all of the transduced adipose tissue reduces local expression of the payload and / or reduces systemic distribution of the payload to an undetectable level.

[0081] In some embodiments of the methods disclosed herein, reduction in local expression of the pay load is observed within about 1 hour of removing the transduced adipose tissue.

[0082] In some embodiments, a gene therapy (e.g., comprising an adeno-associated (AAV) particle) is adjusted by modulating an amount of transduced adipose tissue that is removed.

[0083] In some embodiments of the methods disclosed herein, a method further comprises administering one or more subsequent doses of a gene therapy comprising an adeno-associated (AAV) particle to adipose tissue.

[0084] In some embodiments of the methods disclosed herein, one or more subsequent doses of gene therapy is administered to the same or nearby adipose tissue to which a first dose of gene therapy is administered.

[0085] In some embodiments of the methods disclosed herein, one or more subsequent doses of gene therapy is administered to a different adipose tissue as compared to adipose tissue to which a first dose of gene therapy is administered.

[0086] In some embodiments of the methods disclosed herein, one or more subsequent doses of gene therapy is administered at substantially the same volume as the volume of a first dose of a gene therapy.

[0087] In some embodiments of the methods disclosed herein, one or more subsequent doses of gene therapy is administered at a different volume as compared to the volume of a first dose of a gene therapy.

[0088] In some embodiments of the methods disclosed herein, an AAV particle is characterized in that when administered to adipose tissue the AAV particle is contained in adipose tissue (e.g., contained at or near adipose tissue at a site of administration) as compared to an otherwise similar AAV particle that is not delivered by a method described herein.

[0089] In some embodiments of the methods disclosed herein, an AAV particle is characterized in that when administered to adipose tissue the AAV particle transduces at least 5% more adipocytes in the adipose tissue that is administered the AAV particle as compared to adipocyte transduction by an otherwise similar AAV particle that is not delivered by a method described herein.

[0090] Further provided herein is an AAV particle comprising an AAV capsid protein and a polynucleotide construct, e.g., as described herein.

[0091] This disclosure also provides a composition comprising an AAV particle comprising an AAV capsid protein and a polynucleotide construct, e.g., as described herein.

[0092] In some embodiments, a composition is a pharmaceutical composition.

[0093] In some embodiments, a pharmaceutical composition comprise one or more pharmaceutically acceptable carriers and / or excipients.

[0094] Also provided herein is a composition comprising an AAV particle comprising an AAV capsid protein and a polynucleotide construct, e.g., as described herein, wherein the composition is formulated for delivery to adipose tissue.

[0095] In some embodiments, a composition is suitable for delivery to adipose tissue via subcutaneous injection.

[0096] Other features, objects, and advantages of the present invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWING

[0097] The Figures described below, which together make up the Drawing, are for illustration purposes only, not for limitation.

[0098] FIGS. 1A-C are a series of confocal images (FIG. 1A) and graphs (FIGS. IB and IC) showing that AAV8.CAG.EGFP robustly transduces adipocytes in C57BL / 6J mice by 15d after local delivery into the left epididymal (LE) or inguinal (LI) adipose pads. FIG. 1A is a series of representative micrographs showing the LE fat pad from a mouse that was injected with 2el 1 vg of AAV8.CAG.EGFP (top and middle row) or from a naive mouse that was not treated (bottom row). The top row shows a view of the entire adipose pad, while the bottom and middle rows show higher magnification views that resolve adipocytes (dashed box in top row indicates magnified region in middle row). Individual panels show native GFP signal (left) and immunofluorescence labeling with antibodies against GFP (middle) or the adipocyte marker Perilipin 1 (Plinl, right). FIG. IB is a graph showing quantification of the percentage of Plinl + adipocytes that were positive for anti-GFP within each injected adipose pad. In all four injected pads, >80% of Plinl + adipocytes had detectable levels of GFP. FIG. IC is a graph showing the volume of each adipose pad containing GFP+ / Plinl+ adipocytes, calculated by multiplying the percentage of GFP+ cells by the total volume of each pad.

[0099] FIGS. 2A-C are a series of fluorescence stereomicroscopy images (FIG. 2A) and graphs (FIGS. 2B and 2C) showing that 2el 1 vg of AAV8.CAG.EGFP delivered locally into a single mouse adipose pad can distribute systemically and transduce other un-injected pads and the liver. FIG. 2A is a series of micrographs showing native GFP fluorescence in whole mounts of left and right epididymal (LE, RE) and inguinal (LI, RI) adipose pads and the liver from AAV8-injected mice or from naive mice (n=2 mice per condition). Organs were harvested 15d after viral delivery. The dashed lines demarcate the outline of the entire adipose pad. FIG. 2B is a graph showing the mean GFP fluorescence intensity measured from the stereomicroscope images of each of the organs in FIG. 2A. While some GFP fluorescence could be observed in un-injected pads from treated mice in FIG. 2A, the mean intensity measured throughout the pad was not significantly different from background levels measured in naive pads. The mean GFP intensity in the liver of treated mice was significantly higher than all other organs, including injected LE and LI pads (p<0.01, one-way ANOVA with Tukey’s post hoc test). FIG. 2C is a graph showing quantification of the percentage of Plinl + adipocytes that were positive for anti-GFP within all adipose pads shown in FIG. 2A. Measurements were taken from images captured with a confocal microscope, which has greater resolution and sensitivity compared to the fluorescence stereomicroscope. Using this technique, un-injected adipose pads from treated mice contained significant percentages of GFP+ adipocytes compared to background levels from naive pads (p<0.01, one-way ANOVA with Tukey’s post hoc test).

[0100] FIG. 3 is a series of graphs showing that dPCR detects systemic distribution of AAV8.CAG.EGFP after local delivery of 2el 1 vg into a single adipose pad in mice. Left panel is a graph showing dPCR quantification of vector genomes in DNA extracted from adipose pads and the liver from AAV8-treated (Tx) mice or from naive mice (n=2 mice per condition). Organs were harvested 15 days after local viral delivery into a single adipose pad. DNA assays were directed against the CAG promoter. Right panel is a graph showing dPCR quantification of GFP transcripts in RNA extracted from the same organs shown in the left panel. RNA assays were directed against the EGFP transgene.

[0101] FIG. 4 is a graph showing that most vector genomes (vg’s) are confined to injection sites at 8d after subcutaneous delivery of AAV into the abdominal adipose space in non-human primates. The graph shows quantification of RNAScope labeling of vg’s with probes recognizing the EGFP transgene in histological sections of injection sites and the liver. For the full-thickness section of each injection site, the skin, adipose, and muscle layers were automatically segmented and total vg copies in each layer was quantified. The data shown for each of these layers is pooled from 14 different AAV capsid variants injected individually into subcutaneous sites spaced across the abdomen of Cynomolgus Macaques (lei 1 vg per site; 1 site / capsid / NHP in n=2 NHPs; 28 sites total). The total vg copies in sections from the left lateral lobe of the liver from both animals was also quantified. Total vg copies in the adipose were significantly greater than all other regions examined (p<0.01, one-way ANOVA with Tukey’s post hoc test). The number of copies in the liver was consistent with expected levels for background labeling.

[0102] FIG. 5 is a series of graphs showing that dPCR detects little to no systemic distribution of AAV capsid variants after subcutaneous injection into non-human primates. Left panel is a graph showing dPCR quantification of vector genomes in DNA extracted from dorsal root ganglia (DRG), gastrocnemius muscle (Gastroc), heart, liver, and testes from Cynomolgus Macaques. Organs were harvested 8d after subcutaneous injection of lei 1 vg of 14 different AAV capsid variants into 18 different sites spread across the abdominal, inguinal, and gluteal regions (1.8el2 vg per NHP, n=2 NHPs). DNA assays were directed against the CAG promoter. Punch biopsy explants from pigs that were subcutaneously injected with lei 1 vg of Php.B.CAG.EGFP and cultured for 6d were included as a positive control. Right panel is a graph showing dPCR quantification of GFP transcripts in RNA extracted from the same organs shown in the left panel. RNA assays were directed against the EGFP transgene. DNA from AAV genomes and RNA from EGFP transcripts was near or below the limit of detection in all primate organs tested, whereas robust levels of DNA and RNA were detected in the pig explants.

[0103] FIG. 6 is a graph showing that the vector genomes of many AAV capsid variants efficiently enter the nuclei of adipocytes from nonhuman primates within 8 days after subcutaneous injection. The graph shows quantification of RNAScope labeling of vg’s within adipocyte nuclei using probes directed against the EGFP transgene encoded in the AAV genome. The data was collected from 14 different AAV capsid variants injected individually into subcutaneous sites spaced across the abdomen of Cynomolgus Macaques (lei 1 vg per site; 1 site / capsid / NHP in n=2 NHPs; 28 sites total). For each histological section, the percentage of all nuclei containing one or more vg’s was assessed within an 80 mm2 region centered on the most dense area of vg labeling in adipose (presumed injection site). Automated segmentation was used to identify nuclei and exclude non-adipocyte areas within the region. The 80 mm2 region corresponds to an 800 uL volume, assuming a conservative distribution diameter of 1 cm. Capsid variants were rank ordered along the x-axis according to the maximum percent nuclei labeled within all the sites examined. The size of each dot on the plot corresponds to the RNAScope labeling H-Score for that site, which was calculated by dividing nuclei into 5 bins based on the number of vg copies per nucleus (0, 1-3, 4-9, 10-15, or >15 copies) and then summing the percentage of total nuclei in each bin multiplied by the bin number (0-4). One control (Cntrl) site was taken from a region on the abdomen of each animal that was distant from the injection sites.

[0104] FIG. 7 is a graph showing that most vector genomes (vg’s) are confined to injection sites at lOd days after subcutaneous delivery of AAV into the abdominal adipose space in pigs. The graph shows quantification of RNAScope labeling of vg’s with probes recognizing the EGFP transgene in histological sections of injection sites and the liver. For the full-thickness section of each injection site, the skin, adipose, and muscle layers were automatically segmented and total vg copies in each layer was quantified. The data shown for each of these layers is pooled from 14 different AAV capsid variants injected individually into subcutaneous sites spaced across the abdomen of Yorkshire pigs (lei 1 vg per site; 2 sites / capsid, except AAV6 which was 4 sites / capsid; 30 sites total; n=2 pigs). The total vg copies in sections from the left lateral lobe of the liver from both animals was also quantified. Total vg copies in the adipose were significantly greater than all other regions examined (p<0.01, one-way ANOVA with Tukey’s post hoc test). The number of copies in the liver was consistent with expected levels for background labeling. The break in the y-axis makes the difference in labeling between all the adipose sites and other regions more apparent since there were a small number of adipose sites with disproportionately high levels of labeling (these sites had likely begun to express RNA, as the probes cannot distinguish RNA from DNA).

[0105] FIG. 8 is a series of graphs showing that dPCR detects little to no systemic distribution of AAV capsid variants after subcutaneous injection into pigs. Left panel is a graph showing dPCR quantification of vector genomes in DNA extracted from dorsal root ganglia (DRG), gastrocnemius muscle (Gastroc), heart, liver, and testes from Yorkshire pigs. Organs were harvested 10 days after subcutaneous injection of lei 1 vg of 14 different AAV capsid variants into 18 different sites spread across the abdomen (1.8el2 vg per pig, n=2 pigs). DNA assays were directed against the CAG promoter. Punch biopsy explants from pigs that were subcutaneously injected with lei 1 vg of Php.B.CAG.EGFP and cultured for 6 days were included as a positive control. Right panel is a graph showing dPCR quantification of GFP transcripts in RNA extracted from the same organs shown in the left panel. RNA assays were directed against the EGFP transgene. DNA from AAV genomes and RNA from EGFP transcripts was near or below the limit of detection in all swine organs tested, whereas robust levels of DNA and RNA were detected in the pig explants.

[0106] FIG. 9 is a graph showing that the vector genomes of many AAV capsid variants efficiently enter the nuclei of adipocytes from pigs within lOd after subcutaneous injection. The graph shows quantification of RNAScope labeling of vg’s within adipocyte nuclei using probes directed against the EGFP transgene encoded in the AAV genome. The data was collected from 14 different AAV capsid variants injected individually into subcutaneous sites spaced across the abdomen of Yorkshire pigs (lei 1 vg per site; 2 sites / capsid, except AAV6 which was 4 sites / capsid; 30 sites total; n=2 pigs). For each histological section, the percentage of all nuclei containing one or more vg’s was assessed within an 80 mm2 region centered on the most dense area of vg labeling in adipose (presumed injection site). Automated segmentation was used to identify nuclei and exclude non-adipocyte areas within the region. The 80 mm2 region corresponds to an 800 uL volume, assuming a conservative distribution diameter of 1 cm. Capsid variants were rank ordered along the x-axis according to the maximum percent nuclei labeled within all the sites examined. The size of each dot on the plot corresponds to the RNAScope labeling H-Score for that site, which was calculated by dividing nuclei into 5 bins based on the number of vg copies per nucleus (0, 1-3, 4-9, 10-15, or >15 copies) and then summing the percentage of total nuclei in each bin multiplied by the bin number (0-4). One control (Cntrl) site was taken from a region on the abdomen of each animal that was distant from the injection sites.

[0107] FIG. 10 is a graph comparing the efficiency of vector genome nuclear entry for 14 different AAV capsid variants in nonhuman primates versus pigs. For each capsid variant, the site with the maximum percentage of labeled nuclei was selected from the data in FIGS. 6 and 9. The graph plots the max values in the nonhuman primate against the pig. The best performing capsids in both species are highlighted by the light grey oval, while the capsids that consistently performed poorly in both species are highlighted in dark grey.

[0108] FIG. 11 is a graph showing the transduction efficiency of AAV1, AAV6, AAV8, Php.B, and Rec2 in adipocytes 3 weeks after subcutaneous delivery in nonhuman primates. All vectors encoded an EGFP transgene under control of a ubiquitous promoter. 3 el 1 vg of each capsid was injected into 2-3 separate abdominal sites in a cynomolgus macaque (n=6 NHPs total; 1-2 animals per capsid), and the sites were extracted for histology via a 2 cm x 1 cm elliptical excision biopsy 3 weeks later. In addition, AAV1, Php.B, and Rec2 were injected into 2-3 sites at a 10-fold lower dose of 30 uL x 3el0 vg. The graph shows the percentage of adipocytes throughout each section that labeled positive with antibodies to GFP. Dots represent mean percentages measured in multiple sections taken from each site, and vertical lines are standard deviations. AAV1, Php.B, and Rec2 transduced a significantly greater mean percentage of adipocytes compared to AAV6 and AAV8 (p<0.01, one-way ANOVA with Tukey’s post hoc test). A 10-fold lower dose of AAV1, Php.B, and Rec2 resulted in a significantly lower mean percentage of transduced adipocytes that was similar to the amount observed with AAV6 and AAV8.

[0109] FIG. 12 is a graph of the detected levels of hSEAP in the serum of 4 Yorkshire pigs following subcutaneous injection of hSEAP vector under the control of a ubiquitous promoter and packaged in Php.B. hSEAP expression was assessed with an enzymatic assay and is displayed from 1 week prior to injection to 2 weeks following injection. The points and error bars plotted at each timepoint represent the mean and standard error of the mean of the measured hSEAP levels across all four pigs.

[0110] FIG. 13 is a graph showing the detected levels of hSEAP in the serum of 3 cynomolgous macaques following subcutaneous abdominal injection of hSEAP vector under the control of a ubiquitous promoter and packaged in Php.B. Monkey 1, 2, and 3 received 1.8el2, 9el 1, and 6el 1 vg in 6, 3, and 2 sites, respectively. hSEAP expression was assessed with an enzymatic assay and is displayed from 1 week prior to injection to 3 weeks following injection. The two points plotted for each animal at every timepoint represent repeated measurements of the assay.

[0111] FIGS. 14A-B are a series of microscopic images (FIG. 14A) and a graph (FIG. 14B) showing the extent to which synthetic combinations of regulatory sequences derived from adipocyte-specific genes could drive AAV transgene expression in adipocytes of nonhuman primates. Three different regulatory elements (mFabp4, mAdipoq, hAdipoq) driving EGFP were packaged into Php.B, and vectors were injected subcutaneously (30 uL x 3el 1 vg per site) into superficial adipose of cynomolgous macaques. FIG. 14A shows a microscopic image of a section taken from a site that was injected with Php.B.hAdipoq.EGFP. Images were collected with an RGB color camera, and the blue signal from hematoxylin labeling (middle) and red signal from GFP immunohistochemistry (right) has been digitally separated from the raw RGB image (left) using color deconvolution. Robust GFP signal can be observed in the superficial subcutaneous adipose, but not in the overlying dermis. FIG. 14B is a graph that shows the mean intensity of GFP immunohistochemical labeling in adipocytes (measured as optical density, OD) versus the total number of GFP-positive adipocytes in a single cross-section of each injection site. For comparison to expression from a ubiquitous promoter, data from a lower (3el0 vg) and equivalent (3el 1 vg) dose of a Php.B. smCBA.EGFP vector is also shown. The hAdipoq regulatory element showed significantly more GFP+ cells and higher mean GFP intensity compared to the mFabp4 and mAdipoq regulatory elements (p<0.01, One-way ANOVA with Tukey’s post-hoc test). At equal doses of 3el 1 vg, hAdipoq performed comparably, if not better, than the smCBA ubiquitous promoter.

[0112] FIGS 15A-B are a series of microscopic images (FIG. 15A) and a graph (FIG. 15B) showing that Rec2 efficiently transduces human adipocytes. FIG. 15A shows microscopic images of adipocytes in ~1 mL pieces of subcutaneous adipose tissue that were explanted from humans and cultured for 8d. A representative image from an untreated control and an explant that was injected with 30 uL x 3el 1 vg of Rec2.smCBA.EGFP at the beginning of the culture period are shown. Hematoxylin and GFP signal have been separated digitally using color deconvolution. A high density of intensely GFP-positive adipocytes can be observed throughout the explants, whereas no GFP labeling was observed in the control cultures. FIG. 15B is a graph showing the percentage of GFP-positive adipocytes quantified in cross-sections of each piece of explanted tissue. >80% of the adipocytes in each ~1 mL explant were GFP-positive, whereas no GFP labeling was detected in controls.

[0113] FIG. 16 is a graph showing changes in levels of an exemplary polypeptide (hSEAP) in serum of mice. All mice were administered an AAV particle having a Rec2 capsid and encoding an hSEAP polypeptide in the left inguinal fat pad. For the “no removal” group, the transduced fat pad was not removed and in the “removal” group the transduced fat pad was removed surgically. Serum was collected every 7 days (up to 28 days) from mice that did not have the transduced fat pad (i.e., adipose tissue) removed (circles, solid line). For mice that had the transduced fat pad (i.e., adipose tissue) removed on day 14 (x’s, dashed line) serum was collected every 7 days up-to day 14. Detected levels of hSEAP were normalized to the mean concentration of the group that did not have adipose removal at each timepoint and expressed as a percent change: for example, change in detectable levels of hSEAP on day 21 in mice that had the transduced fat pad removed is approximately a decrease of 0.6, or 60% compared to the levels of hSEAP in mice that did not undergo adipose removal.

[0114] FIG. 17 is a schematic of the Adipo2.BGI promoter comprising an hADIPOQ enhancer, an hADIPOQ promoter, and a human P-Globin intron, located upstream of an exemplary transgene. An exemplary sequence for the Adipo2.BGI promoter is provided as SEQ ID NO: 37.

[0115] FIG. 18 is a graph showing enhanced expression of exemplary transgenes with adipocyte-specific promoter Adipo2.BGI (SEQ ID NO: 37). FIG. 18 shows mRNA expression of exemplary transgenes driven by two adipocyte-specific promoters hAdipo (SEQ ID NO:6) or Adipo2.BGI (SEQ ID NO: 37) relative to transgene expression driven by a constitutive CBA promoter. After transfection of 3T3-L1 cells with plasmids comprising the respective promoters and transgenes, the 3T3-L1 cells were differentiated into adipocytes for 8 days. Quantitative RT-PCR was performed to measure mRNA levels of the exemplary transgenes and mRNA levels were normalized to CBA promoter controls. For each of the exemplary transgenes, the Adipo2.BGI promoter resulted in higher mRNA expression than the hAdipo promoter.

[0116] FIG. 19 is a graph showing expression of an exemplary transgene (SEAP) driven from the different promoters described in FIG. 18, in preadipocytes or adipocytes. Undifferentiated 3T3-L1 cells were transfected with constructs harboring a SEAP transgene driven by CBA promoter, hAdipo promoter (SEQ ID NO: 6), or Adipo2.BGI promoter (SEQ ID NO: 37). Supernatant samples were obtained from preadipocytes prior to differentiation, and from adipocytes after 7 days of differentiation. SEAP activity assay was performed using the same assay used in FIG. 12. DEFINITIONS

[0117] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.

[0118] 5’ and 3’: The terms “5”’ and “3”’ are relative terms to define a spatial relationship or directionality between two or more segment of a nucleic acid sequence. Thus, 3’ of a nucleic acid indicates a segment of the nucleic acid that is downstream of another segment, while 5’ indicates a segment of the nucleic acid that is upstream of another segment. For example, 3 ’ may indicate that a segment is in the 3 ’ half of the nucleic acid sequence or even at the 3’ end of the nucleic acid sequence. Similarly, 5’ may indicate that a segment is in the 3’ half of the nucleic acid sequence or even at the 5’ end of the nucleic acid sequence. Unless indicated otherwise, the directionality of a nucleic acid will be in the 5’ to 3’ direction of translation.

[0119] About or approximately. As used herein, the terms “approximately” or “about” in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0120] Adeno-associated virus (AAV): As used herein, the terms “Adeno-associated virus” and “AAV” refer to viral particles, in whole or in part, of the family Parvoviridae and the genus Dependoparvovirus. AAV is a small replication-defective, nonenveloped virus. AAV includes, but is not limited to, AAV serotype 1, AAV serotype 2, AAV serotype 3 (including serotypes 3A and 3B), AAV serotypes 4, AAV serotypes 5, AAV serotypes 6, AAV serotypes 7, AAV serotypes 8, AAV serotypes 9, AAV serotypes 10, AAV serotypes 11, AAV serotypes 12, AAV serotype 13, AAVrh74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, non-human primate AAV, e.g., from rhesus monkeys, and any variant of any of the foregoing. Wild-type AAV is replication deficient and requires coinfection of cells by a helper virus, e.g., adenovirus, herpes, or vaccinia virus, e.g., an Ad2 or Ad5 virus, or supplementation of helper viral genes, in order to replicate.

[0121] Administration: As used herein, the term “administration” refers to the administration of a composition comprising rAAV particles as described herein to a subject. Administration may be by any appropriate route. For example, in some embodiments, administration may be local or systemic administration (e.g., to a mammal, e.g., to a human, e.g., a patient). A composition of the disclosure may be administered by injection or infusion by any route. For example, a composition may be administered by retinal, subretinal, intravitreal, suprachoroidal, intraspinal, intra-cisterna magna, or intrathecal injection or infusion. Additional exemplary routes of administration may include, but are not limited to, bronchial (e.g., bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., intratracheal instillation), transdermal, vaginal, and vitreal.

[0122] Agent: As used herein, the term “agent”, may refer to a physical entity or phenomenon. In some embodiments, an agent may be characterized by a particular feature and / or effect. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymeric moiety. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantially free of any polymer or polymeric moiety. In some embodiments, an agent may be or comprise a system or device.

[0123] Bioreactor: The term “bioreactor,” as used herein, refers to any vessel used for the growth of a cell culture (e.g., a mammalian cell culture). The bioreactor can be of any size and / or any shape so long as it is useful for culturing a cell culture (e.g., a mammalian cell culture).

[0124] Cap polypeptide: As used herein, the term “Cap polypeptide” refers to the structural proteins that form a functional AAV capsid, which can in turn package DNA and infect or transduce a target cell. In some embodiments, a Cap polypeptide comprises a variant AAV capsid as disclosed herein. In some embodiments, Cap polypeptides will comprise all of the AAV capsid subunits, but less than all of the capsid subunits may be present as long as a functional capsid is produced. In some embodiments, the nucleic acid sequence encoding Cap polypeptides will be present on a single vector (e.g., plasmid). In some embodiments, the Cap polypeptide comprises an AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or AAVrh74 Cap polypeptide, or a variant of any of the foregoing. AAV capsid genes and proteins have been described in, e.g., Knipe el al., Fields Virology, Volume 1, (6th ed., Lippincott-Raven Publishers), which is hereby incorporated by reference in its entirety.

[0125] Cell Density. As used herein, the term “cell density” refers to that number of cells present in a given volume of medium or the number of cells present in a given surface area. For example, cell density may be represented as viable cells (vc) / cm2 of culture medium or vc / mL.

[0126] Culture'. As used herein, the terms “culture” and “cell culture” refer to a cell population (e.g., a eukaryotic cell population) that is suspended in or covered by a medium under conditions suitable to survival and / or growth of the cell population. As will be clear to those of ordinary skill in the art, these terms can also refer to the combination comprising the cell population and the medium.

[0127] Fragment: As used herein, the terms “fragment” or “portion” refers to a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole structure. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a nucleotide fragment comprises or consists of at least3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomeric units (e.g., nucleic acids) as found in the whole nucleotide. In some embodiments, a nucleotide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the monomeric units (e.g., residues) found in the whole nucleotide. The whole material or entity may in some embodiments be referred to as the “parent” of the whole.

[0128] Gene. As used herein, the term “gene” refers to a DNA sequence that codes for a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene includes coding sequence (i.e., a sequence that encodes a particular product). In some embodiments, a gene includes non-coding sequence. In some particular embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequence. In some embodiments, a gene may include one or more regulatory elements that, for example, may control or effect one or more aspects of gene expression (e.g., inducible expression, etc.).

[0129] Gene therapy . As used herein, the term “gene therapy” refers to insertion or deletion of specific genomic DNA sequences to treat or prevent a disorder or condition for which such therapy is sought. In some embodiments, the insertion or deletion of genomic DNA sequences occurs in specific cells (e.g., target cells). Target cells may be from a mammal and / or may be cells in a mammalian subject. Mammals include but are not limited to humans, dogs, cats, cows, sheep, pigs, llamas, etc. In some embodiments, heterologous DNA is transferred to target cells. The heterologous DNA may be introduced into the selected target cells in a manner such that the heterologous DNA is expressed and a therapeutic product encoded thereby is produced. Additionally or alternatively, the heterologous DNA may in some manner mediate expression of DNA that encodes the therapeutic product, or it may encode a product, such as a peptide or RNA that in some manner mediates or modulates, directly or indirectly, expression of a therapeutic product. Genetic therapy may also be used to deliver nucleic acid encoding a gene product that replaces a defective gene or supplements a gene product produced by the mammal or the cell in which it is introduced. The heterologous DNA encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof. Genetic therapy may also involve delivery of an inhibitor or repressor or other modulator of gene expression. Such an inhibitor or repressor or other modulator can be a polypeptide, peptide, or nucleic acid (e.g., DNA or RNA). Gene therapy may include in vivo or ex vivo techniques. In some embodiments, viral and non-viral based gene transfer methods can be used to introduce a nucleic acid encoding a polypeptide of interest or to introduce a therapeutic nucleic acid into mammalian cells or target tissues. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as poloxamers or liposomes. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256: BOSSIS (1992); Miller, Nature 357:455-460 (1992); Feuerbach et al., Kidney International 49:17911794 (1996); Urnov et al., Nature Reviews Genetics 11, 636-646 (2010); and Collins et al., Proceedings Biologicial Sciences / The Royal Society, 282(1821 ):pii 20143003 (2015), each of which is hereby incorporated by reference in its entirety.

[0130] Host Cell. As used herein, the term “host cell” refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. In some embodiments, host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life that are suitable for expressing an exogenous DNA (e.g., a recombinant nucleic acid sequence).

[0131] Identity. As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0132] Improve, increase, inhibit, or reduce: As used herein the terms “improve”, “increase,” “inhibit,” “reduce,” or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single sample, e.g., of a culture medium) under otherwise comparable conditions absent presence of (e.g., prior to and / or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.

[0133] Medium: As used herein, the terms “medium,” “culture medium,” and “growth medium” refer to a solution comprising nutrients to nourish cells (e.g., growing cells, e.g., eukaryotic cells). Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for survival and / or minimal growth. The solution can also comprise components that enhance survival and / or growth above the minimal rate, including hormones and growth factors. The solution can be formulated to a pH and concentration of one or more salts that are optimal for cellular survival and / or proliferation. For example, the medium can also be a “defined medium” or “chemically defined medium,” e.g., a serum-free medium that contains no proteins, hydrolysates, or components of unknown composition. Defined media are free of animal-derived components and all components have a known chemical structure. One of skill in the art understands a defined medium can comprise recombinant polypeptides, for example, but not limited to, hormones, cytokines, interleukins, and / or other signaling molecules.

[0134] Nucleic acid'. The term “nucleic acid” includes any nucleotides, analogs thereof, and polymers thereof. The term “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecules and, thus, include double- and singlestranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated, protected and / or capped nucleotides or polynucleotides. The terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligodeoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and / or modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified phosphorus-atom bridges (also referred to herein as “internucleotide linkages”). The term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified phosphorus atom bridges. Examples include, and are not limited to, nucleic acids containing ribose moieties, the nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. In some embodiments, the prefix poly- refers to a nucleic acid containing 2 to about 10,000, 2 to about 50,000, or 2 to about 100,000 nucleotide monomer units. In some embodiments, the prefix oligo- refers to a nucleic acid containing 2 to about 200 nucleotide monomer units. In accordance with the methods and compositions described herein, in some embodiments, an RNA comprises a short hairpin RNA (shRNA), small interfering RNA (siRNA), mRNA, snRNA, CRISPR / Cas guide RNA, microRNA (miRNA), and / or a precursor thereof.

[0135] Payload: As used herein, the term “payload” refers to a nucleic acid sequence (e.g., comprising a sequence that encodes a payload, such as a polypeptide or RNA) that is desired to be introduced into a cell, tissue, organ, organism, and / or system comprising cells; or a polypeptide. A pay load can be a heterologous protein with a therapeutic purpose, e.g., an enzyme or antibody. The payload can be a heterologous nucleic acid with a therapeutic purpose, e.g., an miRNA, siRNA, shRNA, mRNA, snRNA, or CRISPR / Cas guide RNA, or a precursor thereof. One of skill in the art will recognize that the payload can be selected from any heterologous protein or nucleic acid of interest. As used herein, “encode” or “encodes” means directs the expression of or processed into. For example, as used herein, a nucleic acid encodes a polypeptide sequence if it directs the expression of that polypeptide sequence. As another example, as used herein, a nucleic acid precursor (e.g., a pri-miRNA or pre-miRNA) encodes a further processed version of the nucleic acid (e.g., mature miRNA) if it is processed into the further processed version.

[0136] Pharmaceutical composition. As used herein, the term “pharmaceutical composition” refers to a composition comprising rAAV particles that is suitable for administration to a human or animal subject. In some embodiments, a pharmaceutical composition comprises an active agent formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen. In some embodiments, a therapeutic regimen comprises one or more doses administered according to a schedule that has been determined to achieve a desired therapeutic effect when administered to a subject or population in need thereof (e.g., by a statistically significant probability). A pharmaceutical composition may be specially formulated for administration in solid or liquid form. In some embodiments, a pharmaceutical composition is formulated for administration by parenteral administration, such as by subcutaneous, intramuscular, intravenous or epidural injection. In some embodiments, a pharmaceutical composition is formulated as a sterile solution or suspension, e.g., in a sustained-release formulation. Pharmaceutical compositions of the disclosure may be formulated for administration by injection (e.g., subcutaneous injection). In some embodiments, a pharmaceutical composition is intended and suitable for administration to a human subject. In some embodiments, a pharmaceutical composition is substantially free of contaminants (e.g., sterile and substantially pyrogen-free). Formulations of the pharmaceutical compositions may include, but are not limited to, formulations for administration to adipose tissue; topical application, such as a cream, ointment, or a controlled-release patch or spray applied to the skin,; or transdermally.

[0137] Polypeptide. The term “polypeptide”, as used herein, generally has its art-recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (e.g., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.

[0138] Recombinant. As used herein, the term “recombinant” is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and / or or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and / or direct expression of the polypeptide or one or more component s), portion(s), element(s), or domain(s) thereof; and / or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and / or otherwise generating a nucleic acid that encodes and / or directs expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc).

[0139] Recombinant AAV (rAAV) particle: A “recombinant AAV particle”, or “rAAV particle,” as used herein, refers to an infectious, replication-defective viral particle comprising an AAV protein shell encapsulating a payload that is flanked on both sides by ITRs. An AAV particle is produced in a suitable host cell (e.g., a HEK293 cell). For example, the host cell is transfected with at least one vector encoding one or more helper polypeptides (e.g., Ad2 helper polypeptides), at least one Rep polypeptide, at least one Cap polypeptide, and at least one pay load (e.g., for expression of a polypeptide or a therapeutic nucleic acid), such that the host cell is capable of producing the Rep and Cap polypeptides necessary for packing the rAAV particle. rAAV particles may be used for subsequent gene delivery.

[0140] Rep polypeptide: The term “Rep polypeptide”, as used herein, refers to the AAV non-structural proteins that mediate AAV replication for the production of AAV particles. The AAV replication genes and proteins have been described in, e.g., Knipe el al., FIELDS VIROLOGY, Volume 1, (6th ed., Lippincott-Raven Publishers), which is hereby incorporated by reference in its entirety.

[0141] Subject: As used herein, the term “subject” refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a pig, a cat, a dog). In some embodiments, a subject is not a rodent, e.g., a mouse and / or rat. In some embodiments, a human subject is an adult, adolescent, or pediatric subject. In some embodiments, a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder or condition that can be treated as provided herein, e.g., a neurological disease or disorder or a cancer or a tumor listed herein. In some embodiments, a subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and / or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing the disease, disorder or condition. In some embodiments, a subject displays one or more symptoms of a disease, disorder or condition. In some embodiments, a subject does not display a particular symptom (e.g. clinical manifestation of disease) or characteristic of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0142] Titer: As used herein, the term “titer” refers to the quantity of virus in a given volume. Titer, for example, can be expressed as viral genome copies (vg) per given volume or plaque forming units (pfu) per given volume. In some embodiments, titer can be expressed as number of capsids per given volume.

[0143] Transduction: As used herein, the term “transduction” refers to the introduction of one or more nucleic acid molecules that is encapsulated by a viral particle (e.g., encoding one or more viral components and / or one or more pay loads) into cells, such as eukaryotic cells (e.g., mammalian cells). In some embodiments, a viral particle is an AAV particle. In some embodiments, transduction occurs after a viral particle, e.g., an AAV particle, is targeted to cells, e.g., via binding of a moiety on a viral capsid of a viral particle [e.g., an AAV capsid of an AAV particle] to one or more receptors on the surface of a cell and internalization of a viral particle into cells.

[0144] Transfection: As used herein, the term “transfection” refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g., mRNA) molecules, into cells, such as eukaryotic cells (e.g., mammalian cells). For example, transfection can include vector-based transfection, viral-based transfection, electroporation, lipofection (e.g., with cationic lipids and / or liposomes), calcium phosphate precipitation, nanoparticle-based transfection, and / or transfection based on cationic polymers (e.g., DEAE-dextran or polyethylenimine). In some embodiments, viral-based transfection is also referred to herein as transduction.

[0145] Treating: As used herein, the term “treating” refers to providing treatment, e.g., providing any type of medical or surgical management of a subject. The treatment can be provided in order to reverse, alleviate, inhibit the progression of, prevent or reduce the likelihood of a disease, disorder, or condition, or in order to reverse, alleviate, inhibit or prevent the progression of, prevent or reduce the likelihood of one or more symptoms or manifestations of a disease, disorder or condition. “Prevent” refers to causing a disease, disorder, condition, or symptom or manifestation of such not to occur for at least a period of time in at least some individuals. Treating can include administering an agent to the subject following the development of one or more symptoms or manifestations indicative of a condition, disease, or disorder, e.g., in order to reverse, alleviate, reduce the severity of, and / or inhibit or prevent the progression of the condition and / or to reverse, alleviate, reduce the severity of, and / or inhibit or one or more symptoms or manifestations of the condition. A composition comprising rAAV particles of the disclosure can be administered to a subject who has developed a disorder or is at increased risk of developing such a disorder relative to a member of the general population. A composition of the disclosure can be administered prophylactically or before development of any symptom or manifestation of the condition. Typically, in this case, the subject will be at risk of developing the condition.

[0146] Variant: As used herein in the context of molecules, e.g., nucleic acids, or proteins, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. To give but a few examples, a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and / or contributing to a particular structural motif and / or biological function; a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Often, a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference. In some embodiments, a reference polypeptide or nucleic acid is one found in nature.

[0147] Vector. As used herein, the term “vector” refers to a molecule comprising a nucleic acid molecule, where the vector is capable of transporting the nucleic acid molecule into a cell. By way of non-limiting example, one type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be packaged into a viral capsid and can be transferred into another cell and / or organism. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0148] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference in its entirety. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0149] The present disclosure provides, inter alia, improved compositions (e.g., recombinant AAV vectors) for delivery of payloads to adipose tissue and methods of using the same.

[0150] The present disclosure is based, in part, on the discovery that the methods disclosed herein allow for local delivery of recombinant AAV vectors comprising payloads to adipose tissue e.g., via subcutaneous administration, without substantial distribution of such AAV vector to non-adipose tissue. Without wishing to be bound by any particular theory, in some embodiments, delivery of such recombinant AAV vectors to adipose tissue with a delivery method disclosed herein, substantially restricts distribution of such vectors to a location at or near a site of administration. In some embodiments, delivery of a recombinant AAV vector to adipose tissue with a delivery method disclosed herein, does not result in detectable distribution of such AAV vectors in non-adipose tissue. Further without wishing to be bound by any particular theory, this avoidance of distribution of the AAV vectors to non-adipose tissue is beneficial in a subject administered such AAV vectors to reduce unwanted off-target effects of the vectors, e.g., transduction of non-adipose cells or transduction of any cells that are distant from the site of administration. Targeting Adipose Tissue and / or Adipocytes

[0151] Targeting adipose tissues or adipocytes in culture e.g., with adenovirus, retrovirus, and lentivirus have been reported with various degrees of transduction efficiencies (see, e.g., Bates R., et. al., Adipose Tissue: An Emerging Target for Adeno-associated Viral Vectors. Mol Ther Methods Clin Dev. 2020 Sep 20; 19:236-249, which is incorporated herein by reference in its entirety). In animal models, liver, heart, skeletal muscle, eyes, and the central nervous system have been safely and successfully targeted for gene transfer (see, e.g., Bates R., et. al., Adipose Tissue: An Emerging Target for Adeno-associated Viral Vectors. Mol Ther Methods Clin Dev. 2020 Sep 20; 19:236-249, which is incorporated herein by reference in its entirety). However, adeno-associated (AAV)-mediated gene transfer poses challenges for specifically targeting adipose tissue or adipocytes, as AAV serotypes have a broad-spectrum affinity for tissues such as liver, heart, and muscle, and off-target effects present a challenge for specifically targeting adipose tissue. In addition, prior strategies to locally administer AAV vectors to the adipose tissue of mice resulted in transduction of non-adipose tissue and systemic distribution of the vector and the expression product encoded thereby (see, e.g., Jimenez V., et. al., In vivo adeno-associated viral vector-mediated genetic engineering of white and brown adipose tissue in adult mice. Diabetes. 2013 Dec;62(12):4012-22, which is incorporated herein by reference in its entirety).

[0152] Among other things, the present disclosures provides an insight that, local administration of a recombinant AAV vector to adipose tissue and / or adipocytes in a subject (e.g., non-human primates, pigs, or humans), for example, using methods provided herein, does not result in significant distribution (e.g., none or minimal distribution) of the AAV vector into non-adipose tissue. In some embodiments, when an AAV vector disclosed herein is administered to adipose tissue and / or adipocytes in a subject (e.g., non-human primates, pigs, or humans) the administered vector stays locally confined to a site of administration (e.g., an injection site). In some embodiments, when an AAV vector disclosed herein is administered to adipose tissue and / or adipocytes in a subject (e.g., non-human primates, pigs, or humans), the expression product of interest encoded thereby (e.g., encoded by the payload) the administered vector, stays locally confined to a site of administration (e.g., an injection site). In some embodiments, when an AAV vector disclosed herein is administered to adipose tissue and / or adipocytes in a subject (e.g., non-human primates, pigs, or humans), the expression product of interest encoded by (e.g., encoded by the payload) the administered vector, is distributed, e.g., secreted e.g., into the secretory system, by the transduced adipose tissue and / or adipocytes and may act a site distant from a site of administration (e.g., an injection site). Adipose Tissue

[0153] The present disclosure provides technologies including targeting adipose tissue and / or adipocyte in a subject. Adipose tissue is one of the largest organs and comprises several cell types e.g., adipocytes and stromal vascular fraction (or cells) e.g., preadipocytes, fibroblasts, mesenchymal stem cells (MSCs), endothelial and smooth muscle cells, macrophages, and immune cells (see, e.g., Richard AJ., et al. Adipose Tissue: Physiology to Metabolic Dysfunction. [Updated 2020 Apr 4], In: Feingold KR, Anawalt B, Blackman MR, et al., editors. Endotext, which is incorporated herein by reference in its entirety). Adipose tissue is distributed in various locations, including visceral and subcutaneous fat depots (see, e.g., Richard AJ., et al. Adipose Tissue: Physiology to Metabolic Dysfunction. [Updated 2020 Apr 4], In: Feingold KR, Anawalt B, Blackman MR, et al., editors. Endotext, which is incorporated herein by reference in its entirety). In addition, adipose tissue can be located in mediastinal, abdominal, and pelvic regions. Without wishing to be bound by any particular theory, unique innervation, vascularization, and cell-autonomous mechanisms in each fat depot may contribute to distinctive functions among various fat depots.

[0154] Adipose tissue is typically classified into two types, white adipose tissue (WAT) and brown adipose tissue (BAT), which are distinguishable based on tissue color. However, recently, two additional adipose tissue types - beige and pink - have been described. In some embodiments of any of the technologies described herein, an adipose tissue includes or comprises white adipose tissue, brown adipose tissue, beige adipose tissue, pink adipose tissue, or any combination thereof.

[0155] In some embodiments, an adipose tissue comprises one or more cell type. In some embodiments, an adipose tissue comprises an adipocyte. In some embodiments, an adipocyte is characterized as having a role in lipolysis, lipogenesis, glucose uptake, thermogenesis, or any combination thereof.

[0156] In some embodiments, non-adipose tissue does not comprises adipocytes. For example, a non-adipose tissue may be cardiac, neural, liver, or skeletal tissue. In some embodiments, a non-adipocyte does not comprises a characteristic of an adipocyte, e.g., as described herein. For example, a non-adipocyte may be a cardiac, neural, liver, or bone cell.

[0157] Methods disclosed herein are useful in delivering AAV particles to adipose tissue. In addition to classifying adipose tissue based on tissue color, adipose tissue can also be identified by its location in a subject’s body. For example, superficial subcutaneous adipose tissue can be found between the dermis and Scapa’s fascia. Deep subcutaneous adipose tissue can be found between Scapa's fascia and Camper's fascia. As yet another example, visceral adipose tissue can be found in or around organs.

[0158] In some embodiments, technologies and methods disclosed herein can be used to administer an AAV particle or a composition comprising the same to any adipose tissue in a subject. In some embodiments, adipose tissue comprises superficial subcutaneous adipose tissue. In some embodiments, adipose tissue comprises deep subcutaneous adipose tissue. In some embodiments, adipose tissue comprises visceral adipose tissue.

[0159] In some embodiments, technologies and methods disclosed herein can be used to administer an AAV particle or a composition comprising the same to one or more adipocytes. In some embodiments, adipocytes that can be targeted with methods and technologies disclosed herein include superficial subcutaneous adipocytes. White Adipose Tissue (WAT)

[0160] White adipose tissue (WAT) makes up the largest adipose tissue volume in the human body. WAT is a large lipid and energy storage in mammalian physiology. For example, WAT contributes to storing and releasing energy during fasting and fed intervals. In addition, WAT has been shown to be involved in endocrine action through adipokine secretion, such as leptin and adiponectin, which is important to control food intake and glucose homeostasis (see, e.g., Richard AJ., et al. Adipose Tissue: Physiology to Metabolic Dysfunction. [Updated 2020 Apr 4], In: Feingold KR, Anawalt B, Blackman MR, et al., editors. Endotext, which is incorporated herein by reference in its entirety).

[0161] WAT is comprised of large adipocytes with a single lipid droplet, and this morphology confers on WAT the capacity for energy storage and homeostasis in response to nutritional demands (e.g., see, Reyes-Farias M., et. al., White adipose tissue dysfunction in obesity and aging. Biochem Pharmacol. 2021 Oct; 192:114723, which is incorporated herein by reference in its entirety). In humans, WAT can be classified according to its distribution in two main depots: visceral, which includes omental, mesenteric, retroperitoneal, gonadal, and pericardial WAT, and subcutaneous, which is located under the skin.

[0162] In some embodiments of any of the technologies described herein, an adipose tissue includes or consists of white adipose tissue. In some embodiments, white adipose tissue comprises white adipocytes. In some embodiments, white adipose tissue is located near or at the following locations in a subject: subcutaneous depot and / or organs. Brown Adipose Tissue (BAT)

[0163] Brown adipose tissue (BAT) is largely present in mammals postnatally and during hibernation. While BAT was originally thought to only be present in infant humans, studies have revealed metabolically active BAT in the supraclavicular and thoracic regions of adults (e.g., see, Nedergaard J., et. al., Unexpected evidence for active brown adipose tissue in adult humans. Am J Physiol Endocrinol Metab. 2007 Aug;293(2):E444-52.; Cypess AM., et. al., Identification and importance of brown adipose tissue in adult humans. N Engl J Med. 2009 Apr 9;360(15):1509-17; and Zingaretti MC., The presence ofUCPl demonstrates that metabolically active adipose tissue in the neck of adult humans truly represents brown adipose tissue. FASEB J. 2009 Sep;23(9):3113-20, each of which is incorporated herein by reference in its entirety).

[0164] Studies have also shown that women have increased BAT mass and activity over men, and chance of detecting BAT activity in either sex has been shown to be inversely correlated with age and body mass index (BMI) (see, e.g., Cypess AM., et. al., Identification and importance of brown adipose tissue in adult humans. N Engl J Med. 2009 Apr 9;360(15):1509-17; and Zingaretti MC., The presence ofUCPl demonstrates that metabolically active adipose tissue in the neck of adult humans truly represents brown adipose tissue. FASEB J. 2009 Sep;23(9):3113-20, which is incorporated herein by reference in its entirety). Further correlations have also been reported, e.g., BAT activity can be higher in the winter and lower in the summer, and in healthy humans, BAT activity contributes to whole-body fat oxidation and diet-induced thermogenesis (see, e.g., Hibi M., Oishi S, Matsushita M, Yoneshiro T, Yamaguchi T, Usui C, Yasunaga K, Katsuragi Y, Kubota K, Tanaka S, Saito M. Brown adipose tissue is involved in diet-induced thermogenesis and whole-body fat utilization in healthy humans. Int J Obes (Lond). 2016Nov;40(ll):1655-1661.

[0165] Brown adipocytes contain multiple lipid droplets dispersed throughout a more ellipsoidal-shaped cell that is enriched with iron-containing mitochondria, giving a brown adipocyte (and the BAT as a whole) a brownish hue (see, e.g., Richard AJ., et al. Adipose Tissue: Physiology to Metabolic Dysfunction. [Updated 2020 Apr 4], In: Feingold KR, Anawalt B, Blackman MR, et al., editors. Endotext, which is incorporated herein by reference in its entirety). Thermogenic activity of brown adipocytes can be attributed, in part, by a presence of numerous mitochondria containing uncoupling protein 1 (UCP-1), a proton transporter that short-circuits the ATP (energy)-generating proton gradient and allows for concurrent heat production as protons flow back into the mitochondrial matrix (see, e.g., Richard AJ., et al. Adipose Tissue: Physiology to Metabolic Dysfimction. [Updated 2020 Apr 4], In: Feingold KR, Anawalt B, Blackman MR, et al., editors. Endotext, which is incorporated herein by reference in its entirety).

[0166] BAT in infants and young adults has been described to be localized mainly in the cervical-supraclavicular region as well as in periaortic areas inside the thorax and the abdomen, and in particular in the perirenal fat (see, e.g., Zoico E., et. al., Brown and Beige Adipose Tissue and Aging. Front Endocrinol (Lausanne). 2019 Jun 20;10:368, which is incorporated herein by reference in its entirety). With aging detectable BAT decreases progressively and it remains represented mainly in the supraclavicular and perirenal sites (see, e.g., Zoico E., et. al., Brown and Beige Adipose Tissue and Aging. Front Endocrinol (Lausanne). 2019 Jun 20; 10:368, which is incorporated herein by reference in its entirety).

[0167] In some embodiments of any of the technologies described herein, an adipose tissue includes or consists of brown adipose tissue. In some embodiments, brown adipose tissue comprises brown adipocytes. In some embodiments, brown adipose tissue and / or brown adipocytes are characterized as having a role in metabolizing fat to produce heat and / or energy metabolism. In some embodiments, brown adipose tissue is located near or at the following locations in a subject: neck, kidney, adrenal glands, heart (e.g., aorta), mediastinum, shoulder. Beige Adipose Tissue (BeAT)

[0168] Beige adipocyte is a type of adipose cell described by the ability to induce these cells to produce heat and increase energy expenditure (see, e.g., Lizcano F., The Beige Adipocyte as a Therapy for Metabolic Diseases. Int J Mol Sci. 2019 Oct 12;20(20):5058, which is incorporated herein by reference in its entirety). Beige adipocyte can be considered phenotypically as a fat cell that possesses characteristics between those of the white fat cell, an accumulator of energy, and the brown cell, which produces heat (see, e.g., Lizcano F., The Beige Adipocyte as a Therapy for Metabolic Diseases. Int J Mol Sci. 2019 Oct 12;20(20):5058, which is incorporated herein by reference in its entirety). Without wishing to be bound by any particular theory, beige adipocytes can be derived from the transformation of mature white adipocytes, arise from a distinct adipocyte precursor, or come from de novo differentiation from tissueresident progenitors.

[0169] In some embodiments of any of the technologies described herein, an adipose tissue includes or consists of beige adipose tissue. In some embodiments, beige adipose tissue comprises beige adipocytes. In some embodiments, beige adipose tissue is located near or at the following locations in the subject: abdomen, face, glute, and / or femur. Pink Adipose Tissue (PAT)

[0170] Pink adipocyte can derive from white adipocytes that take on epithelial-like features to form milk-secreting alveoli, giving the tissue a pink hue (see, e.g., Richard AJ., et al. Adipose Tissue: Physiology to Metabolic Dysfunction. [Updated 2020 Apr 4], In: Feingold KR, Anawalt B, Blackman MR, et al., editors. Endotext, which is incorporated herein by reference in its entirety). Pink adipocytes are characterized by compartmentalized lipid droplets, cytoplasmic projections, and abundant organelles including mitochondria, peroxisomes, and rough endoplasmic reticulum, that show a structure more typical of epithelial cells (see, e.g., Richard AJ., et al. Adipose Tissue: Physiology to Metabolic Dysfunction. [Updated 2020 Apr 4], In: Feingold KR, Anawalt B, Blackman MR, et al., editors. Endotext, which is incorporated herein by reference in its entirety).

[0171] In some embodiments of any of the technologies described herein, an adipose tissue includes or consists of pink adipose tissue. In some embodiments, pink adipose tissue comprises pink adipocytes. In some embodiments, pink adipose tissue is located near or at the following locations in the subject: abdomen, breast, bone marrow, and / or dermis. Administration methods

[0172] The present disclosure, among other things, provides methods of administering a composition to adipose tissue and / or adipocytes. In some embodiments, the present disclosure provides methods of administering a composition comprising a vector comprising a payload to adipose tissue and / or adipocytes in a subject. In some embodiments, a subject is a mammal. In some embodiments, a mammal is a pig, cow, dog, cat, non-human primate, or human. In some embodiments, a mammal is a human. In some embodiments, a mammal is a non-rodent mammal (e.g., not a rat or mouse).

[0173] In some embodiments, methods disclosed herein result in local distribution (e.g., at or near a site of administration) of a composition comprising a vector comprising a payload in a subject. In some embodiments, local delivery (e.g., at or near a site of administration) of a composition comprising a vector comprising a payload disclosed herein results in local expression (e.g., at or near a site of administration) of the payload. In some embodiments, local delivery (e.g., at or near a site of administration) of a composition comprising a vector comprising a payload disclosed herein results in local expression (e.g., at or near a site of administration) of the payload. In some embodiments, local delivery (e.g., at or near a site of administration) of a composition comprising a vector comprising a payload disclosed herein results in local expression (e.g., at or near a site of administration) of the pay load and subsequent distribution of a payload to non-adipose tissue (e.g., distant from of an administration site).

[0174] In some embodiments, local delivery of a composition comprising a payload disclosed herein does not result in detectable distribution of the composition systemically (e.g., no systemic distribution). In some embodiments, local delivery of a composition comprising a pay load disclosed herein to adipose tissue and / or adipocytes with a delivery method disclosed herein, does not result in detectable distribution of the composition systemically (e.g., no systemic distribution).

[0175] In some embodiments, local delivery of a composition comprising a vector comprising a payload disclosed herein to adipose tissue and / or adipocytes with a delivery method disclosed herein, results in a payload that is expressed locally, but distributed systemically (e.g., via the secretory system).

[0176] For example, local delivery (e.g., delivery to adipose tissue), of an AAV particle, results in local distribution (e.g., transduction of adipocytes at the site of administration) but no or minimal systemic distribution of the AAV particle (e.g., no or minimal transduction of cells outside the site of administration). As a further example, local delivery (e.g., delivery to adipose tissue), of an AAV particle comprising a payload, results in local expression (e.g., in adipose tissue at a site of administration) of the payload and local distribution of the payload. In some embodiments, local delivery (e.g., delivery to adipose tissue), of an AAV particle comprising a pay load, results in local expression (e.g., in adipose tissue at a site of administration) of the pay load and distribution of the payload to non-adipose tissue (e.g., distant from of an administration site).

[0177] In some embodiments, a payload is expressed and / or distributed at a therapeutically effective level. In some embodiments, a payload can be detected by about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, or about 2 weeks after administration.

[0178] In some embodiments, expression of a payload (e.g., locally at or near an administration site) persists for about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months, about 24 months, about 36 months, about 48 months, about 5 years, about 10 years, or about 20 years after administration.

[0179] In some embodiments, expression of a payload (e.g., locally at or near an administration site) persists for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, at least 5 years, at least 10 years, or at least 20 years after administration.

[0180] In some embodiments, distribution of a payload (e.g., systemically) persists for about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months, about 24 months, about 36 months, about 48 months, about 5 years, about 10 years, or about 20 years after administration.

[0181] In some embodiments, distribution of a payload (e.g., systemically) persists for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, at least 5 years, at least 10 years, or at least 20 years after administration.

[0182] In some embodiments, administration of a composition to a subject does not substantially target (e.g., does not target) non-adipose tissue and / or non-adipocytes in a subject. In some embodiments, administration of a composition to a subject does not substantially target (e.g., does not target) non-adipose tissue and / or non-adipocytes that was not directly administered a composition. In some embodiments, administration of a composition to a subject does not substantially target (e.g., does not target) adipose tissue and / or adipocytes that was not directly administered a composition.

[0183] In some embodiments, administration of a composition to a subject does not substantially transduce (e.g., does not transduce) non-adipose tissue and / or non-adipocytes in a subject. In some embodiments, administration of a composition to a subject does not substantially transduce (e.g., does not transduce) non-adipose tissue and / or non-adipocytes that was not directly administered a composition. In some embodiments, administration of a composition to a subject does not substantially transduce (e.g., does not transduce) adipose tissue and / or adipocytes that was not directly administered a composition.

[0184] In some embodiments, targeting of a composition comprising an AAV particle to adipose tissue and / or adipocytes comprises recognition, e.g., binding, of an AAV particle through one or more moieties on an AAV capsid to one or more receptors or other proteins on a surface of a cell. In some embodiments, targeting of an AAV particle to adipose tissue comprises binding to one or more molecules on a surface of a cell in adipose tissue and internalization into a cell, e.g., via endocytosis. In some embodiments, targeting of adipose tissue and / or adipocytes by an AAV particle does not necessarily comprise transduction of adipose tissue and / or adipocytes by the AAV particle.

[0185] In some embodiments, transduction of an adipose tissue and / or adipocyte with an AAV particle comprises targeting of said adipose tissue and / or adipocyte by an AAV particle, release of one or more nucleic acid components in the AAV particle (e.g., nucleic acid sequence encoding a payload and / or nucleic acid sequence encoding one or more components of an AAV vector genome) into a cell, and expression of one or more nucleic acid components (e.g., nucleic acid sequence encoding a payload and / or nucleic acid sequence encoding one or more components of an AAV vector genome) in a cell.

[0186] In some embodiments, provided methods herein include subcutaneous injection of a composition in a subject. In some embodiments, depth of an injection is between a dermis and Scapa's fascia (e.g., superficial subcutaneous adipose tissue) of a subject. In some embodiments, depth of an injection is between a Scarpa's fascia and Camper's fascia (e.g., deep subcutaneous adipose tissue) of a subject. In some embodiments, depth of an injection is within visceral adipose tissue of a subject. In some embodiments, injection of a composition as described herein is guided by a device. In some embodiments, a device is an imaging device.

[0187] In some embodiments of any of the methods of delivering an AAV particle or a composition comprising the same as disclosed herein, an AAV particle is characterized in that when administered to adipose tissue an AAV particle is contained in adipose tissue as compared to an otherwise similar AAV particle that is not delivered according to a method disclosed herein, e.g., not directly delivered to adipose tissue.

[0188] In some embodiments of any of the methods of delivering an AAV particle or a composition comprising the same as disclosed herein, an AAV particle is characterized in that when administered to adipose tissue, an AAV particle transduces at least 5% adipocytes in adipose tissue administered an AAV particle as compared to adipocyte transduction by an otherwise similar AAV particle that is not delivered according to a method disclosed herein, e.g., not directly delivered to adipose tissue. In some embodiments, an AAV particle when delivered to adipose tissue according to a method disclosed herein transduces at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% adipocytes in adipose tissue administered an AAV particle. In some embodiments, an AAV particle when delivered to adipose tissue according to a method disclosed herein transduces about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% adipocytes in adipose tissue administered an AAV particle.

[0189] Any device known in the art for use in delivering a composition (e.g., pharmaceutical compounds) to a subject may be used. In some embodiments, a composition as described herein may be filled into a device for administration to a subject. In some embodiments, a composition may be administered by the subject or by a third person (e.g. doctor). In some embodiments, a composition may be administered automatically administered to the subject. In some embodiments, a device includes or consists of a syringe and needle, autoinjector, injection pen, or jet injector. In some embodiments, parameters such as needle gauge, needle length, and syringe barrel size can potentially impact injection time and depth.

[0190] A composition (e.g., a pharmaceutical composition) described herein can be administered in a sufficient or effective amount to a subject in need thereof. Doses can vary and depend upon a type, onset, progression, severity, frequency, duration, or probability of disease to which treatment is directed, the clinical endpoint desired, previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject, and other factors that will be appreciated by a skilled artisan. Dose amount, number, frequency, or duration may be proportionally increased or reduced, as indicated by any adverse side effects, complications, or other risk factors of treatment and status of the subject. A skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit.

[0191] A dose to achieve a therapeutic effect will vary based on several factors including, but not limited to: route of administration, level of payload or payload expression required to achieve a therapeutic effect, specific disease treated, any host immune response, and stability of payload or level of payload expression. One skilled in the art can determine a dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors.

[0192] An effective amount or a sufficient amount can (but need not) be provided in a single administration, may require multiple administrations, and, can (but need not) be, administered alone or in combination with another composition. For example, an amount may be proportionally increased as indicated by need of a subject, type, status, and severity of disease treated or side effects (if any) of treatment. Amounts considered effective also include amounts that result in a reduction of use of another treatment, therapeutic regimen, or protocol. Administration Sites

[0193] In some embodiments, a composition described herein is administered (e.g., injected) to a plurality of locations in a subject (e.g., injection sites). In some embodiments, a plurality of locations are non-overlapping (e.g., non-overlapping injection sites). In some embodiments, a plurality of locations are overlapping (e.g., overlapping injection sites).

[0194] In some embodiments, a composition is administered to at least one location (e.g., at least one injection site) in a subject. In some embodiments, a composition is administered between about 1 to about 10 different locations (e.g., injection site), about 1 to about 20 different locations (e.g., injection site), about 1 to about 30 different locations (e.g., injection site), about 1 to about 40 different locations (e.g., injection site), about 1 to about 50 different locations (e.g., injection site), about 1 to about 60 different locations (e.g., injection site), about 1 to about 70 different locations (e.g., injection site), about 1 to about 80 different locations (e.g., injection site), about 1 to about 90 different locations (e.g., injection site), about 1 to about 100 different locations (e.g., injection site) in a subject. In some embodiments, a composition is administer to at least 100 different locations (e.g., injection site) in a subject.

[0195] In some embodiments, a location (e.g., an injection site) in a subject comprises white adipose tissue, brown adipose tissue, beige adipose tissue, pink adipose tissue, or any combination thereof. In some embodiments, a location (e.g., an injection site) in a subject comprises white adipocytes, brown adipocytes, beige adipocytes, pink adipocytes, or any combination thereof.

[0196] In some embodiments, a location (e.g., an injection site) in a subject comprises superficial subcutaneous adipose tissue, deep subcutaneous adipose tissue and / or visceral adipose tissue. In some embodiments, a location (e.g., an injection site) in a subject comprises superficial subcutaneous adipocytes.

[0197] In some embodiments, a location (e.g., an injection site) is in the same tissue. In some embodiments, a location (e.g., an injection site) is in different tissues.

[0198] In some embodiments, a location (e.g., an injection site) is defined in relation to a prior location (e.g., prior injection site). In some embodiments, a location (e.g., an injection site) is defined in relation to a distance from another location (e.g., another injection site). For example, a first location is a first administration site (e.g., first injection site) and a second location is a second administration site (e.g., second injection site) with a specified distance from the first administration site (e.g., first injection site). In some embodiments, a location (e.g., injection site) may be at least about 0.1 cm, about 0.2 cm, about 0.3 cm, about 0.4 cm, about 0.5 cm, about 0.6 cm, about 0.7 cm, about 0.8 cm, about 0.9 cm, about 1.0 cm, about 2.0 cm, about 3.0 cm, about 4.0 cm, about 5.0 cm, about 6.0 cm, about 7.0 cm, about 8.0 cm, about 9.0 cm, about 10.0 cm, about 11.0 cm, about 12.0 cm, about 13.0 cm, about 14.0 cm, about 15.0 cm, about 16.0 cm, about 17.0 cm, about 18.0 cm, about 19.0 cm, or about 20.0 cm in distance from another location (e.g., another injection).

[0199] In some embodiments, a composition disclosed herein is administered at a plurality of sites in a first section of adipose tissue in a subject. In some embodiments, a first section of adipose tissue comprises adipose tissue in a particular location (e.g., a first location) in a subject’s body. In some embodiments, an AAV particle is administered at between 1 and 100 different sites in a first section of adipose tissue.

[0200] In some embodiments, an AAV particle is administered at another section (e.g., a second or subsequent section) of adipose tissue in a subject’s body. In some embodiments, another section (e.g., a second or subsequent section) of adipose tissue is at a different location from a first section of adipose tissue. In some embodiments, an AAV particle is administered at a plurality of sites in another section of adipose tissue (e.g., a different location from a first section of adipose tissue).

[0201] In some embodiments, a first or subsequent section of adipose tissue is selected based on one or more characteristics of adipocytes in a first or subsequent section.

[0202] In some embodiments, a location (e.g., an injection site) is visualized with an agent. In some embodiments, a location (e.g., an injection site) is characterized with an agent. In some embodiments, structures of a location (e.g., an injection site) is visualized with an agent. For example, an agent facilitates visualization of a location (e.g., injection site) and provides guidance of undesired location (e.g., non-injection site, e.g., blood vessels). In some embodiments, an agent includes or consists of a dye or contrast agent. In some embodiments, an agent includes an adipose tissue specific marker and / or adipocyte specific marker. In some embodiments, an agent includes an non-adipose tissue specific marker and / or non-adipocyte specific marker. Dosing

[0203] In accordance with various embodiments, methods provided herein, further include administering one or more doses of a composition. In accordance with various embodiments, methods provided herein, include administering one or more doses of a composition to adipose tissue and / or adipocytes.

[0204] In some embodiments, a subject is administered a volume of a composition. In some embodiments, a subject is administered a volume of a composition to target adipose tissue and / or adipocytes. Dosing volume and dose of AAV particle

[0205] In accordance with various embodiments, any of a variety of application -appropriate volumes may be administered. In some embodiments, a composition disclosed herein (e.g., comprising an AAV particle) is administered to a subject at a volume of about 10 uL, about 20 uL, about 30 uL, about 40 uL, about 50 uL, about 60 uL, about 70 uL, about 80 uL, about 90 uL, about 100 uL, about 120 uL, about 140 uL, about 160 uL, aboutl80 uL, about 200 uL, about 220 uL, about 240 uL, about 260 uL, about 280 uL, about 300 uL, about 350 uL, about 400 uL, about 450 uL, about 500 uL, about 550 uL, about 600 uL, about 650 uL, about 700 uL, about 750 uL, about 800 uL, about 850 uL, about 900 uL, about 950 uL, about 1000 uL, about 1100 uL, about 1200 uL, about 1300 uL, about 1400 uL, about 1500 uL, about 1600 uL, about 1700 uL, about 1800 uL, about 1900 uL, about 2000 uL, about 2100 uL, about 2200 uL, about 2300 uL, about 2400 uL, about 2500 uL, about 2600 uL, about 2700 uL, about 2800 uL, about 2900 uL, or about 3000 uL.

[0206] In some embodiments, a composition disclosed herein (e.g., comprising an AAV particle) is administered to a subject at a volume of at least 10 uL, at least 20 uL, at least 30 uL, at least 40 uL, at least 50 uL, at least 60 uL, at least 70 uL, at least 80 uL, at least 90 uL, at least 100 uL, at least 120 uL, at least 140 uL, at least 160 uL, at least 180 uL, at least 200 uL, at least 220 uL, at least 240 uL, at least 260 uL, at least 280 uL, at least 300 uL, at least 350 uL, at least 400 uL, at least 450 uL, at least 500 uL, at least 550 uL, at least 600 uL, at least 650 uL, at least 700 uL, at least 750 uL, at least 800 uL, at least 850 uL, at least 900 uL, at least 950 uL, at least 1000 uL, at least 1100 uL, at least 1200 uL, at least 1300 uL, at least 1400 uL, at least 1500 uL, at least 1600 uL, at least 1700 uL, at least 1800 uL, at least 1900 uL, at least 2000 uL, at least 2100 uL, at least 2200 uL, at least 2300 uL, at least 2400 uL, at least 2500 uL, at least 2600 uL, at least 2700 uL, at least 2800 uL, at least 2900 uL, or at least 3000 uL.

[0207] In accordance with various embodiments, any of a variety of application -appropriate volumes may be administered. In some embodiments, a composition disclosed herein (e.g., comprising an AAV particle) is administered to a subject at a volume of about 10 uL, about 20 uL, about 30 uL, about 40 uL, about 50 uL, about 60 uL, about 70 uL, about 80 uL, about 90 uL, about 100 uL, about 120 uL, about 140 uL, about 160 uL, aboutl80 uL, about 200 uL, about 220 uL, about 240 uL, about 260 uL, about 280 uL, about 300 uL, about 350 uL, about 400 uL, about 450 uL, about 500 uL, about 550 uL, about 600 uL, about 650 uL, about 700 uL, about 750 uL, about 800 uL, about 850 uL, about 900 uL, about 950 uL, about 1000 uL, about 1100 uL, about 1200 uL, about 1300 uL, about 1400 uL, about 1500 uL, about 1600 uL, about 1700 uL, about 1800 uL, about 1900 uL, about 2000 uL, about 2100 uL, about 2200 uL, about 2300 uL, about 2400 uL, about 2500 uL, about 2600 uL, about 2700 uL, about 2800 uL, about 2900 uL, or about 3000 uL per injection site.

[0208] In some embodiments, a composition disclosed herein (e.g., comprising an AAV particle) is administered to a subject at a volume of at least 10 uL, at least 20 uL, at least 30 uL, at least 40 uL, at least 50 uL, at least 60 uL, at least 70 uL, at least 80 uL, at least 90 uL, at least 100 uL, at least 120 uL, at least 140 uL, at least 160 uL, at least 180 uL, at least 200 uL, at least 220 uL, at least 240 uL, at least 260 uL, at least 280 uL, at least 300 uL, at least 350 uL, at least 400 uL, at least 450 uL, at least 500 uL, at least 550 uL, at least 600 uL, at least 650 uL, at least 700 uL, at least 750 uL, at least 800 uL, at least 850 uL, at least 900 uL, at least 950 uL, at least 1000 uL, at least 1100 uL, at least 1200 uL, at least 1300 uL, at least 1400 uL, at least 1500 uL, at least 1600 uL, at least 1700 uL, at least 1800 uL, at least 1900 uL, at least 2000 uL, at least 2100 uL, at least 2200 uL, at least 2300 uL, at least 2400 uL, at least 2500 uL, at least 2600 uL, at least 2700 uL, at least 2800 uL, at least 2900 uL, or at least 3000 uL per injection site.

[0209] In some embodiments, a composition disclosed herein (e.g., comprising an AAV particle) is administered to a subject at a dose of about 1 x 10(11) vector genomes (vg) to about 3 xl0(14) vg.

[0210] In some embodiments, a composition disclosed herein (e.g., comprising an AAV particle) is administered to a subject at a dose of about 1 x 10(12) vg / ml to about 1 x 10(14) vg / ml. Repeat Dosing

[0211] In accordance with various embodiments, methods provided herein, further include one or more subsequent doses of a composition. In some embodiments, one or more subsequent doses of a composition is administered to the same adipose tissue (e.g., the same adipose deposit) to which the first dose of the gene therapy is administered. In some embodiments, one or more subsequent doses of a composition is administered to a different adipose tissue as compared to the adipose tissue to which the first dose of the composition is administered.

[0212] In some embodiments, one or more subsequent doses of a composition are administered at the same volume as the first dose of the composition. In some embodiments, one or more subsequent doses of a composition are administered at different volume as the first dose of the composition.

[0213] In some embodiments, one or more subsequence doses of a composition are repeatedly administered at different timepoints. In some embodiments, one or more subsequence doses of a composition are repeatedly administered at different timepoints up until a desired therapeutic dose. Combination Therapy

[0214] In some embodiments, one or more compositions described herein are administered in combination with a second agent (e.g., a second composition or other therapeutic agent). In some embodiments, a first composition may be administered simultaneously with a second agent. In some embodiments, a first composition and second agent may be administered sequentially (e.g., within minutes, hours, days, weeks, or months of one another). In some embodiments, one or more compositions and / or agents may be administered via the same route. In some embodiments, one or more compositions and / or agents may be administered via different routes.

[0215] In some embodiments, one or more compositions described herein are administered in combination with an agent. In some embodiments, an agent modulates one or more body systems and / or organs. In some embodiments, an agent modulates activity of a payload. For example, in some embodiments, an agent enhances activity of a payload.

[0216] In some embodiments, an agent comprises one or more of: an immune modulating agent (e.g., an immune suppressing agent or an immune stimulating agent), an agent that reduces immunogenicity, an agent that reduces inflammation, an agent that enhances expression (e.g., locally) and / or activity (e.g., locally and / or systemically) of a payload, an agent that reduces and / or inhibits neutralizing antibodies (e.g., neutralizing antibodies which bind to an AAV capsid).

[0217] In some embodiments, an agent includes or consists of an immune modulating agent. In some embodiments, a first composition comprising a payload described herein is administered in combination with a second composition comprising an immune modulating agent. In some embodiments, a first compositions comprising a payload described herein is administered in combination with a second composition comprising an immune modulating agent that modifies an immune response induced by the payload of the first composition. In some embodiments, an immune modulating agent includes or consists of a corticosteroid (e.g., dexamethasone), rapamycin, tacrolimus, methotrexate, mycophenolate mofetil, rituximab, eculizumab or their derivatives thereof. Gene Therapy

[0218] Gene therapies alter the gene expression profile of a patient’s cells by gene transfer, a process of delivering an exogenous therapeutic gene, called a transgene. Various delivery vehicles are known to be used as vectors to transport transgenes into the nucleus of a cell to alter or augment a cell’s capabilities (e.g., proteome, functionality, etc.).

[0219] There are multiple gene therapy approaches (e.g., viral or non-viral including liposomes). For example, AAV gene therapy, in which a transgene is introduced into a nucleus of a host cell, but is not intended to integrate in chromosomal DNA. The transgene is expressed from a non-integrated genetic element called an episome that exists inside the nucleus. A second type of gene therapy employs the use of a different type of virus, such as lentivirus, that inserts itself, along with the transgene, into the chromosomal DNA but at arbitrary sites. Episomal expression of a gene must be driven by an exogenous promoter, leading to production of a protein that corrects or ameliorates the disease condition.

[0220] In some embodiments, a transgene is provided using a delivery vehicle. In some embodiments, compositions of the present disclosure comprise a delivery vehicle. In some embodiments, a delivery vehicle is or comprises a viral particle (e.g., a viral vector). In some embodiments, a delivery vehicle is a lipid particle (e.g., a lipid nanoparticle).

[0221] In some embodiments, a combination of one or more different payloads may be delivered with one or more delivery systems described herein (e.g., viral vector). In some embodiments, a delivery system (e.g., viral vector) may deliver one payload in combination with a second, distinct pay load. In some embodiments, a first delivery system (e.g., viral vector) may deliver one payload in combination with a second, distinct delivery system (e.g., viral vector), which delivers a second, distinct payload. Viral Vectors

[0222] In some embodiments, a delivery system is or comprises a viral vector. Viral vectors comprise virus or viral chromosomal material, within which a heterologous nucleic acid sequence can be inserted for transfer into a target sequence of interest (e.g., for transfer into genomic DNA within a cell). Various viruses can be used as viral vectors, including, e.g., singlestranded DNA (ssDNA), double-stranded DNA (dsDNA) viruses, and / or RNA viruses with a DNA stage in their lifecycle. In some embodiments, a viral vector is or comprises an adeno-associated virus (AAV) or AAV variant. 11V Particle

[0223] In some embodiments, an AAV particle is a single unit of virus comprising a capsid encapsidating a virus-based polynucleotide (e.g., a wild-type viral genome or a recombinant viral vector). In some embodiments, a vector particle is or comprises an AAV vector particle. In some embodiments, an AAV particle refers to a vector particle comprised of at least one AAV capsid protein and an encapsidated AAV vector. In some embodiments, a vector particle (also referred to as a viral vector) comprises at least one AAV capsid protein and an encapsidated AAV vector, wherein the vector further comprises one or more heterologous polynucleotide sequences. Capsid

[0224] In some embodiments, an AAV particle comprises a capsid protein. In some embodiments, an AAV capsid protein is or comprises an AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 (AAVrhlO), AAV11, AAV12, AAV-PHP.B, AAV8-PHP.eB, AAV-PHP.S, AAV-7m8, AAV-DJ, AAV-OligOOl or Rec2 capsid protein, or a variant or fragment of any of the foregoing.

[0225] In some embodiments, an AAV particle comprises a capsid protein provided in Table 1, or a fragment thereof, or a sequence with at least 90% identity thereto.

[0226] In some embodiments, an AAV particle comprises a capsid protein provided in any one of SEQ ID NO:s 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, or 29, or a sequence with at least 90% identity thereto. Table 1: Exemplary AAV capsid protein sequences SEQ ID NO Description Sequence 16 Rec2 MAADGYLPDWLEDNLSEGIREWWDLI<PGAPI<PI<ANQQI<Q DDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKA YDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAV FQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGI GKTGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGS GTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDR VITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQ VKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQG CLPPFP AD VFMIPQ YGYLTLNNGS Q AVGRS SF YCLE YFPS Q MLRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYL YYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPC YRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIA MATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEE EIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGM VWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPP QILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQ KENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGT RYLTRNL 17 AAV-PHP.B MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQ DNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKA YDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRA VFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAG IGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGS LTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDR VITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQ VKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHE GCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQ MLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYL YYLSRTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYR QQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAM ASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEI KTTNPVATESYGQVATNHQSAQTLAVPFKAQAQTGWVQN QGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGF GMI<HPPPQILII<NTPVPADPPTAFNI<DI<LNSFITQYSTGQVS SEQ ID NO Description Sequence VEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGV YSEPRPIGTRYLTRNL 18 AAV1 MAADGYLPDWLEDNLSEGIREWWDL1<PGAPI<PI<ANQQI<Q DDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKA YDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAV FQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIG KTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPT TMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRV ITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPW GYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQV KEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGC LPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYY LNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYR QQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMAS HKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIK ATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMV WQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQI L1I<NTPVPANPPAEFSATI<FASFITQYSTGQVSVEIEWELQI<E NSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTR YLTRPL 19 AAV2 MAADGYLPDWLEDTLSEGIRQWWI<L1<PGPPPPI<PAERHI<D DSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYD RQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ A K K R VEE PLGLVE E P VKT A PGK K R P VE H S P VE PD S S S GTGK AGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNT MATGS GAPM A D N N EG A DG VGN S S GNWHC D S TWMGD R VI TTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWG YFDFNRFHCHFSPRDWQRL1NNNWGFRPI<RLNFI<LFNIQVI< EVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCL PPFP ADVFMVPQ YGYLTLNNGSQ AVGRS SF YCLEYFPS QML RTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYL SRTNTPTGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQ RVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASH KDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTT NPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQ DRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIK NTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENS KRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLT RNL 20 AAV3B MAADGYLPDWLEDNLSEGIREWWALKPGVPQPKANQQHQ DNRRGLVLPGYKYLGPGNGLDKGEPVNEADAAALEHDKA YDQQLKAGDNPYLKYNHADAEFQERLQEDTSFGGNLGRA SEQ ID NO Description Sequence VFQ AKKRILEPLGLVEEAAKT APGKKRPVDQ SPQEPD S S S G VGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLG SNTM ASGGGAPM ADNNEGADGVGNS SGN WHCDSQ WLGD RVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQ VKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQG CLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQ MLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYL YYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPC YRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPA MASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEE EIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGM VWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPP QIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQ KENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGT RYLTRNL 21 AAV4 MTDGYLPDWLEDNLSEGVREWWALQPGAPKPKANQQHQ DNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKA YDQQLKAGDNPYLKYNHADAEFQQRLQGDTSFGGNLGRA VFQAKKRVLEPLGLVEQAGETAPGKKRPLIESPQQPDSSTGI GKKGKQPAKKKLVFEDETGAGDGPPEGSTSGAMSDDSEMR AAAGGAAVEGGQGADGVGNASGDWHCDSTWSEGHVTTT STRTWVLPTYNNHLYKRLGESLQSNTYNGFSTPWGYFDFN RFHCHFSPRDWQRLINNNWGMRPKAMRVKIFNIQVKEVTT SNGETTVANNLTSTVQIFADSSYELPYVMDAGQEGSLPPFPN DVFMVPQYGYCGLVTGNTSQQQTDRNAFYCLEYFPSQMLR TGNNFEITYSFEKVPFHSMYAHSQSLDRLMNPLIDQYLWGL QSTTTGTTLNAGTATTNFTKLRPTNFSNFKKNWLPGPSIKQQ GFSKTANQNYKIPATGSDSLIKYETHSTLDGRWSALTPGPP MATAGPADSKFSNSQLIFAGPKQNGNTATVPGTLIFTSEEEL AATNATDTDMWGNLPGGDQSNSNLPTVDRLTALGAVPGM VWQNRDIYYQGPIWAKIPHTDGHFHPSPLIGGFGLKHPPPQI FIKNTPVPANPATTFSSTPVNSFITQYSTGQVSVQIDWEIQKE RSKRWNPEVQFTSNYGQQNSLLWAPDAAGKYTEPRAIGTR YLTHHL 22 AAV5 MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQ ARGLVLPGYNYLGPGNGLDRGEPVNRADEVAREHDISYNE QLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQA KKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSK PSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLG DNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLP SYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHS HWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTT SEQ ID NO Description Sequence TIANNLTSTVQVFTDDDYQLPYWGNGTEGCLPAFPPQVFT LPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEF TYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTG GVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNR ASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYAL ENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYN VGGQM ATNNQS SET AP ATGT YNLQEIVPGS VWMERD V YLQ GPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPG NITSFSDVPVS SFITQ YS TGQ VTVEME WELK KENS KRWNPEI QYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL 23 AAV6 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQ DDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKA YDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAV FQAKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIG KTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPT TMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRV ITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPW GYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQV KEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGC LPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQM LRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYY LNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYR QQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMAS HKDDKDKF FPMS GVMIFGKES AG A S NT A LDN VMITDEEEIK ATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMV WQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQI LIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKE NSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTR YLTRPL 24 AAV7 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQ DNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKA YDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAV FQAKKRVLEPLGLVEEGAKTAPAKKRPVEPSPQRSPDSSTGI GKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSSVGS GTVAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDR VITTSTRTWALPTYNNHLYKQISSETAGSTNDNTYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLRFKLFNIQ VKEVTTNDGVTTIANNLTSTIQVFSDSEYQLPYVLGSAHQG CLPPFPADVFMIPQYGYLTLNNGSQSVGRSSFYCLEYFPSQM LRTGNNFEFSYSFEDVPFHSSYAHSQSLDRLMNPLIDQYLYY LARTQSNPGGTAGNRELQFYQGGPSTMAEQAKNWLPGPCF RQQRVSKTLDQNNNSNFAWTGATKYHLNGRNSLVNPGVA MATHKDDEDRFFPS SGVLIFGKTGATNKTTLENVLMTNEEE SEQ ID NO Description Sequence IRPTNPVATEEYGIVSSNLQAANTAAQTQVVNNQGALPGM VWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPP QILIKNTPVPANPPEVFTPAKFASFITQYSTGQVSVEIEWELQ KENSKRWNPEIQYTSNFEKQTGVDFAVDSQGVYSEPRPIGT RYLTRNL 25 AAV8 MAADGYLPDWLEDNLSEGIREWWALI<PGAPI<PI<ANQQI<Q DDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKA YDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAV FQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGI GKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGP NTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDR VITTS TRTW A LPT YNNUL YKQISNGTS GGATNDNTYFGYS T PWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNI QVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQ GCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQ MLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYL YYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPC YRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIA MATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEE EIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGM VWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPP QILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQ KENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGT RYLTRNL 26 AAV9 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQ DNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKA YDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRA VFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAG IGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGS LTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDR VITTS TRTW ALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQ VKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHE GCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQ MLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYL YYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYR QQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAM ASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEI KTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGM VWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPP QILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQ KENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGT RYLTRNL SEQ ID NO Description Sequence 27 AAV10 MAADGYLPDWLEDNLSEGIREWWDLI<PGAPI<PI<ANQQI<Q DDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKA YDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAV FQAKKRVLEPLGLVEEAAKTAPGKKRPVEPSPQRSPDSSTGI GKKGQQPAKKRLNFGQTGESESVPDPQPIGEPPAGPSGLGS GTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDR VITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQ VKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQG CLPPFP AD VFMIPQ YGYLTLNNGS Q AVGRS SF YCLE YFPS Q MLRTGNNFEFS YTFEDVPFHS S Y AHSQSLDRLMNPLIDQ YL YYLSRTQSTGGTQGTQQLLFSQAGPANMSAQAKNWLPGPC YRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVA MATHKDDEERFFPSSGVLMFGKQGAGRDNVDYSSVMLTSE EEIKTTNPVATEQYGWADNLQQANTGPIVGNVNSQGALPG MVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPP PQILIKNTPVPADPPTTFSQAKLASFITQYSTGQVSVEIEWEL QKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIG TRYLTRNL 30 rhlO MAADGYLPDWLEDNLSEGIREWWDLI<PGAPI<PI<ANQQI<Q DDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKA YDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAV FQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGI GKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGS GTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDR VITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQ VKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQG CLPPFP AD VFMIPQ YGYLTLNNGS Q AVGRS SF YCLE YFPS Q MLRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYL YYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPC YRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVA M ATHKDDEERFF PS SGVLMFGKQGAGKDNVD YS S VMLTSE EEIKTTNPVATEQYGWADNLQQQNAAPIVGAVNSQGALP GMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHP PPQILIKNTPVPADPPTTFSQAKLASFITQYSTGQVSVEIEWE LQKENSKRWNPEIQYTSNYYKSTNVDFAVNTDGTYSEPRPI GTRYLTRNL 28 AAV11 MAADGYLPDWLEDNLSEGIREWWDLI<PGAPI<PI<ANQQI<Q DDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKA YDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAV FQAKKRVLEPLGLVEEGAKTAPGKKRPLESPQEPDSSSGIGK KGKQPARKRLNFEEDTGAGDGPPEGSDTSAMSSDIEMRAAP SEQ ID NO Description Sequence GGNAVDAGQGSDGVGNASGDWHCDSTWSEGKVTTTSTRT WVLPTYNNHLYLRLGTTSSSNTYNGFSTPWGYFDFNRFHC HFSPRDWQRLINNNWGLRPKAMRVKIFNIQVKEVTTSNGET TVANNLTSTVQIFADSSYELPYVMDAGQEGSLPPFPNDVFM VPQYGYCGIVTGENQNQTDRNAFYCLEYFPSQMLRTGNNF EMAYNFEKVPFHSMYAHSQSLDRLMNPLLDQYLWHLQSTT SGETLNQGNAATTFGKIRSGDFAFYRKNWLPGPCVKQQRFS KTASQNYKIPASGGNALLKYDTHYTLNNRWSNIAPGPPMA TAGPSDGDFSNAQLIFPGPSVTGNTTTSANNLLFTSEEEIAAT NPRDTDMFGQIADNNQNATTAPITGNVTAMGVLPGMVWQ NRDIYYQGPIWAKIPHADGHFHPSPLIGGFGLKHPPPQIFIKN TPVPANPATTFTAARVDSFITQYSTGQVAVQIEWEIEKERSK RWNPEVQFTSNYGNQSSMLWAPDTTGKYTEPRVIGSRYLT NHL 29 AAV12 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQ DNGRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKA YDKQLEQGDNPYLKYNHADAEFQQRLATDTSFGGNLGRA VFQAKKRILEPLGLVEEGVKTAPGKKRPLEKTPNRPTNPDS GKAPAKKKQKDGEPADSARRTLDFEDSGAGDGPPEGSSSGE MSHDAEMRAAPGGNAVEAGQGADGVGNASGDWHCDSTW SEGRVTTTSTRTWVLPTYNNHLYLRIGTTANSNTYNGFSTP WGYFDFNRFHCHFSPRDWQRLINNNWGLRPKSMRVKIFNI QVKEVTTSNGETTVANNLTSTVQIFADSTYELPYVMDAGQE GSFPPFPNDVFMVPQYGYCGWTGKNQNQTDRNAFYCLEY FPSQMLRTGNNFEVSYQFEKVPFHSMYAHSQSLDRMMNPL LDQYLWHLQSTTTGNSLNQGTATTTYGKITTGDFAYYRKN WLPGACIKQQKFSKNANQNYKIPASGGDALLKYDTHTTLN GRWSNMAPGPPMATAGAGDSDFSNSQLIFAGPNPSGNTTTS SNNLLFTSEEEIATTNPRDTDMFGQIADNNQNATTAPHIANL DAMGIVPGMVWQNRDIYYQGPIWAKVPHTDGHFHPSPLM GGFGLKHPPPQIFIKNTPVPANPNTTFSAARINSFLTQYSTGQ VAVQIDWEIQKEHSKRWNPEVQFTSNYGTQNSMLWAPDN AGNYHELRAIGSRFLTHHL

[0227] In some embodiments, an AAV particle comprises an AAV1 capsid protein, or a variant or fragment thereof. In some embodiments, an AAV particle comprises an AAV1 capsid protein of SEQ ID NO: 18, or a sequence with at least 90% identity thereto.

[0228] In some embodiments, an AAV particle comprises a Rec2 capsid protein, or a variant or fragment thereof. In some embodiments, an AAV particle comprises an Rec2 capsid protein of SEQ ID NO: 16, or a sequence with at least 90% identity thereto.

[0229] In some embodiments, an AAV particle comprises an AAV-PHP.B capsid protein, or a variant or fragment thereof. In some embodiments, an AAV particle comprises an AAV-PHP.B capsid protein of SEQ ID NO: 17, or a sequence with at least 90% identity thereto. Vectors for expressing payloads and components thereof

[0230] Many forms of vectors can be used in compositions and methods described herein. Non-limiting examples of vectors include naked DNA, lentivirus, HSV, adenovirus, retrovirus, long-lasting RNA. A vector genetic element may be delivered by any suitable method known in the art, e.g., to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques (See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y, which is incorporated herein by reference in its entirety).

[0231] In some embodiments, a vector encodes at least one helper polypeptide. In some embodiments, a vector encodes at least one Rep polypeptide and / or at least one Cap polypeptide. In some embodiments, a vector comprises at least one payload (e.g., for expression of polypeptide or as an inhibitory or guide nucleic acid). In some embodiments, a vector encodes at least one helper polypeptide and at least one Rep polypeptide. In some embodiments, a vector encodes at least one Cap polypeptide and at least one payload.

[0232] A vector can include conventional control elements operably linked to a nucleic acid encoding any polypeptide or payload described herein, in a manner that permits transcription, translation and / or expression in a cell (e.g., adipocyte) transfected with a vector described herein. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A number of expression control sequences, including promoters that are native, constitutive, inducible, and / or adipose tissue-specific, are known in the art and may be included in a vector described herein.

[0233] Exemplary inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors, such as temperature, or the presence of a specific physiological state (e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only). Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Examples of inducible promoters regulated by exogenously supplied promoters include a zinc-inducible sheep metallothionine (MT) promoter, a dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, a T7 polymerase promoter system, an ecdysone insect promoter, a tetracycline-repressible system, a tetracycline-inducible system, a RU486-inducible system, and an rapamycin-inducible system.

[0234] In some embodiments, native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic native expression (e.g., in adipose tissue and / or adipocytes).

[0235] In some embodiments, a vector described herein may include at least one miRNA target sequence that limits expression in non-adipocytes. In some embodiments, at least one miRNA target sequence is miRl, miR122, miR142, miR652, or any combination thereof.

[0236] In some embodiments, a vector described herein further includes at least one nucleotide sequence that further suppresses immunogenicity of a vector or an expression products. In some embodiments, a vector including a Toll-Like-Receptor 9 cloaking sequence.

[0237] In some embodiments, a vector is encapsulated in a delivery vehicle (e.g., lipid nanoparticle).

[0238] In some embodiments, methods described herein comprise localized transduction of adipocytes in a subject with an AAV vector comprising a nucleotide sequence encoding a pay load, wherein the method comprises injecting (e.g., subcutaneously) into adipose tissue of a subject the AAV vector.

[0239] In some embodiments, methods described herein comprise increasing the localized expression of a pay load in a subject, wherein the method comprises injecting (e.g., subcutaneously) into adipose tissue of the subject an AAV vector comprising a nucleotide sequence encoding a payload. Payloads

[0240] In some embodiments, one or more vectors or constructs described herein may comprise a nucleotide sequence encoding one or more payloads.

[0241] As described herein, one or more vectors disclosed herein results in local expression of a payload (e.g., by transducing one or more cells such as an adipocyte). A payload that is expressed locally, e.g., at or near a site of administration, can be distributed locally (e.g., at or near a site of administration) and / or systemically (e.g., at one or more distant sites in the body, e.g., at one or more tissues and / or compartments that are not directly administered an AAV particle).

[0242] In some embodiments, an AAV particle disclosed herein is administered at a first site, e.g., adipose tissue. In some embodiments, a payload encoded by a polynucleotide construct in an AAV particle is expressed at a first site. In some embodiments, a payload expressed at a first site can be distributed at a first site (e.g., locally). In some embodiments, a payload distributed at a first site can have one or more activities associated with a payload. In some embodiments, a payload expressed at a first site can be distributed to a second site, e.g., a site other than a first site, e.g., a different tissue and / or compartment in the body (e.g., systemically). In some embodiments, a payload distributed at a second site can have one or more activities associated with a payload.

[0243] In accordance with various aspects, any of a variety of payloads may be used (e.g., those with a diagnostic and / or therapeutic purpose), alone or in combination. In some embodiments, a payload may be or comprise a polynucleotide sequence encoding a peptide or polypeptide. In some embodiments, a payload is a peptide that has cell-intrinsic or cell-extrinsic activity that promotes a biological process to treat a medical condition. In some embodiments, a payload may be or comprise a transgene (also referred to herein as a gene of interest (GOI)). In some embodiments, a payload may be or comprise one or more transgenes with flanking ITR sequences. In some embodiments, a payload may be or comprise one or more heterologous nucleic acid sequences encoding a reporter gene (e.g., a fluorescent or luminescent reporter). In some embodiments, a pay load may be or comprise one or more biomarkers (e.g., proxy for payload expression). In some embodiments, a payload may comprise a sequence for polycistronic expression (including, e.g., a 2A peptide, or intronic sequence, internal ribosomal entry site). In some embodiments, a payload may be or comprise a polynucleotide sequence, which comprises an expression cassette.

[0244] In some embodiments, a transgene is a corrective gene chosen to improve one or more signs and / or symptoms of a disease, disorder, or condition. In some embodiments, a transgene may integrate into a host cell genome through use of vector(s) encompassed by the present disclosure. In some embodiments, transgenes are functional versions of disease associated genes (i.e., gene isoform(s) which are associated with the manifestation or worsening of a disease, disorder or condition) found in a host cell. In some embodiments, transgenes are an optimized version of disease-associated genes found in a host cell (e.g., codon optimized or expression-optimized variants). In some embodiments, transgenes are variants of disease-associated genes found in a host cell (e.g., functional gene fragment or variant thereof). In some embodiments, a transgene is a gene that causes expression of a peptide that is normally expressed in one or more healthy tissues.

[0245] In some embodiments, a transgene may be or comprise a gene encoding a functional nucleic acid. In some embodiments, a therapeutic agent is or comprises an agent that has a therapeutic effect upon a host cell or subject (including, e.g., a ribozyme, guide RNA (gRNA), antisense oligonucleotide (ASO), miRNA, siRNA, and / or shRNA). For example, in some embodiments, a therapeutic agent promotes a biological process to treat a medical condition, e.g., at least one symptom of a disease, disorder, or condition.

[0246] In some embodiments, transgenes are transiently expressed in a subject (e.g., episomal expression from plasmids, mini circle DNAs, viruses, etc.) one or more weeks after treatment at a level comparable to that observed within one or more days after treatment. In some embodiments, transgenes are transiently expressed in a subject (e.g., episomal expression from plasmids, minicircle DNAs, viruses, etc.) one or more months after treatment at a level comparable to that observed within one or more days after treatment.

[0247] In some embodiments, transgenes are transiently expressed in a subject (e.g., episomal expression from plasmids, mini circle DNAs, viruses, etc.) one or more weeks after treatment at a level that is reduced relative to that observed within one or more days after treatment. In some embodiments, transgenes are transiently expressed in a subject (e.g., episomal expression from plasmids, mini circle DNAs, viruses, etc.) one or more months after treatment at a level that is reduced relative to that observed within one or more days after treatment.

[0248] In some embodiments, a nucleotide sequence encoding a transgene is codon-optimized. In some embodiments, a nucleotide sequence encoding a transgene is codon-optimized for a certain cell type (e.g., mammalian, insect, bacterial, fungal, etc.). In some embodiments, a nucleotide sequence encoding a transgene is codon-optimized for a human cell. In some embodiments, a nucleotide sequence encoding a transgene is codon-optimized for a human cell of a particular tissue type (e.g., adipose).

[0249] In some embodiments, one or more vectors described herein comprise a nucleotide sequence encoding GLA, LPL, FVIII, FIX, GLP-1, GIP, ADIPOQ, FGF19, FGF21, PPY, BMP7, LEP, Adalimumab, Etanercept, Pembrolizumab, Ustekinumab, Dupilumab, Nivolumab, Daratumumab, Risankizumab, Secukinumab, Pertuzumab, Emicizumab, Denosumab, Abatocept, Atezolizumab, Durvalumab, Guselkumab, Tocilizumab, Ixekizumab, Infliximab, Eculizumab, Ravulizumab, Insulin. Polypeptide payloads

[0250] An AAV particle disclosed herein can comprise a polynucleotide construct comprising a nucleotide sequence encoding a payload.

[0251] In some embodiments, a payload is or comprises a polypeptide, e.g., encoded by a nucleic acid sequence within an AAV particle.

[0252] In some embodiments, a polypeptide is or comprises an enzyme.

[0253] In some embodiments, a polypeptide is or comprises an antibody.

[0254] In some embodiments, a polypeptide is or comprises a secreted protein.

[0255] In some embodiments, a polypeptide is or comprises GLA, LPL, FVIII, FIX, GLP-1, GIP, ADIPOQ, FGF19, FGF21, PPY, BMP7, LEP, Adalimumab, Etanercept, Pembrolizumab, Ustekinumab, Dupilumab, Nivolumab, Daratumumab, Risankizumab, Secukinumab, Pertuzumab, Emicizumab, Denosumab, Abatocept, Atezolizumab, Durvalumab, Guselkumab, Tocilizumab, Ixekizumab, Infliximab, Eculizumab, Ravulizumab, Insulin, or any combination thereof. Polynucleotide payloads

[0256] In some embodiments, a polynucleotide construct comprises one or more coding sequences. In some embodiments, one or more coding sequences encode a polyribonucleotide (RNA). In some embodiments, one or more coding sequences encode a polypeptide.

[0257] In some embodiments, a polynucleotide construct comprises a transgene.

[0258] In some embodiments, a payload is or comprises a DNA molecule.

[0259] In some embodiments, a payload is or comprises a polyribonucleotide (RNA) molecule. In some embodiments, an RNA molecule is or comprises a messenger RNA, an inhibitory RNA, or a non-coding RNA. Regulatory elements

[0260] Polynucleotide constructs disclosed herein can include one or more additional elements, such as a regulatory element. In some embodiments, a regulatory element comprises one or more promoters or a fragment or variant thereof, one or more enhancers or a fragment or variant thereof, one or more silencers or a fragment or variant thereof, one or more insulators or a fragment or variant thereof, or any combination thereof.

[0261] In some embodiments, a polynucleotide disclosed herein comprises one or more regulatory elements disclosed in Table 2, or a fragment thereof, or a sequence having at least 90% identity thereto. Table 2: Exemplary regulatory elements in polynucleotides disclosed herein. SEQ ID NO Description Sequence 1 mirl / 122 / 142 / 6 52 target sequences CAAACACCATTGTCACACTCCATGAATGGCACCCTCTCCT AGGGTTGATACATACTTCTTTACATTCCATCCATAAAGTA GGAAACACTACACAAACACCATTGTCACACTCCATGAAT GGCACCCTCTCCTAGGGTTGATACATACTTCTTTACATTC CATCCATAAAGTAGGAAACACTACA 2 mirl / 122 target sequences CAAACACCATTGTCACACTCCAATACATACTTCTTTACAT TCCACAAACACCATTGTCACACTCCAATACATACTTCTTT SEQ ID NO Description Sequence ACATTCCACAAACACCATTGTCACACTCCAATACATACTT CTTTACATTCCACAAACACCATTGTCACACTCCAATACAT ACTTCTTTACATTCCA 3 smCBA promoter CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACC GCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTA TGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGT CAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCA GTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTG ACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCC AGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACAT CTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGC CCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCC ACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTG CAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCA GGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAG GCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGC TCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGC GGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTC GCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCC GCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTAC TCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGG GCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTT CTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGCTA GAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTA CAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATC ATTTTGGCAAAGGCGCGCCGAAGTTGGTCGTGAGGCACT GGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGA GACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTC TTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCC ACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGGCG 4 CAG promoter TACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCC CCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTA ACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAG TATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACG GTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTT ATGGGACTTTCCTACTTGGCAGTACATCTACTCGAGGCCA CGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCC CCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGC GATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCG GGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGG AGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGA SEQ ID NO Description Sequence AAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCC TATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGGGATC AGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGA AAAACCAGAAAGTTAACTGGTAAGTTTAGTCTTTTTGTCT TTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAA GAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCT GTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATTG TACCC 5 mouse fabp4(ap2) regulatory element-derived promoter AATTCCAGCAGGAATCAGGTAGCTGGAGAATCGCACAGA GCCATGCGATTCTTGGCAAGCCATGCGACAAAGGCAGAA ATGCACATTTCACCCAGAGAGAAGGGATTGATGTCAGCA GGAAGTCACCACCCAGAGAGCAAATGGAGTTCCCAGATG CCTGACATTTGCCTTCTTACTGGATCAGAGTTCACTAGTG GAAGTGTCACAGCCCAAACACTCCCCCAAAGCTCAGCCC TTCCTTGCCTTGTAACAATCAAGCCGCTCCTGGATGAACT GCTCCGCCCTCTGTCTCTTTGGCAGGGTTGGAGCCCACTG TGGCCTGAGCGACTTCTATGGCTCCCTTTTCTGTGATTTTC ATGGTTTCTGAGCTCTTTTCCCCCGCTTTATGATTTTCTCT TTTTGTCTCTCTCTTGCTAAACCTCCTTCGTATATATGCCC TCTCAGGTTTCATTTCTGAATCATCTACTGTGAACTATTCC CATTGTTTGCCAGAAGCCCCCTGGTTCTTCCTTCTAGACA CCAGGCAAGGGGCAGGAGGTAAGAGGCAGGAGTCCATA AAACAGCCCTGAGAGCCTGCTGGGTCAGTGCCTGCTGTC AGAA 6 Exemplary human adipoq regulatory element-derived promoter 1 CTCTTTCCACATGACGGCCTTTGTGGTGGGTGGCAGATTG CCCTGAGGCCTCGCAAAATGCTAGGCTTTCACAATGTCAC TGACTGACAGCCAGGCCCAGCACAGTCTTGGTGTGATTG TGGGGCTAAAGTTATTCCACCTTGTGCAATAGCTACAGCG GCGCGCCAGCTTTAAGAATTCAGGGCCTTTTTAACTTGCC AAGCCCCACACCACTCCAGGAACTTCCCCACACCCCAGT TCTCAGAATTCATGTGCAAGGTCTTTCCTAAATCCAGGGT CCAGGTCAGAGAGTGGAGGATGTGCTCTATTTCTTACCTG ATTGCAGACCCCTCTGACAGTGCTCCCTTCTGAAGCACTC ACTGTCTGAACGTACACAGTCTCAGACTTAATCATGCACA GTGAGCAAGACTGTGGTGTGATAATTGGCGTCCCTGACTT ATTAGGGCAAATCTATGGGAGGGGGAGACCTCCTGGACC ACTGAGCAATTAATTCATTTACATTAGGAAGTTTCTCCGT CAGATGCAGGAAAAAAATCTTGTTTTCCTGCTGTGGTTTT GACTTTTGCCCCATCTTCTGTTGCTGTTGTAGGAGGCAAA ATAAGGGTCAAGGCCTGGAAACACAAGTGCTTTGACTGA AGCTCCACTTGGCTTCCGAAGCCCAAGCTGGGTTGTACCA GGTTCCCTAGGGTGCAGGCTGTGGGCAACTGCCAGGGAC ATGTGCCTGCCCACCGGCCTCTGGCCCTCACTGAGTTGGC SEQ ID NO Description Sequence CAATGGGAAATGACAATTGTGAGGTGGGGACTGCCTGCC CCCGTGAGTACCAGGCTGTTGAGGCTGGGCCATCTCCTCC TCACTTCCATTCTGACTGCAGTCTGTGGTTCTGATTCCAT ACCAGAGGG 36 Exemplary human adipoq regulatory element-derived promoter 2 CTCTTTCCACATGACGGCCTTTGTGGTGGGTGGCAGATTG CCCTGAGGCCTCGCAAAATGCTAGGCTTTCACAATGTCAC TGACTGACAGCCAGGCCCAGCACAGTCTTGGTGTGATTG TGGGGCTAAAGTTATTCCACCTTGTGCAATAGCTACAGCG GCGCGCCAGCTTTAAGAATTCAGGGCCTTTTTAACTTGCC AAGCCCCACACCACTCCAGGAACTTCCCCACACCCCAGT TCTCAGAATTCATGTGCAAGGTCTTTCCTAAATCCAGGGT CCAGGTCAGAGAGTGGAGGATGTGCTCTATTTCTTACCTG ATTGCAGACCCCTCTGACAGTGCTCCCTTCTGAAGCACTC ACTGTCTGAACGTACACAGTCTCAGACTTAATCATGCACA GTGAGCAAGACTGTGGTGTGATAATTGGCGTCCCTGACTT ATTAGGGCAAATCTATGGGAGGGGGAGACCTCCTGGACC ACTGAGCAATTAATTCATTTACATTAGGAAGTTTCTCCGT CAGATGCAGGAAAAAAATCTTGTTTTCCTGCTGTGGTTTT GACTTTTGCCCCATCTTCTGTTGCTGTTGTAGGAGGCAAA ATAAGGGTCAAGGCCTGGAAACACAAGTGCTTTGACTGA AGCTCCACTTGGCTTCCGAAGCCCAAGCTGGGTTGTACCA GGTTCCCTAGGGTGCAGGCTGTGGGCAACTGCCAGGGAC ATGTGCCTGCCCACCGGCCTCTGGCCCTCACTGAGTTGGC CAATGGGAAATGACAATTGTGAGGTGGGGACTGCCTGCC CCCGTGAGTACCAGGCTGTTGAGGCTGGGCCATCTCCTCC TCACTTCCATTCTGACTGCAGTCTGTGGTTCTGATTCCAT A 37 Exemplary human adipoq regulatory element-derived promoter 2 and human beta globin intron CTCTTTCCACATGACGGCCTTTGTGGTGGGTGGCAGATTG CCCTGAGGCCTCGCAAAATGCTAGGCTTTCACAATGTCAC TGACTGACAGCCAGGCCCAGCACAGTCTTGGTGTGATTG TGGGGCTAAAGTTATTCCACCTTGTGCAATAGCTACAGCG GCGCGCCAGCTTTAAGAATTCAGGGCCTTTTTAACTTGCC AAGCCCCACACCACTCCAGGAACTTCCCCACACCCCAGT TCTCAGAATTCATGTGCAAGGTCTTTCCTAAATCCAGGGT CCAGGTCAGAGAGTGGAGGATGTGCTCTATTTCTTACCTG ATTGCAGACCCCTCTGACAGTGCTCCCTTCTGAAGCACTC ACTGTCTGAACGTACACAGTCTCAGACTTAATCATGCACA GTGAGCAAGACTGTGGTGTGATAATTGGCGTCCCTGACTT ATTAGGGCAAATCTATGGGAGGGGGAGACCTCCTGGACC ACTGAGCAATTAATTCATTTACATTAGGAAGTTTCTCCGT CAGATGCAGGAAAAAAATCTTGTTTTCCTGCTGTGGTTTT GACTTTTGCCCCATCTTCTGTTGCTGTTGTAGGAGGCAAA ATAAGGGTCAAGGCCTGGAAACACAAGTGCTTTGACTGA SEQ ID NO Description Sequence AGCTCCACTTGGCTTCCGAAGCCCAAGCTGGGTTGTACCA GGTTCCCTAGGGTGCAGGCTGTGGGCAACTGCCAGGGAC ATGTGCCTGCCCACCGGCCTCTGGCCCTCACTGAGTTGGC CAATGGGAAATGACAATTGTGAGGTGGGGACTGCCTGCC CCCGTGAGTACCAGGCTGTTGAGGCTGGGCCATCTCCTCC TCACTTCCATTCTGACTGCAGTCTGTGGTTCTGATTCCAT ATCTAGACTGATCCTGAGAACTTCAGGGTGAGTCTATGG GACCCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAG TTCATGTCATAGGAAGGGGAGAAGTAACAGGGTACACAT ATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAA AAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATT TCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATG ATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATA ACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCT GCATATAAATATTTCTGCATATAAATTGTAACTGATGTAA GAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACC ATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTAT TCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATA CCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGG TCTGTGTGCTGGCCCATCACTTTGG 7 mouse adipoq regulatory element-derived promoter AAACCACCCAGCAAAAAACCAAACCGCCTAGCCTCAAGA CATGTGTGGTTGAATGTTTTTCACTTCTAGTCGCTAAGCA AGTGTGTGTTTTTACACAATGCCCTCTGTGGTGAGTGGCG GATTCCCCTGAGAGTTCACCAAATGATAGGCTTTCACAAT GCTCCCGGGTGTCTACCAGACCCAGCAAAGTATTGATGT GGTTTTGGGGTGAAAGTCACTCTGTCTTGTGCAATAGTTA GAATCTGCTGAAACCAGCAGTGTTCCTATATGGGACAGG GGTCCAGAGCTAACCCGGAGGCTATAACTGAGCAGAGGT GAAGACCACGAGGCATTGGGGAGCGTATGCCCTTTGTGG TCAGAGAGATCTAGCTTCGTGCCTTGGGTCTGTGTCTCTC CCTCTTACTGGCTTCTGGCTTCTTCATTAAGTGGGAGACA ACCACAGGTATCTGTATGGGAAGACTCGACTACCCCTTG ACTCAACATTGCTTGTTACTTACTTTGTACAAGATACTAC TTAGTCTAGGGGTTATGGAGCATAACCTCAAGTAGGTAA AGCCCCTGCTCCAGCGTGTTTGCATTCCAGTAAGAAGCG AAAGACAGTAACACACATACAAAATAAGTAAGAAAATG CAACAACAGCAACAACAACAACACACACACACAAAGTA AGCAAAACGCTAAGGGAAAGATAGAGAGTGATACAGCT TTGAGTTGCTGTAGTTCTTCTCTCTCCTTTGCTTCATACAG TTTGCTTGGGAAGTGTCCAGGGCCATGGGGTCACAACTA ACAGCCCTTGGAAATGAGCTTGTGTCCTTAATCTTCATGA CCTAACGTGATTTCTCTAGAAACATCAGTGCATTAACAGG AAGACAAGATGGAAGATCATATTTTGGCTCTCCTTCCTTG SEQ ID NO Description Sequence GTGGGTTGACACTGCTGGTCCTATCCACTAGTAAAAGCAT GACTCTTAGGCTCTGTGTGGCCAGTGGAAGGTGGCAGTT GGAGGAAGCAGATGCTTGGCCAGCCTTTGCCTGGGAGCA GTCTAGCTCTGAGTGTCTTATTGGAGCAGCTGCTGGCATC CAGAGTTCTTTTTGGATTCACGATTTAATTCAAAAGCTTT GTGCTCCCGAGAATCAGCTCTGGTCTTTCAAAAATAAGAT GTGAGTCCGCCGAGAGGCTCCCAAGGTATTGCCTTGCCA ACTGCAAGCCTTTTAGGAGCAGTTTAGTGAGTGGTGACT GCTAGTTGCAGTTGGCTGTTAGCCCAGAGCTAATAATAG ATAGAAAAGGTATATACTTAAGGAGTCTGGAAACTGAGG TTTATCTACTCACAGAAAATGAGTTTCTAAAAAACTAGCT TGAAACTTACCCAGAAAAATCTTAGAACATGGTTCTCCA ATGTCAAGGTAAGTGTTCTGTGACACTGGGCTTGAATTAT GTAGGGACCACAGATTTTAGAATTTGGACCCCTGAACTT GCTTCACACCCCACCAGGAACCTTCCTGTACAACAGCCCT CAGAATTCATCTACATGGTCTTTTCTCAGTATGGGATCCG GTCTAGCAAGTGGAGCACACCTTCTATTGCTTAAAGATTT GTTTATGTATATGGGTATTTTGGCTGCATGCATATTTGCA CACCAAAAGAAGGCAGCGGATCCCATGGAATTACTGTGG GTGCTGGGAATTGAACTCAGGACCTCTGGAAGAATAGCC AGTGCTCTTAACCACTGAGCCATGCCTGCAGTCCATCTAT TTTTTATTCTAGTACAGCCCCTCTTCATTCTTACTGAAATA GTAATGCCTGAACCACACAGCTTCACATTTAGTTACAAA GAAAGAGTGGGAGTATCATGTGACAATTAGTGTTGTTGA CTCTCCAGGACAAACTTATGGGAAAGGGAGGTCTCCTGA CCCCTGAACAATCATTTTACTTGAGGATAATTTTCATTGC ACTCAGAAACATGCTGAATTATTGTCCTTACCCTTGCCCC ATCTCTTGCTCTGGTAGAGAATGGCCAAAGCCTGGAAAC AGGATGGCTTGACAGAAGCTCTACTTGGCTTCCCAGACC CAAGCTGGATTAAACCAGGTTCCCTAAGGAGTCTTAAGG CAGCTGCCAGGAGCAAGGGGCCCACTCATTGGCTATTGG CCTTGACTGGGTTGGCCAATGGTAAGCTGGGGTCTGCCTG TCCCCATGAGTACCAGACTAATGAGACCTGGCCACTTTCT CCTCATTTCTGTCTGTACGATTGTCAGTGGATCTGACGAC ACCAAAAGGTAAGAAC 8 Stuffer 5 CCTAGGTAAGGAGGGAGGGGACAGGAGAGAGCCCTGTA GAAGCCTGCTTGGAGTGGGGTACTTTCAGAGGCCAGGTG TCTACTGCTAAGGTTGCATCTTTAGATGTGTTTAGACCCT TCCCATTACCATTAGGGAGTGGCTGTATGGTATAGACTTC AGAACTTATTCAACCTACCCTTGGAAGTCAGCTGCACTGT GAGTCCCATATTGAAGTCCTTAAACATAGGCATGGAGAT GTGACATTCTGTCTGTAATTTAGCCTTGGCATTGCAAGAC TGATGCTGATGTCTTAGAAGAGTCTCAACCTAACTTCCTG SEQ ID NO Description Sequence TATATGTGGCCTAAGTGCTGACTATCTCTCATGTCGGATA GACCCTATAGCCCTATCCATGGACCTATGTGCTAGAGTGT AAGGCAGTGGGGATTGTACCCTCATCTATCTCCATCACTC TGTGGGTGCAGAGTAACACTTAGCAGCACTGGGTGCCCA GAACCACTGCCACCCCACATGTTGAGACTAGCAACTACA GTACTTGGTAAAGAGGGAGAGCAATCTGAAGGAGGCAA ATCACTGGCTTG 9 Stuffer 4 GTCCTCAGGCTAACAGTAGAGAACCAGGTCTGGCTCAGG ACTACTGTTCTGGGCAAGCCCCACACTAATGGGCTTAGCT ACACACTTGTCCTGCATAAGGTTGGCACACCTTGTTACTT TTCAGAGAGGGTGAGCTAACATAGGCTAGAGGTACACAT GTTTGAGATACCCATGTGTCCAAAAATGACAACCTGACT CAACCAAACCACTGGTGCATTCAAACTGAGGCCAGGTGA cTTTTACTAACCAGATGAGTTAGAGCTTTGGTGGGGTCCA CACTCCTCATGTAGCCATTCAGCCAACTTGGCTGCTAAAG CCAATGGCAGCATTCAACTGCAGGGGCCCTTTGAGTCCA GGGTTGGGAGTGCAGTCTATGGCCCCCAACCCCTGTTAG ACAGACTGCCATCCAGGTACTAGCATGGGATAGTACACA AACAATGATGCCTTAGAGGGCAAAACCATTTGGGTGGCC CAAGAGGCCATTAAAACACTTTCAGATTCACTTTGTGACC AACAATGTCCACATCTGTATTAGCAAGTAGAGCTGTGCA AGCCAGCAGGGGTAGCCCACCATGAGCATACCACATGTC ATATCCCAGTACTGAGTGATATCCTGATCCCACTATTCAT GGAGTAGCTAGGGAGGAGAGGGAATCCCGTGCAACTGAT ACTACAGTGTACTAGGCACACTCCCTACACCCCCATCAG AAACTCAGTGTCTGTTGGTCCCCAGAAAGTTTGGTGTCAA GTCTTATTAGGGAATCTACTAGTACCACCAAATGGCTATG GTGTAGCACCTCTGCACACTATATGTAGGAGCTAGCCTA GGTAAGGAGGGAGGGGACAGGAGAGAGCCCTGTAGAAG CCTGCTTGGAGTGGGGTACTTTCAGAGGCCAGGTGTCTAC TGCTAAGGTTGCATCTTTAGATGTGTTTAGACCCTTCCCA TTACCATTAGGGAGTGGCTGTATGGTATAGACTTCAGAA CTTATTCAACCTACCCTTGGAAGTCAGCTGCACTGTGAGT CCCATATTGAAGTCCTTAAACATAGGCATGGAGATGTGA CATTCTGTCTGTAATTTAGCCTTGGCATTGCAAGACTGAT GCTGATGTCTTAGAAGAGTCTCAACCTAACTTCCTGTATA TGTGGCCTAAGTGCTGACTATCTCTCATGTCGGATAGACC CTATAGCCCTATCCATGGACCTATGTGCTAGAGTGTAAGG CAGTGGGGATTGTACCCTCATCTATCTCCATCACTCTGTG GGTGCAGAGTAACACTTAGCAGCACTGGGTGCCCAGAAC CACTGCCACCCCACATGTTGAGACTAGCAACTACAGTAC TTGGTAAAGAGGGAGAGCAATCTGAAGGAGGCAAATCA CTGGCTTG SEQ ID NO Description Sequence 10 Stuffer 3 TTTTACTAACCAGATGAGTTAGAGCTTTGGTGGGGTCCAC ACTCCTCATGTAGCCATTCAGCCAACTTGGCTGCTAAAGC CAATGGCAGCATTCAACTGCAGGGGCCCTTTGAGTCCAG GGTTGGGAGTGCAGTCTATGGCCCCCAACCCCTGTTAGA CAGACTGCCATCCAGGTACTAGCATGGGATAGTACACAA ACAATGATGCCTTAGAGGGCAAAACCATTTGGGTGGCCC AAGAGGCCATTAAAACACTTTCAGATTCACTTTGTGACCA ACAATGTCCACATCTGTATTAGCAAGTAGAGCTGTGCAA GCCAGCAGGGGTAGCCCACCATGAGCATACCACATGTCA TATCCCAGTACTTGGTAAAGAGGGAGAGCAATCTGAAGG AGGCAAATCACTGGCTTG 11 Stuffer 2 TTTTACTAACCAGATGAGTTAGAGCTTTGGTGGGGTCCAC ACTCCTCATGTAGCCATTCAGCCAACTTGGCTGCTAAAGC CAATGGCAGCATTCAACTGCAGGGGCCCTTTGAGTCCAG GGTTGGGAGTGCAGTCTATGGCCCCCAACCCCTGTTAGA CAGACTGCCATCCAGGTACTAGCATGGGATAGTACACAA ACAATGATGCCTTAGAGGGCAAAACCATTTGGGTGGCCC AAGAGGCCATTAAAACACTTTCAGATTCACTTTGTGACCA ACAATGTCCACATCTGTATTAGCAAGTAGAGCTGTGCAA GCCAGCAGGGGTAGCCCACCATGAGCATACCACATGTCA TATCCCAGTACTGAGTGATATCCTGATCCCACTATTCATG GAGTAGCTAGGGAGGAGAGGGAATCCCATGCAACTGATA CTACAGTGTACTAGGCACACTCCCTACACCCCCATCAGA AACTCAGTGTCTGTTGGTCCCCAGAAAGTTTGGTGTCAAG TCTTATTAGGGAATCTACTAGTACCACCAAATGGCTATGG TGTAGCACCTCTGCACACTATATGTAGGAGCTAGCCTAG GTAAGGAGGGAGGGGACAGGAGAGAGCCCTGTAGAAGC CTGCTTGGAGTGGGGTACTTTCAGAGGCCAGGTGTCTACT GCTAAGGTTGCATCTTTAGATGTGTTTAGACCCTTCCCAT TACCATTAGGGAGTGGCTGTATGGTATAGACTTCAGAAC TTATTCAACCTACCCTTGGAAGTCAGCTGCACTGTGAGTC CCATATTGAAGTCCTTAAACATAGGCATGGAGATGTGAC ATTCTGTCTGTAATTTAGCCTTGGCATTGCAAGACTGATG CTGATGTCTTAGAAGAGTCTCAACCTAACTTCCTGTATAT GTGGCCTAAGTGCTGACTATCTCTCATGTAGGATAGACCC TATAGCCCTATCCATGGACCTATGTGCTAGAGTGTAAGGC AGTGGGGATTGTACCCTCATCTATCTCCATCACTCTGTGG GTGCAGAGTAACACTTAGCAGCACTGGGTGCCCAGAACC ACTGCCACCCCACATGTTGAGACTAGCAACTACAGTACTT GGTAAAGAGGGAGAGCAATCTGAAGGAGGCAAATCACT GGCTTGATACATGTGGGGTGAGCACTAGGGGCTTGCTCC CCCTACTGGTGTTGGATTGATGCCCCAGGTCTTTTGGTCT TGTCCTGGTTACAGGTCCTCAGGCTAACAGTAGAGAACC SEQ ID NO Description Sequence AGGTCTGGCTCAGGACTACTGTTCTGGGCAAGCCCCACA CTAATGGGCTTAGCTACACACTTGTCCTGCATAAGGTTGG CACACCTTGTTACTTTTCAGAGAGGGTGAGCTAACATAG GCTAGAGGTACACATGTTTGAGATACCCATGTGTCCAAA AATGACAACCTGACTCAACCAAACCACTGGTGCATTCAA ACTGAGGCCAGGTGATGCTTGGGAGCAGAGTAATTTTAT GTCCCCTGCCCTGCACACTCCTCTAGCAGTGATCCTGATG AAGAATCTAACTCCTTGTTGGAATGTTGAGCCACTCCCTA CCTCAGGTTTAGAAGTATGGGGATTGTGTGCCCTTGGGG GAAGTGGGTGACTTAGTGGAAACACACAGGCAGTGTGGA ATTGCTGATCCTCCATTGAGAGGGAGACTAAGAGACCAG ATATCCCACCCCAGCATTGTACCCCTCTGAGCCAGGGCCA GACAACATGGTTCTGATGGCATGGGATTATGATTGCCACT TACAGTCCCCAGGCCATATTGGGGTATTCCCCCAACTCCT GAAGTGCCACAGAGGGGCTATTCCAGCTATTATATAGCA CCCAACTAGGCCTATGAGGTGGCCACTGCCTGGGAGGAG GTATGGTTTGTAGTACCCCCATCTTACTCTGGCAGACCTT TTCTATCAGTCCACCCTCTGGGGAAGTGGAAGGATGCAC TAGTAGAGTCCCCACAACCCTCAAGAAGA 12 Stuffer 1 TTTTACTAACCAGATGAGTTAGAGCTTTGGTGGGGTCCAC ACTCCTCATGTAGCCATTCAGCCAACTTGGCTGCTAAAGC CAATGGCAGCATTCAACTGCAGGGGCCCTTTGAGTCCAG GGTTGGGAGTGCAGTCTATGGCCCCCAACCCCTGTTAGA CAGACTGCCATCCAGGTACTAGCATGGGATAGTACACAA ACAATGATGCCTTAGAGGGCAAAACCATTTGGGTGGCCC AAGAGGCCATTAAAACACTTTCAGATTCACTTTGTGACCA ACAATGTCCACATCTGTATTAGCAAGTAGAGCTGTGCAA GCCAGCAGGGGTAGCCCACCATGAGCATACCACATGTCA TATCCCAGTACTGAGTGATATCCTGATCCCACTATTCATG GAGTAGCTAGGGAGGAGAGGGAATCCCGTGCAACTGATA CTACAGTGTACTAGGCACACTCCCTACACCCCCATCAGA AACTCAGTGTCTGTTGGTCCCCAGAAAGTTTGGTGTCAAG TCTTATTAGGGAATCTACTAGTACCACCAAATGGCTATGG TGTAGCACCTCTGCACACTATATGTAGGAGCTAGCCTAG GTAAGGAGGGAGGGGACAGGAGAGAGCCCTGTAGAAGC CTGCTTGGAGTGGGGTACTTTCAGAGGCCAGGTGTCTACT GCTAAGGTTGCATCTTTAGATGTGTTTAGACCCTTCCCAT TACCATTAGGGAGTGGCTGTATGGTATAGACTTCAGAAC TTATTCAACCTACCCTTGGAAGTCAGCTGCACTGTGAGTC CCATATTGAAGTCCTTAAACATAGGCATGGAGATGTGAC ATTCTGTCTGTAATTTAGCCTTGGCATTGCAAGACTGATG CTGATGTCTTAGAAGAGTCTCAACCTAACTTCCTGTATAT GTGGCCTAAGTGCTGACTATCTCTCATGTCGGATAGACCC SEQ ID NO Description Sequence TATAGCCCTATCCATGGACCTATGTGCTAGAGTGTAAGGC AGTGGGGATTGTACCCTCATCTATCTCCATCACTCTGTGG GTGCAGAGTAACACTTAGCAGCACTGGGTGCCCAGAACC ACTGCCACCCCACATGTTGAGACTAGCAACTACAGTACTT GGTAAAGAGGGAGAGCAATCTGAAGGAGGCAAATCACT GGCTTG 31 Mirl ATACATACTTCTTTACATTCCA 32 Mir 122 CAAACACCATTGTCACACTCCA 33 Mir 142 TCCATAAAGTAGGAAACACTACA 34 Mir652 TGAATGGCACCCTCTCCTAGGGTTG 35 Beta globin intron GTGAGTCTATGGGACCCTTGATGTTTTCTTTCCCCTTCTTT TCTATGGTTAAGTTCATGTCATAGGAAGGGGAGAAGTAA CAGGGTACACATATTGACCAAATCAGGGTAATTTTGCATT TGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTT TGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTC AGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCA TTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAA TAGCAATATTTCTGCATATAAATATTTCTGCATATAAATT GTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTA CAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGAT AAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCT AATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTG GGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGG

[0262] In some embodiments, a regulatory element comprises one or more promoters or a fragment or variant thereof; and one or more enhancers or a fragment or variant thereof. In some embodiments, a regulatory element is or is derived from a gene expressed in adipocytes.

[0263] In some embodiments, a regulatory element is or comprises mFabp4. In some embodiments, is also known as mini / aP2. In some embodiments, a regulatory element comprises the sequence of SEQ ID NO: 5, or a sequence having at least 90% identity thereto.

[0264] In some embodiments, a regulatory element is or comprises mAdipoq. In some embodiments, a regulatory element comprises the sequence of SEQ ID NO: 7, or a sequence having at least 90% identity thereto.

[0265] In some embodiments, a regulatory element is or comprises hADIPOQ. In some embodiments, a regulatory element comprises the sequence of SEQ ID NO: 6, or a sequence having at least 90% identity thereto.

[0266] In some embodiments, a regulatory element is or comprises hADIPOQ. In some embodiments, a regulatory element comprises the sequence of SEQ ID NO: 36, or a sequence having at least 90% identity thereto.

[0267] In some embodiments, a regulatory element is or comprises a ubiquitous promoter and / or a ubiquitous enhancer. In some embodiments, a regulatory element comprises the sequence of SEQ ID NO: 3, or a sequence having at least 90% identity thereto. In some embodiments, a regulatory element comprises the sequence of SEQ ID NO: 4, or a sequence having at least 90% identity thereto.

[0268] In some embodiments, a promoter disclosed herein comprises one or more additional regulatory sequences. In some embodiments, a regulatory sequence is or comprises an intron from a beta globin gene, or a variant or fragment thereof. In some embodiments, a regulatory sequence is or comprises a beta globin intron as provided in SEQ ID NO: 35, or a sequence having at least 85% identity thereto.

[0269] In some embodiments, a regulatory element further comprises one or more RNAi elements. In some embodiments, an RNAi is or comprises a micro RNA (miR).

[0270] In some embodiments, a regulatory element comprises one or more: miR122 (miRBase database accession number MI0000442), miRl 52 (MI0000462), miRl 99 (MI0000242), miR215 (MI0000291), miR92 (MI0000234), miR148a (MI0000253), miR194 (MI0000488), miRl (MI0000651), miRT133 (MI0000450), miR206 (MI0000490), miR208 (MI0000251), miR124 (MI0000443), miR125 (MI0000469), miR216 (MI0000292), miR130 (MI0000448), miR142 (MI0000458), miR652 (MI0003667). mIR sequences can be obtained from the miRbase database.

[0271] In some embodiments, a regulatory element comprises a sequence provided in Table 2 or a sequence having at least 90% identity thereto. In some embodiments, a regulatory element comprises a sequence of SEQ ID NO: 1 or a sequence having at least 90% identity thereto. In some embodiments, a regulatory element comprises a sequence of SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

[0272] In some embodiments, a regulatory element comprises a stuffer sequence. In some embodiments, a stuffer sequence comprises a sequence of SEQ ID NO: 8 or a sequence having at least 90% identity thereto. In some embodiments, a stuffer sequence comprises a sequence of SEQ ID NO: 9 or a sequence having at least 90% identity thereto. In some embodiments, a stuffer sequence comprises a sequence of SEQ ID NO: 10 or a sequence having at least 90% identity thereto. In some embodiments, a stuffer sequence comprises a sequence of SEQ ID NO: 11 or a sequence having at least 90% identity thereto. In some embodiments, a stuffer sequence comprises a sequence of SEQ ID NO: 12 or a sequence having at least 90% identity thereto.

[0273] In some embodiments, a regulatory element reduces (e.g., prevents) expression of a payload in non-adipocytes. In some embodiments, a regulatory element reduces (e.g., prevents) expression of a payload in adipocytes which are not present at a site of administration when a AAV particle is administered.

[0274] In some embodiments, a polynucleotide construct disclosed herein comprises a beta-globin intron or a variant or a fragment thereof and one or more additional regulatory elements, such as a promoter or a variant or fragment thereof, e.g., as provided in Table 2.

[0275] In some embodiments, a polynucleotide construct disclosed herein comprises a beta-globin intron provided in SEQ ID NO: 35 or a sequence having at least 90% identity thereto, or a fragment thereof, and one or more additional regulatory elements provided in Table 2, or variants or fragments thereof.

[0276] In some embodiments, a polynucleotide construct disclosed herein comprises the sequence of SEQ ID NO: 37, or a sequence having at least 90% identity thereto. Removal of Modified Adipose Tissue and / or Adipocytes

[0277] Genetic therapies hold promise to treat many diseases, but they still have risks. Potential risks could include certain types of cancer, allergic reactions, or damage to organs or tissues if an injection is involved. In addition, immune system-mediated toxicity present challenges to successful gene transfer by AAV vectors especially when high doses are required to correct the targeted genetic disease (see, e.g., Ertl HCJ. Immunogenicity and toxicity of AAV gene therapy. Front Immunol. 2022 Aug 12;13:975803, which is incorporated herein by reference in its entirety).

[0278] In accordance with various embodiments, the present disclosure provides one or more advantages for modulating an effect of a gene therapy. In some embodiments, methods provided herein reduce a presence of modified adipose tissue and / or adipocytes (e.g., transduced adipose tissue and / or adipocytes). Without wishing to be bound by any theory, modified cells (e.g., transduced cells) can be preferentially targeted (e.g., removed) as modified cells (e.g., transduced cells) are limited to and around a site of administration (e.g., an injection site) of a composition (e.g., AAV particle).

[0279] In some embodiments, methods provided herein further comprise removal of modified adipose tissue and / or adipocytes (e.g., transduced adipose tissue and / or adipocytes) from a subject. Without wishing to be bound by any particular theory, removal of modified adipose tissue and / or adipocytes (e.g., transduced adipose tissue and / or adipocytes) after administration of a therapy reduces efficacy of an initial therapy.

[0280] In some embodiments, removal of all or a portion of modified adipose tissue and / or adipocytes (e.g., transduced adipose tissue and / or adipocytes), reduces expression of a pay load encoded by a polynucleotide construct and / or reduces activity of a payload encoded by a polynucleotide construct (e.g., reduces distribution and / or activity as compared to an otherwise similar subject in whom no removal has been performed, or as compared to the same subject prior to removal). In some embodiments, expression (e.g., locally) and / or activity (e.g., locally and / or systemically) of a payload of a payload is reduced by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%. In some embodiments, expression and / or activity of a payload is reduced by about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, expression (e.g., locally) and / or activity (e.g., locally and / or systemically) of a payload of a payload is reduced to an undetectable level.

[0281] In some embodiments, a reduction in expression (e.g., locally) and / or activity (e.g., locally and / or systemically) of a payload of a payload is observed within about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours or about 96 hours of removing transduced adipose tissue. In some embodiments, a reduction in expression (e.g., locally) and / or activity (e.g., locally and / or systemically) of a payload of a payload is observed within about one half-life of a payload.

[0282] In some embodiments, localized expression of a payload is reduced at a time after delivery of an initial therapy by removing all or a portion of modified adipose tissue and / or adipocytes (e.g., transduced adipose tissue and / or adipocytes). In some embodiments, removal comprises removing at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of modified adipose tissue and / or adipocytes (e.g., transduced adipose tissue and / or adipocytes). In some embodiments, removal comprises removing all or substantially all of the modified adipose tissue and / or adipocytes (e.g., transduced adipose tissue and / or adipocytes) reduces an expression product of interest (e.g., encoded by pay load) to an undetectable level.

[0283] In some embodiments, removal of modified adipose tissue and / or adipocytes (e.g., transduced adipose tissue and / or adipocytes) is achieved by physical excision. In some embodiments, physical removal of modified adipose tissue and / or adipocytes (e.g., transduced adipose tissue and / or adipocytes) is achieved by any one or combination of the following: excision biopsy, liposuction, cryolypolysis.

[0284] In some embodiments, removal of modified adipose tissue and / or adipocytes (e.g., transduced adipose tissue and / or adipocytes) is achieved by chemical ablation. In some embodiments, chemical ablation removal of modified adipose tissue and / or adipocytes (e.g., transduced adipose tissue and / or adipocytes) is achieved by a combination including deoxy cholate injection and / or radioablation.

[0285] In some embodiments, compositions described herein are administered to reduce scarring of removed modified adipose tissue and / or adipocytes (e.g., transduced adipose tissue and / or adipocytes). In some embodiments, compositions described herein are administered in a patterned manner to reduced scarring of removed modified adipose tissue and / or adipocytes (e.g., transduced adipose tissue and / or adipocytes). For example, administering one more doses of a composition described herein in a linear line.

[0286] In some embodiments, removal of modified adipose tissue and / or adipocytes (e.g., transduced adipose tissue and / or adipocytes) is achieved by an agent. In some embodiments, an agent is exogenously provided. In some embodiments, an agent silences an expression product by inactivating one or more elements in a vector. In some embodiments, an agent silences an expression product by inactivating one or more elements in a vector that mediates transcription of the expression product. In some embodiments, an agent silences an expression product by inactivating one or more elements in a vector that inhibits the expression product.

[0287] In some embodiments, an agent silences an expression product by activating one or more elements in a vector. In some embodiments, an agent silences an expression product by activating one or more elements in a vector that inhibits transcription of the expression product. In some embodiments, an agent silences an expression product by activating one or more elements in a vector that inhibits the expression product.

[0288] In accordance with various embodiments, provided methods include controlling and / or reversing an AAV gene therapy. In some embodiments, methods include (1) delivering a first dose of a gene therapy comprising an adeno-associated (AAV) particle to adipose tissue, wherein the AAV particle comprises an AAV capsid and a polynucleotide construct encoding a pay load; (2) reducing or eliminating expression of the pay load in the adipose tissue by removing at least a portion of the adipose tissue transduced by the AAV particle; and / or (3) reducing or eliminating distribution of the payload systemically by removing at least a portion of adipose tissue transduced by the AAV particle. In some embodiments, methods include (1) delivering a first dose of a gene therapy comprising an adeno-associated (AAV) particle to adipocytes, wherein the AAV particle comprises an AAV capsid and a polynucleotide construct encoding a payload; (2) reducing or eliminating expression of the payload in the adipose tissue; and / or (3) reducing or eliminating distribution of the payload systemically by removing at least a portion of adipocytes transduced by the AAV particle.

[0289] In some embodiments, one or more therapies described herein is adjusted by modulating an amount of modified adipose tissue and / or adipocytes (e.g., transduced adipose tissue and / or adipocytes). In some embodiments, one or more therapies described herein is adjusted by modulating an amount of modified adipose tissue and / or adipocytes (e.g., transduced adipose tissue and / or adipocytes) that is removed. ENUMERATED EMBODIMENTS

[0290] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0291] 1. A method of delivering an adeno-associated (AAV) particle to adipose tissue in a subject, wherein the AAV particle comprises an AAV capsid protein and a polynucleotide construct, wherein the subject is a non-rodent mammal.

[0292] 2. The method of embodiment 1, wherein delivery comprises administration of the AAV particle subcutaneously.

[0293] 3. The method of embodiment 2, wherein the AAV particle is administered once.

[0294] 4. The method of embodiment 2, wherein the AAV particle is administered repeatedly, e.g., two times, three times, four times, five times, six times, seven times, eight times, nine times, ten times or twenty times.

[0295] 5. The method of any one of embodiments 2-4, wherein the AAV particle is administered at a pre-determined tissue depth in a subject.

[0296] 6. The method of embodiment 5, wherein the AAV particle is administered between the dermis and Scapa’s fascia.

[0297] 7. The method of embodiment 5 or 6, wherein the AAV particle is administered to superficial subcutaneous adipose tissue.

[0298] 8. The method of embodiment 5, wherein the AAV particle is administered between Scapa's fascia and Camper's fascia.

[0299] 9. The method of embodiment 5 or 6, wherein the AAV particle is administered to deep subcutaneous adipose tissue.

[0300] 10. The method of any one of embodiments 2-5, wherein the AAV particle is administered to visceral adipose tissue.

[0301] 11. The method of any one of embodiments 2-4, wherein the AAV particle is administered at a pre-determined depth from the exterior surface of the skin.

[0302] 12. The method of embodiment 11, wherein the depth is between at least about 2mm to about 6mm.

[0303] 13. The method of embodiment 11, wherein the depth is between at least about 3mm to about 5mm.

[0304] 14. The method of embodiment 12, wherein the depth is at least about 2mm.

[0305] 15. The method of embodiment 12, wherein the depth is at least about 3mm.

[0306] 16. The method of embodiment 12, wherein the depth is at least about 4mm.

[0307] 17. The method of embodiment 12, wherein the depth is at least about 5mm.

[0308] 18. The method of embodiment 12, wherein the depth is at most about 6mm.

[0309] 19. The method of any one of embodiments 1-18, wherein the adipose tissue is or comprises brown adipose tissue, beige adipose tissue, white adipose tissue, pink adipose tissue, or any combination thereof.

[0310] 20. The method of embodiment 19, wherein the adipose tissue is or comprises brown adipose tissue.

[0311] 21. The method of embodiment 20, wherein the brown adipose tissue is located near or at the following locations in the subject: neck, kidney, adrenal glands, heart (e.g., aorta), mediastinum, shoulder, neck, and / or back.

[0312] 22. The method of embodiment 20 or 21, wherein brown adipose tissue is characterized as metabolizing fat to produce heat and / or has a role in energy metabolism.

[0313] 23. The method of embodiment 19, wherein the adipose tissue is or comprises beige adipose tissue.

[0314] 24. The method of embodiment 23, wherein the beige adipose tissue is located near or at the following locations in the subject: abdomen, face, glute, and / or femur.

[0315] 25. The method of embodiment 19, wherein adipose tissue is or comprises white adipose tissue.

[0316] 26. The method of embodiment 25, wherein the white adipose tissue is located near or at the following locations in the subject: bone marrow, abdomen, and / or organs.

[0317] 27. The method of embodiment 25 or 26, wherein white adipose tissue is characterized by its location.

[0318] 28. The method of embodiment 27, wherein white adipose tissue is present in a subject near or next to an organ in the form of a visceral depot.

[0319] 29. The method of any one of embodiments 25-28, wherein the white adipose tissue comprises subcutaneous white adipose and / or visceral white adipose tissue.

[0320] 30. The method of any one of embodiments 25-29, wherein the white adipose tissue is characterized as storing fat.

[0321] 31. The method of embodiment 19, wherein adipose tissue is or comprises pink adipose tissue.

[0322] 32. The method of embodiment 31, wherein the pink adipose tissue is located near or at the following locations in the subject: abdomen, breast, bone marrow, and / or dermis.

[0323] 33. The method of any one of embodiments 1-32, wherein the adipose tissue comprises adipocytes.

[0324] 34. The method of any one of embodiments 1-33, wherein the AAV particle does not substantially target (e.g., does not target) non-adipose tissue, non-adipocytes, and / or adipose tissue (e.g., adipose tissue at a different location) which was not directly administered the AAV particle.

[0325] 35. The method of embodiment 34, wherein the AAV particle does not substantially target (e.g., does not target) non-adipose tissue.

[0326] 36. The method of embodiment 34, wherein the AAV particle does not substantially target (e.g., does not target) non-adipocytes.

[0327] 37. The method of embodiment 34, wherein the AAV particle does not substantially target (e.g., does not target) adipose tissue (e.g., adipose tissue at a different location) which was not directly administered the AAV particle.

[0328] 38. The method of any one of embodiments 1-33, wherein the AAV particle does not substantially transduce (e.g., does not transduce) non-adipose tissue, non-adipocytes, and / or adipose tissue (e.g., adipose tissue at a different location) which was not directly administered the AAV particle.

[0329] 39. The method of embodiment 38, wherein the AAV particle does not substantially transduce (e.g., does not transduce) non-adipose tissue.

[0330] 40. The method of embodiment 38, wherein the AAV particle does not substantially transduce (e.g., does not transduce) non-adipocytes.

[0331] 41. The method of embodiment 38, wherein the AAV particle does not substantially transduce (e.g., does not transduce) adipose tissue (e.g., adipose tissue at a different location) which was not directly administered the AAV particle.

[0332] 42. The method of any one of embodiments 1-33, wherein the AAV particle is substantially not detected (e.g., not detectable) in non-adipose tissue (e.g., liver, skin, muscle), non-adipocytes, and / or adipose tissue (e.g., adipose tissue at a different location) which was not directly administered the AAV particle.

[0333] 43. The method of embodiment 42, wherein the AAV particle is substantially not detected (e.g., not detectable) in non-adipose tissue (e.g., liver, skin, muscle)

[0334] 44. The method of embodiment 42, wherein the AAV particle is substantially not detected (e.g., not detectable) in adipose tissue (e.g., adipose tissue at a different location) which was not directly administered the AAV particle.

[0335] 45. The method of any one of embodiments 1-33, wherein the AAV particle is substantially not detectable (e.g., not detectable) in a non-adipose tissue sample from the subject or adipose tissue sample (e.g., adipose tissue sample at a different location) which was not directly administered the AAV particle.

[0336] 46. The method of any one of embodiments 1-33, wherein expression of a pay load encoded by the polynucleotide construct is substantially not detectable (e.g., not detectable) in non-adipose tissue (e.g., liver, skin, muscle) non-adipocytes, and / or adipose tissue (e.g., adipose tissue at a different location) which was not directly administered the AAV particle.

[0337] 47. The method of embodiment 46, wherein expression of a payload encoded by the polynucleotide construct is substantially not detected (e.g., not detectable) in non-adipose tissue (e.g., liver, skin, muscle).

[0338] 48. The method of embodiment 46, wherein expression of a payload encoded by the polynucleotide construct is substantially not detected (e.g., not detectable) in adipose tissue (e.g., adipose tissue at a different location) which was not directly administered the AAV particle.

[0339] 49. The method of any one embodiments 1 -48, wherein administration of the AAV particle to adipose tissue results in: (1) local expression and / or distribution of a payload encoded by the polynucleotide construct; (2)local expression and / or systemic distribution of a payload encoded by the polynucleotide construct, or (3) both (1) and (2).

[0340] 50. The method of any one of the preceding embodiments, wherein a pay load encoded by the polynucleotide construct is expressed and / or distributed at a therapeutically effective level, e.g., at a level that treats, prevents and / or reduces the severity and / or frequency of one or more symptoms of a disease or disorder.

[0341] 51. The method of embodiment 49 or 50, wherein systemic distribution of the payload is detected at least about 24 hours after administration.

[0342] 52. The method of any one of embodiments 49-51, wherein systemic distribution of the pay load persists for at least about 1 week after administration.

[0343] 53. The method of any one of embodiments 49-52, wherein systemic distribution of the pay load of the pay load is dose dependent.

[0344] 54. The method of any one of embodiments 49-53, wherein the payload is a secreted polypeptide.

[0345] 55. The method of any one of embodiments 49-54, wherein the pay load acts systemically.

[0346] 56. The method of any one of embodiments 49-53, wherein the payload acts locally, e.g., at or near the site of administration.

[0347] 57. The method of any one of embodiments 49-56, wherein detection of expression of the pay load is performed in a biological sample from a subject.

[0348] 58. The method of embodiment 57, wherein the biological sample is or comprises cells, tissue, and / or bodily fluid.

[0349] 59. The method of embodiment 58, wherein the biological sample is or comprises cells.

[0350] 60. The method of embodiment 58, wherein the biological sample is or comprises tissue.

[0351] 61. The method of embodiment 58, wherein the biological sample is or comprises bodily fluid.

[0352] 62. The method of any one of embodiments 1-61, wherein the AAV particle is administered at a plurality of sites in the subject.

[0353] 63. The method of any one of embodiments 1-62, wherein the AAV particle is administered at a plurality of sites in a first section of adipose tissue.

[0354] 64. The method of embodiment 63, wherein the first section of adipose tissue comprises adipose tissue in a pre-selected location in a subject’s body.

[0355] 65. The method of embodiment 63 or 64, wherein the AAV particle is administered at between 1 and 100 different sites in the first section of adipose tissue.

[0356] 66. The method of embodiment 65, wherein the AAV particle is administered at between 1 and 100 different sites in the first section of adipose tissue.

[0357] 67. The method of embodiment 66, wherein the AAV particle is administered at between 1 and 50 different sites in the first section of adipose tissue.

[0358] 68. The method of embodiment 66, wherein the AAV particle is administered at between 1 and 20 different sites in the first section of adipose tissue.

[0359] 69. The method of embodiment 66, wherein the AAV particle is administered at between 1 and 10 different sites in the first section of adipose tissue.

[0360] 70. The method of embodiment 66, wherein the AAV particle is administered at between 1 and 6 different sites in the first section of adipose tissue.

[0361] 71. The method of embodiment 66, wherein the AAV particle is administered at between 1 and 5 different sites in the first section of adipose tissue.

[0362] 72. The method of embodiment 66, wherein the AAV particle is administered at between 1 and 4 different sites in the first section of adipose tissue.

[0363] 73. The method of embodiment 66, wherein the AAV particle is administered at between 1 and 3 different sites in the first section of adipose tissue.

[0364] 74. The method of embodiment 66, wherein the AAV particle is administered at between 1 and 2 different sites in the first section of adipose tissue.

[0365] 75. The method of any one of embodiments 62-74, wherein the plurality of sites are non-overlapping.

[0366] 76. The method of any one of embodiments 62-75, wherein the plurality of sites are at a pre-specified distance from one another.

[0367] 77. The method of embodiment 76, wherein the pre-specified distance is about 0.1 cm, about 0.2 cm, about 0.3 cm, about 0.4 cm, about 0.5 cm, about 0.6 cm, about 0.7 cm, about 0.8 cm, about 0.9 cm, or about 1.0 cm.

[0368] 78. The method of embodiment 77, wherein the pre-specified distance is about 0.1 cm.

[0369] 79. The method of embodiment 77, wherein the pre-specified distance is about 0.2 cm.

[0370] 80. The method of embodiment 77, wherein the pre-specified distance is about 0.3 cm.

[0371] 81. The method of embodiment 77, wherein the pre-specified distance is about 0.4 cm.

[0372] 82. The method of embodiment 77, wherein the pre-specified distance is about 0.5 cm.

[0373] 83. The method of embodiment 77, wherein the pre-specified distance is about 0.6 cm.

[0374] 84. The method of embodiment 77, wherein the pre-specified distance is about 0.7 cm.

[0375] 85. The method of embodiment 77, wherein the pre-specified distance is about 0.8 cm.

[0376] 86. The method of embodiment 77, wherein the pre-specified distance is about 0.9 cm.

[0377] 87. The method of embodiment 77, wherein the pre-specified distance is about 1.0 cm.

[0378] 88. The method of any one of embodiments 1-87, wherein the AAV particle is administered at another (e.g., a second) section of adipose tissue in the subject’s body.

[0379] 89. The method of embodiment 88, wherein the another (e.g., a second) section of adipose tissue is at a different location from a first section of adipose tissue.

[0380] 90. The method of embodiment 88 or 89, wherein the AAV particle is administered at a plurality of sites in the another (e.g., a second) section of adipose tissue.

[0381] 91. The method of any one of embodiments 63-90, wherein a first or subsequent (e.g., a second) section of adipose tissue is selected based on one or more characteristics of adipocytes in the first or subsequent section.

[0382] 92. The method of any one of embodiments 1-91, wherein the AAV particle is administered at a volume of at least about lOuL.

[0383] 93. The method of any one of embodiments!-92, wherein the method further comprises administering one or more doses of the AAV particle.

[0384] 94. The method of any one of embodiments 1-93, wherein the AAV particle is administered at a dose about 1 x 10(11) vector genomes (vg) to about 3 xl0(14) vg.

[0385] 95. The method of embodiment 94, wherein the AAV particle is administered at a dose of about 1 x 10(11) vg.

[0386] 96. The method of embodiment 94, wherein the AAV particle is administered at a dose of about 1 x 10(12) vg.

[0387] 97. The method of embodiment 94, wherein the AAV particle is administered at a dose of about 1 x 10(13) vg.

[0388] 98. The method of embodiment 94, wherein the AAV particle is administered at a dose of about 1 x 10(14) vg.

[0389] 99. The method of embodiment 94, wherein the AAV particle is administered at a dose of 3 x 10(14) vg.

[0390] 100. The method any one of embodiments 1 -99, wherein the AAV particle is administered at a concentration of about 1 x 10(12) vg / mL to about 1 x 10(14) vg / ml.

[0391] 101. The method of embodiment 100, wherein the AAV particle is administered at a concentration of about 1 x 10(12) vg / mL.

[0392] 102. The method of embodiment 100, wherein the AAV particle is administered at a concentration of about 1 x 10(13) vg / mL.

[0393] 103. The method of embodiment 100, wherein the AAV particle is administered at a concentration of about 1 x 10(14) vg / mL.

[0394] 104. The method of any one of embodiments 1-103, wherein the polynucleotide construct comprises one or more coding sequences.

[0395] 105. The method of embodiment 104, wherein the one or more coding sequences encodes a payload.

[0396] 106. The method of embodiment 105, wherein the payload is or comprises a polypeptide.

[0397] 107. The method of embodiment 105 or 106, wherein the payload is effective in treating a disease and / or disorder.

[0398] 108. The method of any one of embodiments 104-107, wherein the pay load is or comprises a therapeutic payload.

[0399] 109. The method of embodiment 108, wherein the therapeutic payload comprises: GLA, LPL, FVIII, FIX, GLP-1, GIP, ADIPOQ, FGF19, FGF21, PPY, BMP7, LEP, Adalimumab, Etanercept, Pembrolizumab, Ustekinumab, Dupilumab, Nivolumab, Daratumumab, Risankizumab, Secukinumab, Pertuzumab, Emicizumab, Denosumab, Abatocept, Atezolizumab, Durvalumab, Guselkumab, Tocilizumab, Ixekizumab, Infliximab, Eculizumab, Ravulizumab, Insulin, or fragments or variants or any of the foregoing, or any combination thereof.

[0400] 1 10. The method of embodiment 109, wherein the therapeutic pay load comprises GLA or a variant or fragment thereof.

[0401] 111. The method of embodiment 109, wherein the therapeutic pay load comprises LPL or a variant or fragment thereof.

[0402] 1 12. The method of embodiment 109, wherein the therapeutic pay load comprises FVIII or a variant or fragment thereof.

[0403] 1 13. The method of embodiment 109, wherein the therapeutic pay load comprises FIX or a variant or fragment thereof.

[0404] 1 14. The method of embodiment 109, wherein the therapeutic pay load comprises GLP-1 or a variant or fragment thereof.

[0405] 1 15. The method of embodiment 109, wherein the therapeutic payload comprises GIP or a variant or fragment thereof.

[0406] 1 16. The method of embodiment 109, wherein the therapeutic pay load comprises ADIPOQ or a variant or fragment thereof.

[0407] 1 17. The method of embodiment 109, wherein the therapeutic pay load comprises FGF19 or a variant or fragment thereof.

[0408] 1 18. The method of embodiment 109, wherein the therapeutic payload comprises FGF21 or a variant or fragment thereof.

[0409] 1 19. The method of embodiment 109, wherein the therapeutic pay load comprises PPY or a variant or fragment thereof.

[0410] 120. The method of embodiment 109, wherein the therapeutic pay load comprises BMP7 or a variant or fragment thereof.

[0411] 121. The method of embodiment 109, wherein the therapeutic payload comprises LEP or a variant or fragment thereof.

[0412] 122. The method of embodiment 109, wherein the therapeutic pay load comprises Adalimumab or a variant or fragment thereof.

[0413] 123. The method of embodiment 109, wherein the therapeutic pay load comprises Etanercept or a variant or fragment thereof.

[0414] 124. The method of embodiment 109, wherein the therapeutic pay load comprises Pembrolizumab or a variant or fragment thereof.

[0415] 125. The method of embodiment 109, wherein the therapeutic payload comprises Ustekinumab or a variant or fragment thereof.

[0416] 126. The method of embodiment 109, wherein the therapeutic pay load comprises Dupilumab or a variant or fragment thereof.

[0417] 127. The method of embodiment 109, wherein the therapeutic pay load comprises Nivolumab or a variant or fragment thereof.

[0418] 128. The method of embodiment 109, wherein the therapeutic pay load comprises Daratumumab or a variant or fragment thereof.

[0419] 129. The method of embodiment 109, wherein the therapeutic pay load comprises Risankizumab or a variant or fragment thereof.

[0420] 130. The method of embodiment 109, wherein the therapeutic payload comprises Secukinumab or a variant or fragment thereof.

[0421] 131. The method of embodiment 109, wherein the therapeutic pay load comprises Pertuzumab or a variant or fragment thereof.

[0422] 132. The method of embodiment 109, wherein the therapeutic payload comprises Emicizumab or a variant or fragment thereof.

[0423] 133. The method of embodiment 109, wherein the therapeutic payload comprises Denosumab or a variant or fragment thereof.

[0424] 134. The method of embodiment 109, wherein the therapeutic payload comprises Abatocept or a variant or fragment thereof.

[0425] 135. The method of embodiment 109, wherein the therapeutic payload comprises Atezolizumab or a variant or fragment thereof.

[0426] 136. The method of embodiment 109, wherein the therapeutic payload comprises Durvalumab or a variant or fragment thereof.

[0427] 137. The method of embodiment 109, wherein the therapeutic payload comprises Guselkumab or a variant or fragment thereof.

[0428] 138. The method of embodiment 109, wherein the therapeutic pay load comprises Tocilizumab or a variant or fragment thereof.

[0429] 139. The method of embodiment 109, wherein the therapeutic payload comprises Durvalumab or a variant or fragment thereof.

[0430] 140. The method of embodiment 109, wherein the therapeutic pay load comprises Ixekizumab or a variant or fragment thereof.

[0431] 141. The method of embodiment 109, wherein the therapeutic payload comprises Infliximab or a variant or fragment thereof.

[0432] 142. The method of embodiment 109, wherein the therapeutic pay load comprises Eculizumab or a variant or fragment thereof.

[0433] 143. The method of embodiment 109, wherein the therapeutic pay load comprises Ravulizumab or a variant or fragment thereof.

[0434] 144. The method of embodiment 109, wherein the therapeutic pay load comprises Insulin or a variant or fragment thereof.

[0435] 145. The method of embodiment 105, wherein the payload is or comprises a polyribonucleotide.

[0436] 146. The method of embodiment 145, wherein the polyribonucleotide is or comprises a messenger RNA.

[0437] 147. The method of embodiment 145, wherein the polyribonucleotide is or comprises an inhibitory RNA

[0438] 148. The method of embodiment 145, wherein the polyribonucleotide is or comprises a non-coding RNA.

[0439] 149. The method of embodiment 104, wherein the polynucleotide construct comprises a transgene.

[0440] 150. The method of any one of embodiments 1-149, wherein the method further comprises removal of transduced adipose tissue and / or adipocytes from the subject.

[0441] 151. The method of embodiment 150, wherein all or substantially all of the transduced adipose tissue and / or adipocytes is removed.

[0442] 152. The method of embodiment 150 or 151, wherein removal comprises physical excision, biopsy, cryolypolysis, surgical removal, liposuction, laser mediated removal, radioablation, imaging-based ablation, or any combination thereof.

[0443] 153. The method of embodiment 152, wherein removal comprises physical excision.

[0444] 154. The method of embodiment 152, wherein removal comprises biopsy.

[0445] 155. The method of embodiment 152, wherein removal comprises cryolypolysis.

[0446] 156. The method of embodiment 152, wherein removal comprises surgical removal.

[0447] 157. The method of embodiment 152, wherein removal comprises liposuction.

[0448] 158. The method of embodiment 152, wherein removal comprises laser mediated removal.

[0449] 159. The method of embodiment 152, wherein removal comprises radioablation.

[0450] 160. The method of embodiment 152, wherein removal comprises imaging-based ablation.

[0451] 161. The method of any one of embodiments 150-160, wherein removal results in: (1) a reduction (e.g., at least 15% reduction) in local expression of the delivered payload; (2) a reduction (e.g., at least 15% reduction) in systemic distribution of the delivered payload; or (3) both (1) and (2).

[0452] 162. The method of any one of embodiments 1-161, wherein the mammal is a pig, cow, dog, cat, non-human primate, or human.

[0453] 163. The method of embodiment 162, wherein the mammal is a pig.

[0454] 164. The method of embodiment 162, wherein the mammal is a cow.

[0455] 165. The method of embodiment 162, wherein the mammal is a dog

[0456] 166. The method of embodiment 162, wherein the mammal is a cat

[0457] 167. The method of embodiment 162, wherein the mammal is a non-human primate

[0458] 168. The method of embodiment 162, wherein the mammal is a human.

[0459] 169. The method of any one of embodiments 1-168, wherein the AAV particle comprises a regulatory element.

[0460] 170. The method of embodiment 169, wherein the regulatory element is or comprises one or more promoters or a fragment or variant thereof, one or more enhancers or a fragment or variant thereof, one or more silencers or a fragment or variant thereof, one or more insulators or a fragment or variant thereof, or any combination thereof, optionally wherein the regulatory element comprises one or more elements provided in Table 2 or variants or fragments thereof.

[0461] 171. The method of embodiment 170, wherein the regulatory element is or comprises one or more promoters or a fragment or variant thereof.

[0462] 172. The method of embodiment 170, wherein the regulatory element is or comprises one or more enhancers or a fragment or variant thereof.

[0463] 173. The method of embodiment 170, wherein the regulatory element is or comprises one or more silencers or a fragment or variant thereof.

[0464] 174. The method of embodiment 170, wherein the regulatory element is or comprises one or more insulators or a fragment or variant thereof.

[0465] 175. The method of any one of embodiments 169-172, wherein the regulatory element comprises one or more promoters or a fragment or variant thereof; and one or more enhancers or a fragment or variant thereof.

[0466] 176. The method of embodiment 175, wherein the regulatory element is or is derived from a gene expressed in adipocytes.

[0467] 177. The method of embodiment 176, wherein the regulatory element comprises: mFabp4, mAdipoq, hADIPOQ, or combinations thereof.

[0468] 178. The method of embodiment 177, wherein the regulatory element comprises mFabp4.

[0469] 179. The method of embodiment 177, wherein the regulatory element comprises mAdipoq.

[0470] 180. The method of embodiment 177, wherein the regulatory element comprises hADIPOQ, optionally wherein the regulatory elements comprises the sequence of SEQ ID NO: 6 or a sequence having at least 90% identity thereto, or the sequence of SEQ ID NO: 36 or a sequence having at least 90% identity thereto.

[0471] 181. The method of any one of embodiments 176-180, wherein the regulatory element comprises a ubiquitous promoter and / or a ubiquitous enhancer.

[0472] 182. The method of embodiment 181, wherein the regulatory element comprises a ubiquitous promoter.

[0473] 183. The method of embodiment 181, wherein the regulatory element comprises a ubiquitous enhancer.

[0474] 184. The method of any one of embodiments 169-183, wherein the regulatory element reduces (e.g., prevents) expression of a payload in non-adipocytes.

[0475] 185. The method of any one of embodiments 169-183, wherein the regulatory element reduces (e.g., prevents) expression of a payload in adipocytes which are not present at a site of administration when the AAV particle is administered.

[0476] 186. The method of any one of embodiments 1-185, wherein the AAV capsid protein is or comprises an AAV1 capsid protein, an AAV2 capsid protein, an AAV3B capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 (AAVrhlO) capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV-PHP.B capsid protein, an AAV8-PHP.eB capsid protein, an AAV-PHP.S capsid protein, an AAV-7m8 capsid protein, an AAV-DJ capsid protein, an AAV-OligOOl capsid protein or a Rec2 capsid protein, or a variant or fragment of any of the foregoing.

[0477] 187. The method of embodiment 186, wherein the AAV capsid protein is or comprises an AAV1 capsid protein, or a variant or fragment thereof.

[0478] 188. The method of embodiment 186, wherein the AAV capsid protein is or comprises an AAV2 capsid protein, or a variant or fragment thereof.

[0479] 189. The method of embodiment 186, wherein the AAV capsid protein is or comprises an AAV3B capsid protein, or a variant or fragment thereof.

[0480] 190. The method of embodiment 186, wherein the AAV capsid protein is or comprises an AAV4 capsid protein, or a variant or fragment thereof.

[0481] 191. The method of embodiment 186, wherein the AAV capsid protein is or comprises an AAV5 capsid protein, or a variant or fragment thereof.

[0482] 192. The method of embodiment 186, wherein the AAV capsid protein is or comprises an AAV6 capsid protein, or a variant or fragment thereof.

[0483] 193. The method of embodiment 186, wherein the AAV capsid protein is or comprises an AAV7 capsid protein, or a variant or fragment thereof.

[0484] 194. The method of embodiment 186, wherein the AAV capsid protein is or comprises an AAV8 capsid protein, or a variant or fragment thereof.

[0485] 195. The method of embodiment 186, wherein the AAV capsid protein is or comprises an AAV9 capsid protein, or a variant or fragment thereof.

[0486] 196. The method of embodiment 186, wherein the AAV capsid protein is or comprises an AAV10 (AAVrhlO) capsid protein, or a variant or fragment thereof.

[0487] 197. The method of embodiment 186, wherein the AAV capsid protein is or comprises an AAV11 capsid protein, or a variant or fragment thereof.

[0488] 198. The method of embodiment 186, wherein the AAV capsid protein is or comprises an AAV12 capsid protein, or a variant or fragment thereof.

[0489] 199. The method of embodiment 186, wherein the AAV capsid protein is or comprises a PHP.B capsid protein, or a variant or fragment thereof.

[0490] 200. The method of embodiment 186, wherein the AAV capsid protein is or comprises a PHP.eB capsid protein, or a variant or fragment thereof.

[0491] 201. The method of embodiment 186, wherein the AAV capsid protein is or comprises a PHP.S capsid protein, or a variant or fragment thereof.

[0492] 202. The method of embodiment 186, wherein the AAV capsid protein is or comprises a 7m8 capsid protein, or a variant or fragment thereof.

[0493] 203. The method of embodiment 186, wherein the AAV capsid protein is or comprises a DJ capsid protein, or a variant or fragment thereof.

[0494] 204. The method of embodiment 186, wherein the AAV capsid protein is or comprises a OligOO 1 capsid protein, or a variant or fragment thereof.

[0495] 205. The method of embodiment 186, wherein the AAV capsid protein is or comprises a Rec2 capsid protein, or a variant or fragment thereof, optionally wherein the Rec2 capsid comprises the sequence of SEQ ID NO: 16 or a sequence having at least 90% identity thereto.

[0496] 206. A method for controlling and / or reversing a gene therapy, the method comprising: (1) delivering a first dose of a gene therapy comprising an adeno-associated (AAV) particle to adipose tissue in a non-rodent mammal, wherein the AAV particle comprises an AAV capsid and a polynucleotide construct encoding a payload; (2) reducing or eliminating expression of the pay load in the adipose tissue by removing at least a portion of the adipose tissue transduced by the AAV particle; and / or (3) reducing or eliminating distribution of the pay load systemically by removing at least a portion of adipose tissue transduced by the AAV particle.

[0497] 207. An adeno-associated (AAV) particle or a composition comprising the same, for use in controlling and / or reversing a gene therapy, the use comprising: (1) delivering a first dose of a gene therapy comprising the AAV particle to adipose tissue in a non-rodent mammal, wherein the AAV particle comprises an AAV capsid and a polynucleotide construct encoding a pay load; (2) reducing or eliminating expression of the pay load in the adipose tissue by removing at least a portion of the adipose tissue transduced by the AAV particle; and / or (3) reducing or eliminating distribution of the payload systemically by removing at least a portion of adipose tissue transduced by the AAV particle.

[0498] 208. The method or AAV particle for use of embodiment 207, wherein at least 10% of the transduced adipose tissue is removed.

[0499] 209. The method or AAV particle for use of embodiment 207 or 208, wherein all or substantially all of the transduced adipose tissue is removed.

[0500] 210. The method or AAV particle for use of any one of embodiments 207-209, wherein removing the transduced adipose tissue reduces local expression of the payload.

[0501] 211. The method or AAV particle for use of any one of embodiments 207-210, wherein removing the transduced adipose tissue reduces systemic distribution of the payload.

[0502] 212. The method or AAV particle for use of any one of embodiments 209-211, wherein removing all or substantially all of the transduced adipose tissue reduces local expression of the payload and / or reduces systemic distribution of the pay load to an undetectable level.

[0503] 213. The method or AAV particle for use of any one of embodiments 209-212, wherein the reduction in local expression and / or systemic distribution of the payload is observed within about 1 hour of removing the transduced adipose tissue.

[0504] 214. The method or AAV particle for use of any one of embodiments 207-213, wherein the gene therapy is adjusted by modulating the amount of transduced adipose tissue that is removed.

[0505] 215. The method or AAV particle for use of any one of embodiments 207-214, wherein the method further comprises administering one or more subsequent doses of a gene therapy comprising an adeno-associated (AAV) particle to the adipose tissue.

[0506] 216. The method or AAV particle for use of embodiment 215, wherein the one or more subsequent doses of gene therapy is administered to the same or nearby adipose tissue to which the first dose of the gene therapy is administered.

[0507] 217. The method or AAV particle for use of embodiment 215, wherein the one or more subsequent doses of gene therapy is administered to a different adipose tissue as compared to the adipose tissue to which the first dose of the gene therapy is administered.

[0508] 218. The method or AAV particle for use of any one of embodiments 215-217, wherein the one or more subsequent doses of gene therapy is administered at substantially the same volume as the first dose of the gene therapy.

[0509] 219. The method or AAV particle for use of any one of embodiments 215-218, wherein the one or more subsequent doses of gene therapy is administered at a different volume as compared to the first dose of the gene therapy.

[0510] 220. The method or AAV particle for use of any one of embodiments 1-219, wherein the AAV particle is characterized in that when administered to adipose tissue the AAV particle is contained in adipose tissue as compared to an otherwise similar AAV particle that is not delivered by the method of embodiment 1.

[0511] 221. The method or AAV particle for use of any one of embodiments 1-220, wherein the AAV particle is characterized in that when administered to adipose tissue the AAV particle transduces at least 5% more adipocytes in the adipose tissue that is administered the AAV particle as compared to adipocyte transduction by an otherwise similar AAV particle that is not delivered by the method of embodiment 1.

[0512] 222. An AAV particle comprising an AAV capsid protein and a polynucleotide construct.

[0513] 223. A composition comprising an AAV particle of embodiment 221.

[0514] 224. The composition of embodiment 223, wherein the composition is a pharmaceutical composition.

[0515] 225. A pharmaceutical composition comprising an AAV particle of embodiment 222.

[0516] 226. The pharmaceutical composition of embodiment 225, wherein pharmaceutical composition comprise one or more pharmaceutically acceptable carriers and / or excipients.

[0517] 227. The composition of embodiment 222 or 223, or the pharmaceutical composition of embodiment 225 or 226, wherein the composition or the pharmaceutical composition is formulated for delivery to adipose tissue.

[0518] 228. The composition or the pharmaceutical composition of embodiment 226, wherein the composition or the pharmaceutical composition is suitable for delivery to adipose tissue via subcutaneous injection.

[0519] 229. The AAV particle of embodiment 222, the composition of embodiment 222 or 223, or the pharmaceutical composition of embodiment 225 or 226 for use in delivering the AAV particle to a subject.

[0520] 230. The AAV particle for use, or the composition for use, or the pharmaceutical composition for use of embodiment 229, wherein delivering comprises administering to the subject the AAV particle.

[0521] 231. The use of embodiment 230, wherein the use comprises treating a subject having a disease or disorder disclosed herein.

[0522] 232. Use of, the AAV particle of embodiment 222, the composition of embodiment 223 or 224, or the pharmaceutical composition of embodiment 225 or 226, for manufacture of a medicament for delivering the AAV particle to a subject.

[0523] 233. Use of embodiment 232, wherein the use comprises administering to the subject the composition comprising the AAV particle.

[0524] 234. Use of embodiment 232 or 233, wherein the use comprises treating a subject having a disease or disorder disclosed herein. EXAMPLES

[0525] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and is not intended to limit the scope of what the inventors regard as their discovery nor are they intended to represent that the experiments below are all or the only experiments performed. Example 1: AAV8 Transduces >80% of Adipocytes after Local Delivery to Murine Adipose Depots

[0526] This Example shows adipocyte transduction after local delivery of AAV to murine adipose depots.

[0527] Methods'.

[0528] To test AAV8’s tropism for adipocytes after local delivery in mice, we directly injected AAV8.CAG.EGFP vectors into the epididymal and inguinal adipose pads and then examined the extent of GFP expression 14 days later. 12-week-old male C57BL / 6J mice weighing 22-28g were anesthetized with ketamine (100 mg / kg) and xylazine (10 mg / kg), and the left epididymal (visceral) or inguinal (subcutaneous) adipose pad was exposed via incision. The adipose pad was grasped with forceps, and then injected with vector using a 100 uL Hamilton syringe and 32G, 0.5 in beveled needle. The left epididymal pad was injected twice with 50 uL of vector solution formulated in PBS to a concentration of 2x1012 vg / mL (for a total of 2x1011 vg in 100 uL of PBS delivered to each pad, n=2 mice). The left injected pad was injected four times with 10 uL of vector solution formulated in PBS to a concentration of 5x1012 vg / mL (for a total of 2xlOn vg in 40 uL of PBS delivered to each pad, n=2 mice). Adipose pads were replaced, incisions were sutured, and mice were monitored for 14d until euthanasia and necropsy. Naive mice (no surgery) were included in the study as negative controls (n=2). At necropsy, the left and right epididymal and inguinal adipose pads and the liver were removed from each animal and fixed in 10% neutral buffered formalin for 48h. Adipose pads were weighed to assess volume and then processed for whole mount immunofluorescence. Briefly, pads were permeabilized and blocked in 0.5% Triton X-100 in PBS (PBS-T) with 10% normal goat serum (NGS), co-labeled with primary antibodies against GFP (Rabbit anti-GFP, Cell Signaling Technology, 1:200) and the pan-adipocyte marker Perilipin 1 (Goat anti-Plinl, Cell Signaling Technology, 1:200) in PBS-T with 2% NGS, washed with PBS-T, and labeled for fluorescence detection with donkey anti-goat Alexa Fluor 568 and donkey anti-rabbit Alexa Fluor 647 secondary antibodies in PBS-T (Thermo Fisher Scientific, 1:500). Pads were washed in PBS and then mounted in SlowFade Glass (Thermo Fisher Scientific) sandwiched between two coverslips taped together to flatten the organs as much as possible. The mounted organs were then imaged on a Zeiss LSM 980 confocal microscope by acquiring z-stacks with a 10x / 0.5 NA objective at multiple regions sampled along their length. HALO image analysis software was used to automatically segment the Plinl-positive outlines of adipocytes and measure the intensity of native GFP and GFP immunolabeling in the cells. Adipocytes with immunolabeled GFP intensity above levels measured in cells from naive mice in at least 50% of the segmented cellular outline were considered positive. The left and right inguinal pads from one of the naive mice were excluded from analysis because they were stained with different antibodies.

[0529] Results'.

[0530] Representative confocal images of the injected left epididymal pad in an AAV8.CAG.EGFP-treated mouse and a corresponding uninjected left epididymal pad from a naive mouse are shown in FIG. 1A. Native GFP and immunolabeled GFP were observed in most Plinl+ adipocytes throughout the entire pad (top row shows tiled view of the entire pad, middle row shows zoomed in region demarcated by dashed box in top row). In contrast, no GFP was detectable in the naive pads. Quantification of the percentage of GFP+ / Plinl+ adipocytes throughout each injected pad is graphed in FIG. IB. AAV8 is highly tropic for adipocytes in mice as >80% of the cells were transduced in the four injected pads. The weighed volumes of each pad were used to convert the adipocyte transduction efficiency into a transduced volume, which is graphed in FIG. IC. The results show that injecting a single pad with AAV8 can result in transduction of 43 to 90 uL of adipose volume in mice. Example 2: Local Delivery of AAV8 to a Single Adipose Depot Results in Systemic Distribution and Transduction of Off-Target Organs in Mice

[0531] This Example shows that AAV delivered to a single adipose depot in mice results in systemic distribution and transduction of non-injected organs including liver and other adipose tissue.

[0532] To assess the systemic distribution of AAV8.CAG.EGFP after local administration to a single fat pad, we first examined the extent of GFP expression throughout all organs collected by taking low resolution, low sensitivity images of native GFP fluorescence in full organs with a Zeiss Axiozoom fluorescence stereomicroscope. The images are shown in FIG. 2A. Dashed lines indicate the outlines of each organ. Note that the display settings for the adipose pads and the livers are different. Brightness and contrast has been enhanced equivalently for all adipose organs to make signal visible, but the same settings were not applied to liver to avoid saturation. Consistent with confocal analysis, GFP signal can be observed throughout the full extent of the injected adipose pads. However, the fluorescence intensity in the inguinal pads is greater than the epididymal pads. Lower levels of GFP signal that were above background could be observed in discrete regions of uninjected pads from treated mice (for example, see the right inguinal pad from left epididymal injection, mouse 2). In addition, the livers of all treated mice displayed intense, even GFP fluorescence throughout all lobes, indicating the entire organ was transduced. FIG. 2B shows quantification of the mean fluorescence intensity measured from each of the organs displayed in FIG. 2A. Results were consistent with previous observations; however, the mean GFP signal in the uninjected organs from treated mice did not rise above background levels from naive mice. Interestingly, the livers of treated mice displayed higher GFP intensity than the injected adipose pads. The uninjected adipose pads from treated mice were also examined with confocal microscopy, which has greater resolution and sensitivity. Images were collected and pads were analyzed in the same way described in Example 1. Quantification of these images is graphed in FIG. 2C. The data showed that there was significant transduction of adipocytes in uninjected pads compared to pads from naive mice(p<0.01, Oneway ANOVA with Tukey’s post-hoc test).

[0533] As an additional measure of vector distribution, digital PCR (dPCR) was performed on DNA and RNA extracted from all the organs after they were the fixed, stained, and imaged. Taqman probe assays targeting the CMV enhancer within the CAG promoter were used to detect the extracted DNA, and probe assays against the EGFP transgene were used to detect transgene mRNA. FIG. 3 shows graphs of the numbers of vector genome copies and mRNA transcripts measured in each organ. The results are consistent with those observed with microscopy. Significant levels of vector genomes and EGFP mRNA were observed in the injected pads and livers of treated mice (Tx) compared to the naive controls (p<0.01, One-way ANOVA with Tukey’s post-hoc test). While the mean levels in the uninjected pads were higher than naive controls, they did not reach statistical significance. The livers had the highest vector genome and EGFP mRNA levels, followed by the injected pads. Thus, AAV8 shows broad systemic distribution after local delivery to a single adipose pad in mice. Example 3: AAV Stays Locally Confined to Injection Sites, Enters Adipocyte Nuclei, and Does Not Distribute Systemically to Vital Organs after Subcutaneous Injection in NonHuman Primates

[0534] This Examples shows that AAVs administered to particular adipose tissue in NHPs, successfully transduce adipocytes at the site of administration and stay confined to the site of administration with no spread to other organs or non-injected adipose tissue.

[0535] Methods:

[0536] The local and systemic distribution of 14 different AAV capsid variants that were administered subcutaneously into the adipose space in nonhuman primates was assessed. Two male cynomolgus macaques (Macaca Fascicularis) weighing 7-9 kg were used. One week prior to dosing, serum was taken from both animals and titers of pre-existing neutralizing antibodies to AAV2, AAV6, AAV8, and AAV9 were assessed using published methods at the University of Pennsylvania Immunology Core. Titers were at or near the limit of detection in both animals. On the day of dosing, animals were sedated, and eighteen subcutaneous injections of AAV were administered to each in non-overlapping locations. A grid of 14 injection sites was marked with indelible marker on the abdomen, with 7 on the left and 7 on the right side (1.25 cm from the mid-line), each spaced ~1.5 cm apart along the cranial-caudal axis spanning from just below the costal margin to the lower quadrant. In addition, one site was marked on each of the left and right inguinal regions and on the left and right rump. 10 uL of the following capsids were separately administered into one of each of the 14 injection sites on the abdomen at a dose of lei 1 vg (dose concentration of lel3 vg / mL): AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, RhlO, AAV.DJ, AAV2.7m8, Php.B, and OligOOl. 100 uL Hamilton syringes with 32G, 0.5 in beveled needles were used for injections. Each vector encoded the same CAG.EGFP.WPRE.SV40pA transgene. AAV1 and AAV8 were administered into the left and right inguinal sites, respectively; and AAV2 and AAV9 were administered into the left and right rump sites. The dose and volume for these sites was the same as for the abdominal injections, except the injection volume was 100 uL for the left and right inguinal sites in one animal, and it was 100 uL for the left and right rump sites in the other animal (total dose was held constant at lei 1 vg; dose concentration was lel2 vg / mL). After injections, animals were allowed to recover and then were monitored daily.

[0537] The animals were euthanized after 8 days, and 1.5 cm x 1.5 cm square regions around all injection sites were excised (full-thickness excisions spanning from epidermis to muscle underlying Camper’s and Scarpa’s fascia) and fixed in 10% neutral buffered formalin for 48h. For controls, a region on the abdomen 3 cm outside of the injection sites was excised from both animals. Both frozen and fixed biopsies were also taken of the left lateral lobe of the liver, apex of the heart, cervical dorsal root ganglia, testes, and gastrocnemius muscle.

[0538] Fixed specimens were washed in PBS, embedded in paraffin, and sectioned. Fullthickness cross-sections of injection sites and liver biopsies were labeled on a Leica Bond automated staining system with RNAScope probes against EGFP, which target both vector DNA and RNA. Because 8d was likely too early for significant levels of GFP RNA or protein to have accumulated, the majority of labeling is expected to correspond to vector genomes (vg’s). Sections were counterstained with hematoxylin. HALO analysis software (Indica Labs) was used to quantify the extent of RNAScope labeling in macroscopic, relevant anatomical locations. For this, an artificial intelligence (AI) classifier was trained to automatically segment the skin, adipose, and muscle regions in the injection site sections. The total amount of probe within each of these regions and the liver was quantified using the ISH module in HALO, which performs color deconvolution and segmentation to automatically detect the number of probe copies (a probe copy consists of an individual labeled puncta presumed to be a single copy of DNA or RNA) within a classified region.

[0539] In addition, the extent of RNAScope labeling in adipocyte nuclei was quantified as a proxy measure of adipocyte transduction since transgene expression was assumed to be still pending or nascent at such an early timepoint in vivo. An AI classifier was first trained to recognize adipocytes, and any nucleus associated with an adipocyte was automatically segmented and assessed for RNAScope labeling using the ISH-IHC module in HALO. Any nucleus that had one or more labeled vg copy was considered positive. An H-score was also calculated for each nucleus, which was calculated by dividing nuclei into 5 bins based on the number of vg copies per nucleus (0, 1-3, 4-9, 10-15, or >15 copies) and then summing the percentage of total nuclei in each bin multiplied by the bin number (0-4). To control for variability in section dimensions, an equal-sized region of interest (ROI) was analyzed for each injection site. For this, the analysis was restricted to an 80 mm2 ROI centered on the area containing the densest labeling in the adipose layer (presumed injection site). Assuming equal spread of virus in all dimensions, this ROI corresponded to an 800 uL volume. Only abdominal injection sites were compared to keep dose, volume, and anatomical location constant.

[0540] For digital PCR (dPCR) measurements, frozen tissues were homogenized, and the homogenate was split equally for isolation of DNA and poly-adenylated mRNA. The concentration of vector genomes in the DNA isolated from each organ was then assessed using TaqMan probes that recognize the CMV enhancer present within the CAG regulatory element of all the vectors. Likewise, the concentration of EGFP transcripts in the mRNA isolated from each organ was measured using TaqMan probes recognizing EGFP. As a positive control, dPCR was also performed on cultured, intact explants from pigs that had been transduced with the same PHP.B.CAG.EGFP virus used in NHPs. For this, full-thickness punch biopsies spanning from skin to adipose were harvested from Yorkshire pigs and then placed free floating into DMEM / F-12 supplemented with 10% fetal bovine serum, 4 ug / mL Ciprofloxacin, and 0.25 ug / mL Fungizone. The next day, the punch explants were grasped with forceps, subcutaneously injected with 10 uL of 1x1011 vg of AAV, and then placed back into culture. The culture was continued for five more days to allow for AAV transduction and GFP expression, and then the explants were frozen and processed for dPCR as described above.

[0541] Results:

[0542] FIG. 4 is a graph that shows the quantification of total vector genomes detected via RNAScope labeling in the classified skin, adipose, and muscle regions of the injection site biopsies collected from the abdomen (10 uL x lei 1 vg for each capsid). Box plots and dot plots show pooled data from all injection sites (14 capsids) for each region. Quantification across the whole section of the liver biopsy from both animals is also included. Vg quantity was significantly greater in the adipose space compared to all other regions examined (p<0.01, ANOVA with Tukey’s test). Higher mean levels of labeling were also detected in the skin (comprising epidermis and dermis); however, they were less than adipose. In contrast, vg labeling in the muscle underlying injection sites and the liver was comparable to control background labeling.

[0543] FIG. 5 is a graph showing the concentration of vector DNA and transgene mRNA measured with dPCR in various organs harvested from the two NHPs at necropsy. In contrast to the significant systemic distribution observed in the mouse (FIG. 3), the levels are at or near zero in every organ examined in NHPs after subcutaneous injection. This data demonstrates that subcutaneous administration of AAVs into the adipose space in nonhuman primates does not result in significant systemic biodistribution. Cultured pig explants transduced with Php.B.CAG.EGFP that were included as positive controls show high concentrations of vector DNA and GFP mRNA.

[0544] FIG 6 is a graph showing the percentage of adipocyte nuclei containing > 1 labeled vg in an 800 uL volume of adipose centered on each abdominal injection site. Capsids are rank ordered based on the maximal percentage of nuclei labeled across the sites examined. Nine (9) of 14 sites showed >40% of nuclei labeled in at least one of the two sites. The H-score (indicated by dot size in the graph) also tended to correlate with the percentage of nuclei labeled, demonstrating that the number of vg’s per nucleus increased as the total number of nuclei vg’s entered increased. This data demonstrates that all of the tested capsids, other than AAV2, AAV3B, AAV4 and OligOOl, efficiently transduced adipocytes, further supporting the development of AAV particles for administration to adipose tissue. Example 4: AAV Stays Locally Confined to Injection Sites, Enters Adipocyte Nuclei, and Does Not Distribute Systemically to Vital Organs after Subcutaneous Injection in Pigs

[0545] This Example shows that AAVs administered to particular adipose tissue in pigs, successfully transduce adipocytes at the site of administration and stay confined to the site of administration with no spread to other organs or non-injected adipose tissue.

[0546] Methods'.

[0547] Similar comparisons were also performed in pigs to look for capsids that performed efficiently across both large animal models. Two female Yorkshire swine aged 3 months and weighing 34-36 kg were used. On the day of dosing, animals were sedated, and eighteen subcutaneous injections of AAV were administered to the abdomen of each in nonoverlapping locations. A grid of 18 injection sites was tattooed on the abdomen, with 9 on the left and 9 on the right side, each spaced ~2 cm apart along the cranial-caudal axis spanning from just below the costal margin to the lower quadrant. 30 uL of the following capsids were separately administered into one of each of 14 injection sites on the abdomen at a dose of lei 1 vg (dose concentration of 3.3el2 vg / mL): AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, RhlO, AAV.DJ, AAV2.7m8, Php.B, and OligOOl. 100 uL Hamilton syringes with 32G, 0.5 in beveled needles were used for injections. Each vector encoded the same CAG.EGFP.WPRE.SV40pA transgene. In the remaining four sites in each animal, we injected different doses and volumes of AAV6: 1) 30 uL x lei 1 vg; 2) 30 uL x lelO vg; 3) 100 uLxlell vg; 4) 1000 uLx lei 1 vg. After injections, animals were allowed to recover and then were monitored daily. The animals were euthanized after lOd, and tissues were harvested and processed exactly as described for nonhuman primates in Example 3. The excised tissue from injection sites was larger in pigs compared to NHPs owing to the larger size of the adipose layer in pigs.

[0548] Results:

[0549] FIG. 7 is a graph that shows the quantification of total vector genomes detected via RNAScope labeling in the classified skin, adipose, and muscle regions of the injection site biopsies collected for capsids dosed at 30 uL x lei 1 vg. Box plots and dot plots show pooled data from all injection sites (14 capsids) for each region. Quantification across the whole section of the liver biopsy from both animals is also included. Vg quantity was greater in the adipose space compared to all other regions examined. Vg labeling in the skin, the muscle underlying injection sites, and the liver was comparable to control background labeling.

[0550] FIG. 8 is a graph showing the concentration of vector DNA and transgene mRNA measured with dPCR in various organs harvested from the two pigs at necropsy. Similar to NHPs, the levels were at or near zero in every organ examined. This data demonstrates that subcutaneous administration of AAVs into the adipose space in pigs does not result in significant systemic biodistribution. Cultured pig explants transduced with Php.B.CAGEGFP that were included as positive controls show high concentrations of vector DNA and GFP mRNA.

[0551] FIG. 9 is a graph showing the percentage of adipocyte nuclei containing > 1 labeled vg in an 800 uL volume of adipose centered on each injection site receiving 30 uL x lei 1 vg. Capsids were rank ordered based on the maximal percentage of nuclei labeled across the sites examined. Significant labeling was detected for a majority of the capsids, showing that many capsids were able to transduce adipocytes in pigs. The H-score also tended to correlate with the percentage of nuclei labeled in pigs.

[0552] Taken together, this data demonstrates that AAVs administered to adipose tissue in pigs successfully transduced adipocytes at the site of administration with minimal systemic biodistribution or spread to non-injected adipose tissue. Example 5. Php.B, AAV1, AAV5, AAV6, AAV7, AAV8 Show the Highest Cross-Species Rate of Adipocyte Nuclear Entry in a Defined Volume of Adipose Centered at the Injection Site

[0553] This Example shows a cross-species comparison of efficiency of capsid nuclear entry in NHPs and pigs.

[0554] FIG. 10 shows a cross-species of comparison of vg entry into adipocyte nuclei in NHPs and pigs using the max values obtained for each capsid shown in FIGS. 6 and 9. AAV2, AAV3B, AAV4, and OligOOl performed consistently worse in both species. Visual inspection of the RNAScope labeling showed most of these capsids tended to have very dense membrane labeling that was highly confined to a small region, suggesting they possess properties that significantly limit their distribution within adipose. Without wishing to be bound by any particular theory, in some embodiments, intensity of membrane labeling may be indicative of the ability of a particular AAV capsid to be targeted and / or distributed to a lipophilic tissue. In some embodiments, AAV capsids having dense membrane labeling may be less likely to be targeted and / or distributed to lipophilic tissue, e.g., adipose. In some embodiments, AAV capsids having less dense membrane labeling may be more likely to be targeted and / or distributed to lipophilic tissue, e.g., adipose. In some embodiments, the intensity of membrane labeling of an AAV capsid may be performed using any of the methods disclosed herein and / or as compared to a reference AAV capsid.

[0555] All other capsids had broader distributions with less intense membrane labeling, indicating they were able to partition further within adipose while still remaining sufficiently confined to limit systemic distribution. These capsids all performed comparably well in NHPs, while there was some variability in performance in pigs. All of the other capsids (other than AAV2, AAV3B, AAV4 and OligOOl) were categorized as “top performing,” and they include several like Php.B, RhlO, 7m8, and AAV.DJ whose tropism for adipocytes has never previously been examined.

[0556] Without wishing to be bound by any particular theory, in some embodiments, 7m8, AAV9, RhlO, AAV.DJ, AAV1, AAV5, Php.B, AAV7, AAV8, and AAV6 have adipose tissue tropism and can transduce adipocytes in mammals. This Data supports the development of AAV particles comprising a capsid protein from 7m8, AAV9, RhlO, AAV.DJ, AAV1, AAV5, Php.B, AAV7, AAV8, or AAV6 for adipose tissue delivery in mammals, e.g., humans. Example 6. AAV1, Php.B and Reel Exhibit Superior Adipocyte Tropism Compared to AAV6 and AAV8 after Subcutaneous Delivery in Non-Human Primates

[0557] This Example shows a comparison of adipocyte tropism of AAV6, AAV8, PHP.B, Rec2 and AAV1 in NHPs following subcutaneous administration of the AAVs.

[0558] Based on the data presented in Example 5, we chose a subset of “top performing” capsids and examined their ability to transduce adipocytes and express a reporter gene at 3 weeks after subcutaneous dosing in nonhuman primates. The capsid Rec2 was included in the comparison because published work has shown it has tropism for adipocytes in mice; however whether or not this capsid has adipose tropism in high order mammals such as non-human primates or humans, has not yet been shown in the field. Six male cynomolgus macaques (Macaca Fascicularis) weighing 8-10 kg were injected subcutaneously in multiple, nonoverlapping sites with 30 uL x 3el 1 vg of AAV1 (n=2 injection sites), AAV6 (n=2 injection sites), AAV8 (n=2 injection sites), Php.B (n=3 injection sites), or Rec2 (n=3 injection sites) vectors expressing GFP under control of ubiquitous promoters. To test the effect of dose on adipocyte transduction, the AAV1 (n=2 injection sites), Php.B (n=3 injection sites), and Rec2 (n=3 injection sites) vectors were also injected into additional non-overlapping sites at a dose of 30 uL x 3el0 vg. Three weeks later, injection sites were harvested via a 2 cm x 1 cm elliptical excision biopsy and fixed in 10% neutral buffered formalin for 48h. Three regions of tissue that were distant from the injection sites were also removed and processed as controls. The biopsies were then embedded in paraffin and sectioned as described in Example 3. GFP protein was detected via automated chromogenic immunohistochemistry on a Leica Bond system. Sections were first incubated with mouse anti-GFP primary antibodies, and subsequently with an antimouse Bond Polymer Refine Red Detection kit. Sections were counterstained with hematoxylin. The percentage of GFP-positive adipocytes in each histological section was quantified in an automated fashion using HALO software.

[0559] FIG. 11 is a graph that demonstrates the transduction efficiency of each capsid. The mean percentage of GFP-positive adipocytes across sections from the injection sites were plotted for each capsid. While AAV6 and AAV8 were able to transduce adipocytes to some extent (7-22% GFP-positive cells), AAV1, Php.B, and Rec2 displayed substantially higher tropism, transducing >60% of adipocytes across the sections examined. AAV1, Php.B, and Rec2’s transduction efficiencies of adipocytes were significantly greater than AAV6 and AAV8 (p<0.01, One-way ANOVA with Tukey’s post-hoc test). Given that AAV1 has previously been shown to have poor adipocyte tropism compared to AAV6 and AAV8 in mice, these results were unexpected and demonstrate that AAV capsid tropism for adipocytes in mammals cannot be inferred from data generated in mice.

[0560] The graph in FIG. 11 also shows the mean percentage of GFP-positive adipocytes across sections from sites injected with a 10-fold lower dose of AAV1, Php.B, or Rec2 (3el0 vg). For all three capsids, GFP-positive adipocytes were still detectable at the lower dose; however, the mean percentage was significantly lower compared to the higher dose (p<0.01, One-way ANOVA with Tukey’s post-hoc test). The infectivity of AAV1, Php.B, and Rec2 at 3el0 vg was similar to the infectivity of AAV6 and AAV8 at 3el 1 vg. Example 7. Subcutaneous Injection of Php.B Vectors Encoding Secreted Proteins Results in Serum Protein Levels in Pigs

[0561] This Example shows that administration of AAV particles comprising a payload via subcutaneous injection in pigs to adipose tissue results in systemic distribution of an exemplary polypeptide payload.

[0562] To test whether transduced adipocytes can secrete vector-derived proteins into the systemic circulation in pigs, four female Yorkshire swine aged 3 months and weighing 30-35 kg were dosed subcutaneously with a Php.B vector encoding human secreted alkaline phosphatase (hSEAP) under the control of a ubiquitous promoter. Each animal was dosed in three separate sites with 30 uL of 3el 1 vg of AAV (9el Ivg total dose per animal). Serum was collected one week before and on the day of dosing, then every 7 days for two weeks thereafter. The detected levels of hSEAP in the collected serum were measured using NovaBright™ Phospha-Light™ EXP Assay Kit for SEAP (Thermo Fisher) and a Thermo Fisher Varioskan LUX multimode plate reader.

[0563] FIG. 12 is a graph showing the detected levels of hSEAP in each animal over time. By 7 days post-dosing, the detected levels of hSEAP in all animals were significantly above baseline levels, then continued to rise thereafter, demonstrating in a large animal model that AAV-transduced adipocytes, e.g., AAV-based adipocyte depots, can secrete exogenous proteins into blood. This data supports the development and utility of AAV particle delivery to adipose tissue for localized expression and systemic distribution of a secretory protein payload in large animals including humans. Example 8: Subcutaneous Injection of Php.B Vectors Encoding Secreted Proteins Results in Serum Protein Levels that Are Controllable via the Number of Dosing Sites in NonHuman Primates

[0564] This Example shows that administration of AAV particles via subcutaneous injection in NHPs to adipose tissue results in systemic distribution of an exemplary polypeptide payload and that expression level of the polypeptide payload can be controlled by the number of doses administered.

[0565] To test whether transduced adipocytes can secrete vector-derived proteins into the systemic circulation, three of the four animals described in Example 6 were also dosed with a Php.B vector encoding human secreted alkaline phosphatase (hSEAP) under the control of a ubiquitous promoter. Each animal received a different number of subcutaneous injections of 30 uL x 3el 1 vg of vector in non-overlapping sites. One animal received two injections (6el 1 vg total dose; monkey 3), another received three injections (9el 1 vg total dose; monkey 2), and the third received six injections (1.8el2 vg total dose; monkey 1). Serum was collected one week before and on the day of dosing, then every 7 days for three weeks thereafter. The detected levels of hSEAP in the collected serum were measured using NovaBright™ Phospha-Light™ EXP Assay Kit for SEAP (Thermo Fisher) and a Thermo Fisher Varioskan LUX multimode plate reader.

[0566] FIG. 13 is a graph showing the detected levels of hSEAP in each animal over time. By 7 days post-dosing, the detected levels of hSEAP in all animals were >50-fold above baseline levels, then continued to rise thereafter. Based on the half-life of hSEAP and the average blood volume of cynomolgus macaques, the peak blood levels of hSEAP achieved in this experiment demonstrates that proteins encoded by exogenous gene therapy were secreted from adipocytes, e.g., at least to a similar level as endogenous adipokines like adiponectin. A dose response was also observed at all timepoints: monkey 1 which received the highest dose of AAV particles showed the highest hSEAP detected levels in the blood, monkey 2 which received the intermediate dose of AAV particles had hSEAP detected levels in the blood which were lower than monkey 1 but higher than monkey 3 (which received the lowest dose of AAV particles). This data demonstrates that the concentration of secreted protein in blood can be controlled, e.g., by varying the number of confined subcutaneous depots, and / or altering the total dose of AAV particles administered.

[0567] This data supports the development and utility of AAV particle delivery to adipose tissue for localized expression and systemic distribution of a secretory payload in large animals including humans. Example 9. Human ADIPOQ Gene Regulatory Elements Drive Strong AAV Transgene Expression in Subcutaneous Adipocytes of Nonhuman Primates

[0568] This Example shows that an exemplary combinatorial sequence of the distal enhancer and proximal promoter of the human ADIPOQ gene drives high levels of transgene expression in subcutaneous adipocytes of nonhuman primates.

[0569] The use of ubiquitous gene regulatory elements derived from CMV (e.g. CMV, CAG, CBA, smCBA) in AAV vectors can be associated with cellular toxicity in various contexts. To examine whether synthetic combinations of regulatory sequences derived from adipocyte-specific genes could drive AAV transgene expression in adipocytes of nonhuman primates, vectors encoding three different regulatory sequences driving an EGFP reporter were constructed and dosed subcutaneously. The three regulatory sequences were the murine Fabp4 enhancer linked to its basal promoter (i.e. mFabp4\ the putative cis-regulatory region for murine Adipoq (i.e. mAdipoq), and the combined distal enhancer and proximal promoter of human ADIPOQ (i.e. hADIPOQ).

[0570] Two male cynomolgus macaques (Macaca Fascicular^ weighing 8-10 kg were injected subcutaneously in 2 non-overlapping sites with 30 uL x 3el 1 vg of Php.B.mFabp4.EGFP, Php.B.mAdipoq.EGFP, and Php.B.hAdipoq.EGFP (n=4 injection sites total per vector). To facilitate across-animal comparison to the ubiquitous promoter data described in Example 6, one of the aforementioned monkeys also received a single subcutaneous injection of 30 uL x 3el 1 vg of Php.B.smCBA.EGFP (n=l injection site total) and three subcutaneous injections of 30 uL x 3el0 vg of Php.B.smCBA.EGFP (n=3 injection sites total). The data from these sites was included in Example 6 and FIG. 11. Three weeks later, injection sites were harvested via a 2 cm x 1 cm elliptical excision biopsy and fixed in 10% neutral buffered formalin for 48h. The biopsies were then embedded in paraffin and sectioned as described in Example 3. GFP protein was labeled via immunohistochemistry as described in Example 6. The number of GFP-positive adipocytes and the average intensity of adipocyte GFP labeling (measured as an optical density) in each histological section was quantified in an automated fashion using HALO software.

[0571] A microscopic image of a section taken from a site that was injected with Php.B.hAdipoq.EGFP is shown in FIG. 14A. The combined RGB image and digitally separated hematoxylin and GFP immunohistochemistry labeling is shown in each panel. Robust GFP signal that is highly confined to the superficial subcutaneous adipose was observed. FIG. 14B is a graph that shows the mean intensity of GFP immunohistochemical labeling versus the number of GFP-positive adipocytes in a single cross-section from each injection site. The control, smCBA Low (3el0 vg dose), and smCBA (3el 1 vg dose) data are the same as shown in Example 6 and FIG. 11. The hAdipoq regulatory element showed significantly more GFP+ cells and higher mean GFP intensity compared to the mFabp4 and mAdipoq regulatory elements (p<0.01, One-way ANOVA with Tukey’s post-hoc test). At equal doses of 3el 1 vg, hAdipoq performed comparably, if not better, than the smCBA ubiquitous promoter. While mFabp4 and mAdipoq did show some activity, it was similar to a 10-fold lower dose of smCBA. Of the regulatory elements derived from adipocyte-specific genes that were tested, the activity from hAdipoq was better than the other regulatory elements that were tested.

[0572] This data demonstrates that adipocyte specific promoter(s) and gene regulatory elements can be used to drive enhanced expression of payloads delivered via AAV to adipose tissue. Example 10. Reel transduces human adipocytes

[0573] This Example shows that Rec2 is able to efficiently transduce subcutaneous adipocytes in cultures of explanted human adipose tissue.

[0574] To test whether Rec2 adipocyte tropism was similar between nonhuman primates and humans, examined GFP expression was examined in intact human adipose explants transduced with Rec2.smCBA.EGFP. A full-thickness skin specimen (including subcutaneous adipose) was obtained from a 46 year old female donor that had undergone panniculectomy. Intact pieces of superficial subcutaneous adipose ~1 mL in size were cut away from the dermis, injected with 30uLx3ell vg of Rec2.smCBA.EGFP (n=2 explants) to mimic in vivo injections performed previously in nonhuman primates, and cultured for 8 days at 37 deg C and 5% CO2 to allow for onset of GFP expression. Two un-injected explants were cultured in parallel as controls. After 8 days, specimens were fixed in 10% NBF for 48h, and processed for sectioning and GFP immunohistochemistry as described in Example 9. The percentage of GFP-positive adipocytes in each histological section was quantified in an automated fashion using HALO software.

[0575] FIG. 15A shows microscopic images of adipocytes in control and Rec2-transduced cultures. A high density of intensely GFP-positive adipocytes was observed throughout the explants, whereas no GFP labeling was observed in the control cultures. Quantification showed that >80% of the adipocytes in each ~1 mL explant were GFP-positive (FIG. 15B). Thus, the adipocyte tropism of Rec2 is comparable between nonhuman primates and humans. Example 11. Exemplary Model System for Removal of Transduced Adipose Significantly Reduces Serum Levels of a Secreted Protein Payload

[0576] This Example shows reduction in serum levels of an exemplary payload by removing adipose tissue into which AAV comprising a transgene encoding the payload was administered an exemplary model system (e.g., in mice).

[0577] To test whether removal of adipocytes transduced with an AAV encoding an exemplary secreted polypeptide reduced the corresponding serum polypeptide levels, 10 uL of a preparation of AAV particles comprising a Rec2 capsid and a sequence encoding human SEAP (referred to herein as Rec2.hAdipoq.hSEAP.miRl / 122) was administered into the left inguinal fat pad of adult mice at a dose of 1 x 10(12) vg / ml (n=12 mice). To further restrict expression of the payload to adipocytes in the injected adipose pad, the hAdipoq promoter was used to drive expression, and the miRl and miR122 target sites were added at the 3’ end of the expression construct to limit off-target expression in muscle and liver. Fourteen days after AAV administration, half of the mice (n=6) were anesthetized, and the left inguinal fat pad was surgically excised. Mice were allowed to recover, and the study was continued for another fourteen days. Serum was collected every seven days, and the detected levels of hSEAP were measured using NovaBrightTM Phospha-LightTM EXP Assay Kit for SEAP (Thermo Fisher).

[0578] Serum hSEAP detected levels were similar in all mice for up to fourteen days after administration of the AAV particles (FIG. 16). Fourteen days later, mice that had adipose tissue removed showed a significant, approximately 70% reduction in serum hSEAP detected levels compared to animals that did not have surgical removal of the left inguinal fat pad (p<0.01, One-way ANOVA with Tukey’s post-hoc test).

[0579] This data demonstrates that removal of adipose tissue transduced with specifically engineered AAV particles and encoding a pay load can be used to reduce levels, e.g., systemic levels, of the payload. One of skill in the art would understand that similar or enhanced payload reduction can be expected when performing a similar adipose removal procedure in a higher order mammal (e.g., pig, cow, dog, cat, non-human primate, or human). In some embodiments, enhanced reduction (e.g., a complete reduction) in levels (e.g., systemic levels) of a payload after removal of adipose tissue, to which an AAV particle was delivered, can be observed in a mammal in which the delivered AAV particle is contained at or near the delivered adipose tissue with minimal systemic biodistribution or spread of the AAV particle to non-injected adipose tissue. Exemplary containment of delivered AAV particles at or near delivered adipose tissue in higher order mammals is shown in Example 3, and FIG. 4, and Example 4 and FIG. 7. Example 12. Human Adipol.BGI Promoter Enhances Expression Of Exemplary Transgenes in Preadipocytes and Adipocytes.

[0580] This Example shows that an engineered adipocyte-specific promoter comprising a human P-Globin intron drives enhanced expression of exemplary transgenes in adipocytes.

[0581] An engineered adipocyte promoter comprising a human P-Globin intron (BGI) was constructed for enhanced transgene expression in adipocytes (referred to herein as Adipo2.BGI). The Adipo2.BGI promoter was designed to avoid potential off-target splicing by removing 8 C-terminal nucleotides predicted to constitute a splice donor site. The Adipo2.BGI promoter incorporates nucleotides 1-875 of the combined distal enhancer and proximal promoter of human ADIPOQ (i.e. hADIPOQ) and a human P-globin intron provided as SEQ ID NO: 35 (FIG. 17). Three exemplary transgenes were expressed in differentiating 3T3 LI adipocytes with expression of the transgenes driven by: (1) a CBA ubiquitous promoter, (2) adipocytespecific hAdipo promoter (SEQ ID NO: 6), or (3) adipocyte-specific Adipo2.BGI promoter (SEQ ID NO: 37). Expression of the exemplary transgenes was measured by PCR eight days after the start of adipocyte differentiation. The Adipo2.BGI promoter drove more robust (approximately 5-8 times more robust) transgene expression than transgene expression from the hAdipo promoter in the exemplary differentiated adipocyte model (FIG. 18).

[0582] To test adipocyte specificity of expression from the Adipo2.BGI promoter, an exemplary transgene (SEAP) was expressed from: (1) a CBA ubiquitous promoter, (2) adipocyte-specific hAdipo promoter (SEQ ID NO: 6), or (3) adipocyte-specific Adipo2.BGI promoter (SEQ ID NO: 37). The various constructs were introduced into 3T3 LI cells prior to differentiation. Cell culture supernatant was collected to determine SEAP detected levels either before (preadipocyte stage) or after (adipocyte stage) differentiation (FIG. 19). Although the CBA ubiquitous promoter drove SEAP expression at both timepoints, the hAdipo and Adipo2.BGI promoters drove expression only after adipocyte differentiation, indicating specificity of both adipocyte-specific promoters for differentiated adipocytes.

[0583] This data demonstrates that the Adipo2.BGI promoter described herein can drive enhanced expression of exemplary transgenes. Expression of transgenes by such promoter is also specific to differentiated adipocytes. Based on the data provided in this example, one of skill in the art would appreciate that further enhancement of adipocyte-specific transgene expression can be anticipated in vivo with delivery of an AAV vector comprising a promoter of interest (e.g., an adipocyte-specific promoter as described herein) and a beta globin intron sequence or a fragment or variant thereof to adipose tissue. INCORPORATION BY REFERENCE

[0584] Each publication, including scientific references to the scientific literature, patent references, and electronic databases, websites and other references accessible through the internet, are hereby incorporated by reference herein, in their entirety, for their disclosure relevant to this specification and as cited herein. EQUIVALENTS

[0585] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to embodiments of invention(s) described herein. The scope of the present invention(s) is not intended to be limited to the above Description, and is set forth in the following claims:

Claims

1. A method of delivering an adeno-associated (AAV) particle to adipose tissue in a subject, wherein the AAV particle comprises an AAV capsid protein and a polynucleotide construct, wherein the subject is a non-rodent mammal.

2. The method of claim 1, wherein delivery comprises administration of the AAV particle subcutaneously.

3. The method of claim 1 or 2, whereina) the AAV particle is administered once or repeatedly; and / orb) the AAV particle is administered at a pre-determined tissue depth in a subject.

4. The method of any one of claims 1-3, wherein the AAV particle is administered:a) between the dermis and Scapa’s fascia; and / orb) between Scapa's fascia and Camper's fascia.

5. The method of claim any one of claims 1-4, wherein the AAV particle is administered to superficial subcutaneous adipose tissue or to deep subcutaneous adipose tissue.

6. The method of claim 1 or 3, wherein the AAV particle is administered to visceral adipose tissue.

7. The method of any one of claims 1-5, wherein the AAV particle is administered at a predetermined depth from the exterior surface of the skin, wherein the depth is between at least about 2mm to about 6mm.

8. The method of any one of the preceding claims, wherein the adipose tissue is or comprises brown adipose tissue, beige adipose tissue, white adipose tissue, pink adipose tissue, or any combination thereof.

9. The method of claim 8, wherein:a) the brown adipose tissue:i) is located near or at the following locations in the subject: neck, kidney, adrenal glands, heart (e.g., aorta), mediastinum, shoulder, neck, and / or back, and / orii) is characterized as metabolizing fat to produce heat and / or has a role in energy metabolism; and / orb) the beige adipose tissue is located near or at the following locations in the subject: abdomen, face, glute, and / or femur; and / orc) the white adipose tissue:i) is located near or at the following locations in the subject: bone marrow, abdomen, and / or organs,ii) is present in a subject near or next to an organ in the form of a visceral depot,iii) comprises subcutaneous white adipose and / or visceral white adipose tissue, and / oriv) is characterized as storing fat; and / ord) the pink adipose tissue is located near or at the following locations in the subject: abdomen, breast, bone marrow, and / or dermis.

10. The method of any one of the preceding claims, wherein the adipose tissue comprises adipocytes.

11. The method of any one of the preceding claims, wherein the AAV particle:a) does not substantially target (e.g., does not target) non-adipose tissue, non-adipocytes, and / or adipose tissue (e.g., adipose tissue at a different location) which was not directly administered the AAV particle; and / orb) does not substantially transduce (e.g., does not transduce) non-adipose tissue, nonadipocytes, and / or adipose tissue (e.g., adipose tissue at a different location) which was not directly administered the AAV particle.

12. The method of any one of claims 1-11, wherein:a) expression of a payload encoded by the polynucleotide construct is substantially not detectable (e.g., not detectable) in non-adipose tissue (e.g., liver, skin, muscle) non-adipocytes, and / or adipose tissue (e.g., adipose tissue at a different location) which was not directly administered the AAV particle; and / orb) the AAV particle is substantially not detected (e.g., not detectable) ini) non-adipose tissue (e.g., liver, skin, muscle), non-adipocytes, and / or adipose tissue (e.g., adipose tissue at a different location) which was not directly administered the AAV particle; and / orii) a non-adipose tissue sample from the subject or adipose tissue sample (e.g., adipose tissue sample at a different location) which was not directly administered the AAV particle.

13. The method of any one of the preceding claims, whereina) administration of the AAV particle to adipose tissue results in local expression and / or systemic distribution of a pay load encoded by the polynucleotide construct; and / orb) a payload encoded by the polynucleotide construct is expressed and / or distributed at a therapeutically effective level.

14. The method of claim 13, wherein systemic distribution of the pay load is dose dependent, and wherein the payload acts systemically or locally, e.g., at or near the site of administration.

15. The method of claim 13 or 14, wherein the payload is a secreted polypeptide.

16. The method of any one of claims 13-15, wherein detection of the payload occurs in a biological sample from a subject, wherein the biological sample is or comprises cells, tissue, and / or bodily fluid.

17. The method of any one of the preceding claims, wherein the AAV particle is administered at a plurality of sites:a) in the subject; and / orb) in a first section of adipose tissue, wherein the first section of adipose tissue comprises adipose tissue in a pre-selected location in a subject’s body.

18. The method of claim 17, wherein the AAV particle is administered at between 1 and 100 different sites in the first section of adipose tissue.

19. The method of claim 17 or 18, wherein the plurality of sites area) non-overlapping; and / orb) at a pre-specified distance from one another, wherein the pre-specified distance is at least about 0.1 cm.

20. The method of any one of claims 17-19, wherein the AAV particle is administered: a) at another (e.g., a second) section of adipose tissue in the subject’s body, wherein the another (e.g., a second) section of adipose tissue is at a different location from the first section of adipose tissue; and / orb) at a plurality of sites in the another (e.g., a second) section of adipose tissue.

21. The method of any one of claims 17-20, wherein a first or subsequent (e.g., a second) section of adipose tissue is selected based on one or more characteristics of adipocytes in the first or subsequent section.

22. The method of any one of the preceding claims, wherein the AAV particle is administered at a volume of at least lOuL.

23. The method of any one of the preceding claims, wherein the method further comprises administering one or more doses of the AAV particle, optionally wherein the AAV particle is administered at a dose of at least 1 x 10(11) vector genomes (vg) or at a concentration of at least 1 x 10(12) vg / mL.

24. The method of any one of the preceding claims, wherein the polynucleotide construct comprises one or more coding sequences, optionally wherein the one or more coding sequences encodes a payload, wherein the payload is or comprises:a) a polypeptide; orb) a polyribonucleotide, wherein the polyribonucleotide is or comprises a messenger RNA, an inhibitory RNA, a non-coding RNA, and / or a transgene.

25. The method of claim 24, wherein the payload is effective in treating, preventing and / or reducing the frequency and / or severity of one or more symptoms of a disease and / or disorder, optionally wherein the payload is or comprises a therapeutic payload.

26. The method of claim 25, wherein the therapeutic payload comprises: GLA, LPL, FVIII, FIX, GLP-1, GIP, ADIPOQ, FGF19, FGF21, PPY, BMP7, LEP, Adalimumab, Etanercept, Pembrolizumab, Ustekinumab, Dupilumab, Nivolumab, Daratumumab, Risankizumab, Secukinumab, Pertuzumab, Emicizumab, Denosumab, Abatocept, Atezolizumab, Durvalumab, Guselkumab, Tocilizumab, Ixekizumab, Infliximab, Eculizumab, Ravulizumab, Insulin, or a fragment or variant of any of the foregoing, or any combination thereof.

27. The method of any one of the preceding claims, wherein the method further comprises removal of transduced adipose tissue and / or adipocytes from the subject, optionally wherein the removal:a) comprises physical excision, biopsy, cryolypolysis, surgical removal, liposuction, laser mediated removal, radioablation, imaging-based ablation, or any combination thereof; and / orb) results in a reduction in local expression and / or activity of the delivered pay load.

28. The method of claim 27, wherein:(i) all or substantially all of the transduced adipose tissue and / or adipocytes is removed;(ii) removal results in a reduction in local expression and / or distribution of the delivered payload, and / or a reduction in systemic distribution of the delivered payload.

29. The method of any one of the preceding claims, wherein the mammal is a pig, cow, dog, cat, non-human primate, or human.

30. The method of any one of the preceding claims, wherein the AAV particle comprises a regulatory element,optionally wherein the regulatory element is or comprises one or more promoters or a fragment or variant thereof, one or more enhancers or a fragment or variant thereof, one or more silencers or a fragment or variant thereof, one or more insulators or a fragment or variant thereof, or any combination thereof.

31. The method of claim 30, wherein:a) the regulatory element is or is derived from a gene expressed in adipocytes; and / orb) the regulatory element comprises:i) mFabp4, mAdipoq, hADIPOQ, or fragments, or variants, or combinations thereof; orii) a ubiquitous promoter and / or a ubiquitous enhancer.

32. The method of claim 30 or31, wherein the regulatory element reduces (e.g., prevents) expression of a payload ina) non-adipocytes; and / orb) adipocytes which are not present at a site of administration when the AAV particle is administered.

33. The method of any one of the preceding claims, wherein the AAV capsid protein is or comprises an AAV1 capsid protein, an AAV2 capsid protein, an AAV3B capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 (AAVrhlO) capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV-PHP.B capsid protein, an AAV8-PHP.eB capsid protein, an AAV-PHP.S capsid protein, an AAV-7m8 capsid protein, an AAV-DJ capsid protein, an AAV-OligOOl capsid protein or a Rec2 capsid protein, or a variant or fragment of any of the foregoing.

34. A method for controlling and / or reversing a gene therapy, the method comprising:(1) delivering a first dose of a gene therapy comprising an adeno-associated (AAV) particle to adipose tissue in a non-rodent mammal, wherein the AAV particle comprises an AAV capsid and a polynucleotide construct encoding a payload; and(2) reducing or eliminating expression of the pay load in the adipose tissue by removing at least a portion of the adipose tissue transduced by the AAV particle.

35. The method of claim 34, whereina) at least 10% of the transduced adipose tissue is removed, and optionally wherein removing the transduced adipose tissue reduces local expression and / or systemic expression of the payload; and / orb) all or substantially all of the transduced adipose tissue is removed, wherein removing all or substantially all of the transduced adipose tissue reduces local expression and / or systemic expression of the payload to an undetectable level.

36. The method of claims 34 or 35, wherein the gene therapy is adjusted by modulating the amount of transduced adipose tissue that is removed.

37. The method of any one of claims 34-36, wherein the method further comprises administering one or more subsequent doses of a gene therapy comprising an adeno-associated (AAV) particle to the adipose tissue, wherein the one or more subsequent doses of gene therapy is administered:a) to the same or nearby adipose tissue to which the first dose of the gene therapy is administered or to a different adipose tissue as compared to the adipose tissue to which the first dose of the gene therapy is administered; andb) at substantially the same volume as the first dose of the gene therapy or at a different volume as compared to the first dose of the gene therapy.

38. The method of any one of the preceding claims, wherein the AAV particle is characterized in that when administered to adipose tissue the AAV particle:a) is contained in adipose tissue as compared to an otherwise similar AAV particle that is not delivered by the method of claim 1; and / orb) transduces at least 5% more adipocytes in the adipose tissue that is administered the AAV particle as compared to adipocyte transduction by an otherwise similar AAV particle that is not delivered by the method of claim 1.

39. An AAV particle comprising an AAV capsid protein and a polynucleotide construct.

40. A composition comprising an AAV particle of claim 39, optionally wherein the composition is a pharmaceutical composition.

41. A pharmaceutical composition comprising an AAV particle of claim 39.

42. The pharmaceutical composition of claim 41, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers and / or excipients, optionally wherein the composition is formulated for delivery to adipose tissue and is suitable for delivery to adipose tissue via subcutaneous injection.

43. The AAV particle of claim 39, the composition of claim 40, or the pharmaceutical composition of claim 41 or 42, for use in delivering the AAV particle, composition or pharmaceutical composition to a subject.

44. The AAV particle for use, composition for use or pharmaceutical composition for use of claim 43, wherein delivering comprises administering the composition to the subject.

45. Use of the AAV particle of claim 39, use of the composition of claim 40, or use of the pharmaceutical composition of claim 41 or 42, for the manufacture of a medicament for delivering the AAV particle to a subject.

46. The use of claim 45, wherein the use comprises administering the AAV particle, composition or pharmaceutical composition to the subject.

47. The AAV particle for use, composition for use or pharmaceutical composition for use of claim 43 or 44, wherein the use comprises treating, preventing and / or reducing the severity and / or frequency of one or more symptoms of a disease or disorder.

48. A method of treating, preventing and / or reducing the severity and / or frequency of one or more symptoms of a disease or disorder in a subject, comprising administering to a subject the AAV particle of claim 39, the composition of claim 40, or the pharmaceutical composition of claim 41 or 42.

49. Use of a gene therapy comprising an adeno-associated (AAV) particle for the manufacture of a medicament for use in controlling and / or reversing a gene therapy, the use comprising:(1) delivering a first dose of a gene therapy comprising the AAV particle to adipose tissue in a non-rodent mammal, wherein the AAV particle comprises an AAV capsid and a polynucleotide construct encoding a payload; and(2) reducing or eliminating expression and / or distribution of the pay load in the adipose tissue (e.g., locally) by removing at least a portion of the adipose tissue transduced by the AAV particle; and / or(3) reducing or eliminating distribution of the payload systemically by removing at least a portion of the adipose tissue transduced by the AAV particle.

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