DOI: 10.5061/dryad.cjsxksnh6
title: Data from: Development of an adipose-tropic AAV capsid ablating liver tropism

description: AAV vectors are mainstream delivery platforms in gene therapy, yet
 AAV-mediated gene transfer to adipose tissue is underdeveloped due to low
 efficiency of natural AAVs. We previously demonstrated that an engineered
 capsid Rec2 displayed improved adipo-tropism but with the caveat of liver
 transduction. To generate highly adipo-tropic capsid, we modified Rec2
 capsid by site-specific mutagenesis and found the variant V7 with F503Y,
 Y708D and K709I substitution to harbor highly selective adipo-tropism
 while diminishing liver transduction. Intraperitoneal injection favored
 transduction to visceral fat while intravenous administration favored
 subcutaneous fat.  Intraperitoneal administration of V7 vector
 harboring human leptin and adiponectin as single transcript normalized the
 metabolic dysfunction of ob/ob mice at a low dose. Moreover, introducing
 the same mutagenesis to AAV8 capsid diminished liver transduction
 suggesting F503, Y708 and K709 critical for liver transduction. The
 Rec2.V7 vector may provide a powerful tool for basic research and potent
 vehicle for adipose-targeting gene therapy.
description: <strong>Rec2 capsid V7 mutagenesis</strong>
 The Rec2 capsid was mutagenized at sites indicated in Figure1A
 and full capsid sequence is listed in Figure 1B. We name Rec2 variants as
 V1 to V8 in numerical order. GeneArt site-directed mutagenesis Plus kit
 (A14604, Invitrogen) was used to carry out the designated mutagenesis.
  Rec2 capsid sequence is based on published data<sup>6</sup>.
 Primers for mutagenesis was synthesized by Sigma-Aldrich: F503Y
 (ACAACAATAGCAACTATGCCTGGACTGCTGG,CCAGCAGTCCAGGCATAGTTGCTATTGTTGT); Y708D
 and K709I (TACACCTCCAACTACGACATATCTACAAGTGTGGA,
 TCCACACTTGTAGATATGTCGTAGTTGGAGGTGTA). Mutagenesis was
 set up and carried out according to the manufacturer’s instruction, and
 the nucleotide replacement at each site was confirmed by sequencing at the
 Core Facility of The Ohio State University, Comprehensive Cancer Center.
 The whole capsid was also sequenced and confirmed without unintended
 mutation. <strong>AAV8 capsid
 mutation</strong> Rec2 capsid was generated from
 capsid domain shuffling between rh20 and AAV8 <sup>6</sup>
 whose C-terminal including those three amino acids mutagenized for V7 is
 identical to AAV8 capsid C-terminal. As such, AAV8 capsid mutagenesis of
 F503Y, Y708D and K709I was conducted as described for V7
 mutagenesis. <strong>AAV vectors
 packaging</strong> The rAAV vector backbone
 contains CBA (hybrid cytomegalovirus–chicken β-actin) promoter, woodchuck
 hepatitis virus posttranscriptional regulatory element (WPRE) and bovine
 growth hormone polyadenylation signal flanked by AAV2-inverted terminal
 repeats. Reporter gene—enhanced green fluorescent protein (referred as
 GFP) or therapeutic gene—human leptin cDNA and human adiponectin cDNA
 linked with a 2A sequence (referred as LEP/ADIPOQ) was cloned into
 polylinker sites of cis-plasmid as described previously
 <sup>7</sup>. LEP/ADIPOQ sequence was synthesized by gBlock of
 IDT. Plasmids used for viral packaging were prepared by using EndoFree
 plasmid Maxi and Mega Kit (Qiagen). Human embryonic kidney 293 cells were
 co-transfected with three plasmids—rAAV plasmid or cis-plasmid, AAV
 trans-plasmid encoding rep and cap genes including Rec2, Rec2.V1 to V8,
 AAV8, and AAV8 mutant, and adenoviral helper pF Δ6—using standard
 CaPO<sub>4</sub> transfection. AAV was purified from the cell
 lysate by ultracentrifugation through an iodixanol density gradient
 (OptiPrep Density Gradient Medium, D1556, Sigma). AAV was tittered using
 quantitative PCR using Step OnePlus Real-Time PCR System (Applied
 Biosystems) with the Power SYBR Green PCR Master Mix (Applied
 Biosystems#A25742) <sup>47</sup>.
 <strong>Administration of AAV vectors
 </strong> AAV vectors were administered into mice
 either through i.p. injection in 150 µL AAV dilution buffer or via tail
 vein injection in 50 µL AAV dilution buffer. Doses ranging from 2E10 to
 8E10 vg/mouse were used and specified in each experiment in figure
 legend. <strong>GFP content measurement
 </strong> GFP content was measured as previously
 described <sup>9</sup>. In brief, tissues were homogenized in
 ice-cold RIPA buffer (25mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1%
 sodium deoxycholate, 0.1% SDS) supplemented with proteinase inhibitors
 cocktail, followed by briefly sonication. The mixture was then spun at
 13,000 rpm for 15 min at 4 °C. The supernatant was collected. GFP
 fluorescence in 150 µL supernatant was measured with microreader (Synergy
 H1, Bio Tek Instrument) using a 485-nm excitation wavelength and cut-off
 528-nm emission wavelength. GFP content was represented as readings after
 subtracting auto-fluorescence from GFP-null corresponding tissue and
 calibrated by either protein content in the samples or wet tissues
 weight.
 <strong>ob</strong><strong>/ob mice
 experiment</strong> Male homozygous
 <em>ob/ob</em> mice (JAX 000632
 B6.Cg-Lep<sup>ob</sup>/J), seven weeks old, were randomly
 assigned to receive V7-GFP or V7-LEP/ADIPOQ. Sex and age-matched
 heterozygous <em>ob/+</em> mice received AAV buffer as another
 control. Body weight and food intake were monitored weekly over the period
 of 9 weeks. One mouse from V7-LEP/ADIPOQ 4E10 group and one mouse from
 V7-LEP/ADIPOQ 8E10 group were excluded due to technical errors of
 administration. <strong>Glucose tolerance test
 (GTT)</strong> Eight weeks after i.p.
 administration of AAV vectors, mice were i.p. injected with glucose
 solution (1mg glucose per gram body weight) after overnight fast with
 water <em>ad libitum</em>. Blood was collected from the tail
 at various time points and the blood glucose concentrations were measured
 with a portable glucose meter (Bayer Contour Next).
 <strong>Rectal temperature measurement
 </strong> Temperature probe (Physitemp, model
 BAT-12) topped with lubricant (white petroleum jelly) was used to measure
 the rectal temperature of <em>ob</em>/<em>ob</em>
 mice at 8-weeks after i.p. administration of AAV vectors.
 <strong>Body composition by
 EchoMRI</strong> EchoMRI was used to measure body
 composition of fat and lean masses in live mice without anesthesia at
 9-weeks post AAV injection. EchoMRI imaging was performed with a 3-in-1
 Analyzer (EchoMRI LLC, Houston, TX) according to manufacturer instructions
 at Small Animal Imaging Core of The Dorothy M. Davis Heart & Lung
 Research Institute, Ohio State University. Mice were subjected to a
 5-Gauss magnetic field and whole-body masses of fat, lean, free water, and
 total water were determined during separate cycles by manufacturer
 software comparison to a canola oil standard.
 <strong>Serum harvest and analysis</strong>
 Trunk blood was collected at euthanasia, clotted on ice, and
 centrifuged at 12,000 rpm for 15 min at 4°C. The serum was collected and
 stored at -20°C until further analysis. R&D Systems DuoSet ELISA
 kits were used to assay serum human leptin (R&D Systems, #DY398)
 and human adiponectin/Acrp30 (R&D Systems, # DY1065). Mouse serum
 insulin was measured with ELISA kit from ALPCO (ALPCO #80-INSMSU-E01). A
 Cayman Chemical colorimetric assay was used to determine serum glucose
 levels (#10009582). <strong>Liver lipid
 extraction and triglycerides assay</strong>
 Hepatic lipid was extracted from 50 mg of liver tissue from each
 animal by chloroform/methanol (2:1 v/v), followed by rinse in 50 mM NaCl
 and CaCl<sub>2</sub> (0.36M)/Methanol (1:1 v/v). An aliquot of
 the extract was mixed with Triton X100 and cold acetone, then was dried up
 and redissolved in PBS. Hepatic triglyceride quantification was performed
 with a Caymen Chemical triglyceride assay kit (Caymen Chemical,
 #10010303).
 <strong>Immunoblotting</strong>
 Tissues were homogenized in ice-cold Pierce RIPA buffer
 containing 1x Roche Phosstop and Calbiochem protease inhibitor cocktail
 III, then was subjected to brief sonication on ice. Tissues lysates were
 spun at 13,000 rpm for 15 min at 4 °C. The supernatant was collected, and
 the protein concentration was assayed with BCA protein assay kit (Piece). 
 20 µg of protein from each sample was loaded and separated by gradient gel
 (4-20%, Mini-PROTEAN TGX, Bio-Rad), then transferred to a nitrocellulose
 membrane (BIO-RAD). Blots were incubated overnight at 4 °C with the
 following primary antibodies, b-actin (Cell Signaling #4970, 1:1000), GFP
 (Santa Cruz Biotechnology, sc-9986 HRP, 1:1000). Chemiluminescence signal
 was detected and visualized by LI-COR Odyssey Fc imaging system (LI-COR
 Biotechnology, Lincoln, NE). Quantification analysis was carried out with
 image studio software version 5.2 (LI-COR Biotechnology).
 <strong>Quantitative RT-PCR</strong>
 Total RNA was isolated using the RNeasy Mini kit (QIAGEN) with
 RNase-free DNase treatment. cDNA was reverse transcribed using Taqman
 Reverse Transcription Reagents (Applied Biosystems). qPCR was carried out
 on StepOnePlus Real-Time PCR System using Power SYBR Green PCR Master Mix
 (Applied Biosystems). Primers were designed to detect GFP
 (GACTTCTTCAAGTCCGCCAT, TGTGGCTGTTGTAGTTGTACTC), ADIPOQ
 (AGGCAGGAAAGGAGAACCT, GTGGAGCCATCATAGTGGT), <em>Gapdh</em>
 (TCCCACTCTTCCACCTTCGA, TGCTGTAGCCGTATTCATTGTCA), and <em>Actinb
 </em>(ACCCGCGAGCACAGCTT, ATATCGTCATCCATGGCGAACT). Data were
 calibrated to <em>Actinb</em> and quantified using the 2
 <sup>-</sup><sup>DD</sup><sup>CT</sup>
 method. <strong>Viral vector copy number
 measurement</strong> To assess vector
 biodistribution, viral vector copy number was measured as previously
 described. Briefly, total DNA from various tissues was isolated using
 DNesay Blood and Tissue kit (Qiagen). GFP fragment was amplified and
 quantified by qPCR to determine the copy number of viral particles. 50 ng
 of DNA from each sample was used for real-time PCR with either duplicate
 or triplicate. Mouse nucleic genomic DNA fragment of the GAPDH gene was
 used as a control for mouse genetic DNA. Genomic DNA of untreated animals
 was also used as negative control for the comparison. Data are expressed
 as copies of V7-GFP/50 ng of genomic DNA.
 <strong>Quantification and Statistical
 analysis</strong> Data are expressed as means ±
 SEM. GraphPad Prism 9 was used to analyze data. One-way ANOVAs with
 Tukey’s <em>post hoc</em> test were utilized for comparisons
 between three or four groups. Time course data (BWs, GTT) were analyzed
 using a mixed ANOVA and area under the curve calculations were performed
 where applicable. Normality was tested using the Shapiro-Wilk method. *
 Indicates p value &lt; 0.05; ** indicates p value &lt; 0.01 in
 figures.
description: # Data from: Development of an adipose-tropic AAV capsid ablating liver
 tropism
 [https://doi.org/10.5061/dryad.cjsxksnh6](https://doi.org/10.5061/dryad.cjsxksnh6) ## Description of the data and file structure Raw data for the paper entitled "Development of an adipose-tropic AAV capsid ablating liver tropism" AAV vectors targeting adipose tissue are still underdeveloped. The F503Y/Y708D/K709I substitution in AAV8 and Rec2 capsids eliminates liver tropism, while the Rec2 variant V7 with this substitution is highly adipo-tropic. V7 vector-based gene therapy effectively rescues metabolic defects in ob/ob mice at a low dose. Tissue types: epididymal (male) or gonadal (female) white adipose tissue (eWAT or gWAT), mesentery white adipose tissue (mWAT), retroperitoneal white adipose tissue (rWAT), inguinal white adipose tissue (iWAT) and intrascapular brown adipose tissue (BAT). ### Files and variables #### File: V7\_Fig2\_Data.csv **Description:** raw data for Fig 2 ##### Variables * Animal ID * AAV vectors and dose * Tissue type (liver, inguinal white adipose tissue (iWAT),  * GFP fluorescence count * viral genome copy number #### File: V7\_Fig5\_Data.csv **Description:** raw data for Fig 5 ##### Variables * Animal ID * genotype * Treatment including AAV vectors and doses * Rectal temperature * Relative fat mass * Relative lean mass * Relative tissue weight (% BW) #### File: V7\_Fig3C\_Data.csv **Description:** raw data for Fig 3C ##### Variables * Animal ID * AAV vectors and doses * Tissue type * Viral genome copy number #### File: V7\_Fig6\_Data.csv **Description:** raw data for Fig 6 ##### Variables * Animal ID * Genotype * Treatment including AAV vectors and doses * Serum human leptin level * Serum human adiponectin level * Serum insulin level * Serum glucose level * Liver triglyceride level #### File: V7\_Fig7C\_Data.csv **Description:** raw data for Fig 7C ##### Variables * Animal ID * AAV vectors * Tissue type * GFP fluorescence count #### File: V7\_Fig4\_Data.csv **Description:** raw data for Fig 4 ##### Variables * Animal ID * Genotype * Treatment including AAV vectors and doses * Body weight * Body weight gain * Blood glucose levels during a glucose tolerance test