Construction, Preparation, and Quantification of AAV5 Vectors Carrying the Ex-4 Minigene
Recombinant AAV particles were produced using a four-plasmid procedure as previously described in Di Pasquale G et al
. The AAV5 vector cassette was designed to contain the cytomegalovirus (CMV) promoter, the mouse nerve growth factor (NGF) signal peptide, which has been shown to mediate secretory expression of polypeptides in vitro and in vivo 
and the minigene coding for Ex-4. Briefly, semiconfluent human embryonic kidney 293T cells obtained from the American Type Culture Collection (ATCC, Manassas, VA) were transfected by calcium phosphate with four plasmids: an adenovirus helper plasmid (pAd12) containing VA RNA and coding for the E2 and E4 proteins; two AAV helper plasmids containing either the AAV2rep or the AAV5 capsid gene and a vector plasmid including the AAV inverted terminal repeats flanking the Ex-4 expression cassette. The cells were harvested 48 hours post-transfection and a crude viral lysate was obtained after three freeze-thaw cycles. The clarified lysate (obtained by further low-speed centrifugation) was treated with 0.5% deoxycolic acid (DOC) and 100 U/ml DNase (Benzonase) for 30 minutes at 37°C. After purification of viruses by CsCl gradient ultracentrifugation, vector titer was determined by quantitative real-time PCR (qPCR) (Applied Biosystems, Foster City, CA). Immediately before experiments, vector doses were dialyzed against 0.9% NaCl.
Cell Cultures and in vitro Transfection
The generated recombinant AAV5 virus encoding the Ex-4 minigene was then tested in vitro. 293T human renal epithelial cells were grown at 37°C under a 5% CO2 humidified atmosphere in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U of penicillin/ml, and 0.1 mg of streptomycin/ml. 293T cells were transfected with the AAV5 Ex-4 vector at 103 DNase Resistant Particles (DRP)/ml per cell. Ex-4 levels into the culture medium was assayed using a specific enzyme immunoassay kit (Phoenix Europe GmbH, Germany).
For in vitro
bioactivity assay, supernatant medium from 293T cells transduced with AAV5 Ex-4 (after 96 hours of incubation) was tested on Chinese hamster ovary cell line obtained from American Type Culture Collection (ATCC, Manassas, VA) stably transfected with rat GLP-1 receptor (CHO-GLP1R) accordingly to previously reported study from Egan JM et al
Animal studies were carried out in strict accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) for experimental animal care. The study protocol was approved by the Italian National Health Institute Committee on Animal experiments. All surgeries were performed under anesthesia, and all efforts were made to minimize suffering.
Male 4 week old CD1 mice (n
20) were purchased from Harlan Laboratories (Udine, Italy), housed at five animals per group and fed High Fat Diet ad libitum
(Dottori Piccioni Laboratories Srl, Milan, Italy). The HFD supplied 60% of energy as fat and 20% as carbohydrate. Fatty acid composition was as follows: 42.0% saturated fatty acids (palmitic and stearic acids), 43% monounsaturated fatty acids (oleic acid), and 15% polyunsaturated fatty acid (linoleic acid and linolenic acid). The HFD contained 300 mg cholesterol/kg and its energy density was 21.10 kJ/g. The HFD fed mice are recognized as an efficient and robust animal model for obesity, early prone to impaired glucose tolerance and T2DM development 
Male Zucker fa/fa rats (n
10) purchased from Charles River laboratories (Lecco, Italy) were housed in single cage and received standard chow ad libitum
(Purina Rodents Laboratory Diet). Zucker fa/fa rats are conversely a spontaneous monogenic obesity model, characterized by a missense mutation on the leptin receptor gene 
Submandibulary salivary glands of 8 week old HFD mice (n
10) and of 9 week old Zucker fa/fa rats (n
5) were transduced by single percutaneous injection of 50 µl of AAV5 Ex-4 (5×1012
DRP/ml) vector. Control animals (n
10 HFD mice; n
5 Zucker fa/fa rats) received 50 µl of AAV-5 vector devoid of Ex-4 transgene (empty vector). On a weekly basis weight, food, water intake, urine volume, glycaemia were monitored throughout the study. In order to evaluate effects of treatment on short term food consumption, a 120 minutes food intake evaluation, after overnight fasting, was also performed in rats. A fixed amount of standard chow was given in individual cages and rodents food intake (evaluated as the difference between the baseline amount and the residual food, including spillage) was measured every 15 minutes.
An intraperitoneal insulin tolerance test (ITT) was performed in HFD mice, 6 weeks following vector administration. Each animal was fasted for 4 hours. Following intraperitoneal insulin (Humulin R Regular, Lilly) injection (1 UI/kg), blood samples from the lateral tail vein were collected to measure glycaemia at 0, 15, 30, 60, 90 and 120 minutes. The glucose areas under the curve (AUC, mM per minute) were calculated according to formula: AUC0–120 min
) x 15/2+(G15
) x 15/2+(G30
) x 30/2+(G60
) x 60/2.
Blood samples were withdrawn at baseline and week 6 in HFD mice in order to detect Ex-4, glycaemia, insulinemia, HbA1c, leptin and adiponectin circulating levels and at 0, 4, and 8 week in Zucker fa/fa rats to detect Ex-4 and glucose values. HbA1c and insulinemia were determined at baseline and 8 weeks after vector administration. Blood samples were obtained through jugular sampling conducted in isoflurane-anesthetized animals. At study end, rats and mice were respectively euthanized by CO2 (80%) inhalation.
SG, liver, spleen, and pancreas tissues were collected for DNA extraction and vector distribution assay.
Ex-4 exhibits 53% structural homology to native GLP-1. Circulating Ex-4 levels were determined by a specific enzyme immunoassay kit (Phoenix Europe GmbH, Germany), unable to detect endogenous GLP-1 values, according to the manufacturers’ instructions. Minimum detectable concentration was 2.6 pmol/L.
In order to assess vector biodistribution at the end point of the study, a DNA isolation kit was used to purify total genomic DNA from SG, liver, spleen and pancreas (Wizard DNA purification kit, Promega Corporation, Madison, WI, USA). Quantitative PCR amplification (20 µl final volume) of genomic DNA (100 ng) was performed with ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) by using the SYBR Green PCR Master Mix and a specific 5′ and 3′ primer pair appropriate (0.3 µM; CMV forward 5′-CATCTACGTATTAGTCATCGCTATTACCAT- 3′, CMV reverse 5′-TGGAAATCCCCGTGAGTCA-3′) for CMV promoter. Amplification and detection were performed with ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). A PCR cycling reaction involved an initial hold at 95° for 10 minutes, followed by cycling conditions of 95°C for 15 seconds, 60°C for 1 min for 40 cycles. The viral DNA in each sample was quantified by comparing the fluorescence amplification profiles with a set of DNA standards using AAV5 vector and 100 ng of genomic DNA of untreated animals for each specific tissue. Each measurement was carried out in duplicate. Data are expressed in copies of AAV5 for 100 ng of genomic DNA.
Salivary Glands Immunohistochemical Assay
At the end of the study, SG were removed from treated (n
5) and control (n
5) HFD mice, fixed in 4% paraformaldehyde for 24 hours at room temperature, cryoprotected in 30% sucrose in phosphate-buffered saline (PBS) for approximately 12 hours at 4°C and then embedded in Killik cryostat embedding medium (Bio-Optica, Milan Italy). Cryosections, 10 µm thick, were collected on polylysine-coated slides.
The slides were pre-incubated in 0.5% Triton (Sigma Aldrich, Milan, Italy) and 1.5% Bovine Serum Albumin (BSA) (Sigma Aldrich, Milan, Italy) in PBS for 15 minutes at room temperature to saturate non specific sites. Then the sections were incubated 24 hours at 4°C with primary antibody against Ex-4 (Phoenix Europe GmbH, Germany) at final dilution of 1
Subsequently, the sections were incubated with an Alexa Fluor 488 secondary Donkey anti Rabbit antibody (Invitrogen, San Diego, CA, USA) at a final dilution of 1
333 for 2 hours at room temperature. The immunoreaction products were observed under an epifluorescence Zeiss Axioskop microscope (Zeiss, Germany) at x40 magnification.
Adipokines Circulating Levels Assay
Serum leptin and adiponectin levels were assayed only in the polygenic model of obesity prone to im paired glucose tolerance and T2DM such as HFD mice using commercially available kit according to manufacturer’s instructions. Sandwich enzyme immunoassay (ELISA) was used for the quantitative measurement of mouse proteins (Biovendor, Heidelberg, Germany and B-Bridge International Inc., CA, USA, for leptin and adiponectin respectively). Intra- and inter-assay coefficient of variation were less than 5%.
Visceral Adipose Tissue Adipokines Profile: RNA Extraction and Real Time PCR Determinations
Total RNA was extracted from 50 mg of mice visceral adipose tissue. Briefly, tissue samples were collected, immediately snap frozen in liquid nitrogen and disrupted by homogenization in QIAzol Lysis Reagent using the TissueLyser (QIAGEN GmbH, Hilden, Germany). RNA was extracted using RNeasy Lipid Tissue Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. One µg of RNA was treated with TURBO DNA-free™ DNase Kit (Ambion, Inc, Austin, TX, USA) and reverse-transcribed into cDNA for 1 h at 37°C in a 50 µl reaction containing 1× RT buffer, 150 ng random hexamers, 0.5 mmol/l dNTPs, 20 units of RNAsin Ribonuclease Inhibitor (Promega Corporation, Madison, WI, USA) and 200 units of M-MLV RT (Promega Corporation, Madison, WI, USA).
Real Time quantitative PCR was carried out on DNA Engine Opticon™ 2 Continuous Fluorescence Detection System (MJ Research, MA, USA), using Platinum® SYBR® Green qPCR SuperMix-UDG (Invitrogen Corporation, CA, USA) and 300 nM specific primers for each gene : 18 s forward 5′-CGG CTA CCA CAT CCA AGG AA-3′, reverse 5′-GCT GGA ATT ACC GCG GCT-3′; leptin forward: 5′-TCC AGA AAG TCC AGG ATG ACA C-3′, reverse: 5′-CAC ATT TTG GGA AGG CAG G-3′; adiponectin forward: 5′-ACA ATG GCA CAC CAG GCC GTG A-3′, reverse: AGC GGC TTC TCC AGG CTC TCC TTT-3′. Each cDNA sample was assayed in duplicate and a no template control was included in every reaction. For each sample, gene expression values were normalized by 18s RNA content and reported as AU ratio.
Blood and Urine Analysis, HOMA-IR Index
Glycaemic values were determined in the morning, after overnight fasting. Blood was obtained via tail vein and tested, using an Accu-Chek Aviva Nano meter (Roche). HbA1c percentage values were measured on 5 µl of whole blood using an A1CNow +
test Kit (Bayer). Insulin levels were determined using a commercially available insulin RIA kit (Linco Research, Inc., St. Charles, MO); the intra-assay coefficient of variation was 1.4%. Insulin resistance was evaluated by the homeostasis model assessment of insulin resistance (HOMA-IR) index calculated as: (fasting glycaemia, mmol/L x fasting serum insulin, pmol/L/135) 
. Urine analysis was performed by a colorimetric method (AUTION Sticks 10TA; Arkray, Inc, Kyoto, Japan) in order to detect glucose levels.
The statistical significance of differences between experimental and control groups was analyzed by Student’s t-test. p<0.05 was considered statistically significant. Values are presented as mean ± standard error (SE).