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• W2065821427 abstract "Adipose tissue plays an essential role in metabolic homeostasis and holds promise as an alternative depot organ in gene therapy. However, efficient methods of gene transfer into adipose tissue in vivo have yet to be established. Here, we assessed the transduction efficiency to fat depots by a family of novel engineered hybrid capsid serotypes (Rec1∼4) recombinant adeno-associated viral (AAV) vectors in comparison with natural serotypes AAV1, AAV8, and AAV9. Rec2 serotype led to widespread transduction in both brown fat and white fat with the highest efficiency among the seven serotypes tested. As a proof-of-efficacy, Rec2 serotype was used to deliver Cre recombinase to adipose tissues of insulin receptor floxed animals. Insulin receptor knockdown led to decreased fat pad mass and morphological and molecular changes in the targeted depot. These novel hybrid AAV vectors can serve as powerful tools to genetically manipulate adipose tissue and provide valuable vehicles to gene therapy targeting adipose tissue. Adipose tissue plays an essential role in metabolic homeostasis and holds promise as an alternative depot organ in gene therapy. However, efficient methods of gene transfer into adipose tissue in vivo have yet to be established. Here, we assessed the transduction efficiency to fat depots by a family of novel engineered hybrid capsid serotypes (Rec1∼4) recombinant adeno-associated viral (AAV) vectors in comparison with natural serotypes AAV1, AAV8, and AAV9. Rec2 serotype led to widespread transduction in both brown fat and white fat with the highest efficiency among the seven serotypes tested. As a proof-of-efficacy, Rec2 serotype was used to deliver Cre recombinase to adipose tissues of insulin receptor floxed animals. Insulin receptor knockdown led to decreased fat pad mass and morphological and molecular changes in the targeted depot. These novel hybrid AAV vectors can serve as powerful tools to genetically manipulate adipose tissue and provide valuable vehicles to gene therapy targeting adipose tissue. Adipose tissue has been recognized as a multifunctional organ that plays important roles in modulating metabolic, endocrine, and immunological processes and disease pathology.1Spiegelman BM Enerbäck S “The adipocyte: a multifunctional cell”.Cell Metab. 2006; 4: 425-427Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar Approaches to genetically manipulate adipose tissue are essential to mechanistic studies of adipose function. Up to date, genetic manipulation of adipose tissue is primarily relied on transgenic mice. However, transgenic technology can be costly, time consuming, difficult to distinguish development effects from maintenance effects, and unable to apply to species other than mouse. Viral vectors can serve as an attractive alternative. Targeting adipose tissues or adipocyte in culture with adenovirus, retrovirus, and lentivirus have been investigated with various degrees of efficiency.2Nagamatsu S Nakamichi Y Ohara-Imaizumi M Ozawa S Katahira H Watanabe T et al.Adenovirus-mediated preproinsulin gene transfer into adipose tissues ameliorates hyperglycemia in obese diabetic KKA(y) mice.FEBS Lett. 2001; 509: 106-110Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar,3Morizono K De Ugarte DA Zhu M Zuk P Elbarbary A Ashjian P et al.Multilineage cells from adipose tissue as gene delivery vehicles.Hum Gene Ther. 2003; 14: 59-66Crossref PubMed Scopus (137) Google Scholar The limitations of these viral vectors include adverse immunogenic effects, or tropism to dividing cells. In contrast, adeno-associated viral (AAV) vectors have been extensively used for gene delivery for experimental and therapeutic purposes in a range of animal models and clinical trials.4Mingozzi F High KA Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges.Nat Rev Genet. 2011; 12: 341-355Crossref PubMed Scopus (708) Google Scholar,5Asokan A Schaffer DV Samulski RJ The AAV vector toolkit: poised at the clinical crossroads.Mol Ther. 2012; 20: 699-708Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar AAVs are nonpathogenic and low immunogenic and can transduce both dividing and postmitotic tissues and generally sustain long-term transgene expression in animal models and humans. In animal models, multiple organs have been successfully and safely transduced by recombinant AAV (rAAV) vectors including liver, heart, skeletal muscle, brain, and eye.4Mingozzi F High KA Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges.Nat Rev Genet. 2011; 12: 341-355Crossref PubMed Scopus (708) Google Scholar However, AAV-mediated gene delivery to adipose tissue has not been extensively investigated. A few recent reports show that AAV1 vector is more efficient than AAV2, 3, 4, and 5. However, the feasible transduction requires excipients such as Pluronic F88 (ref. 6Mizukami H Mimuro J Ogura T Okada T Urabe M Kume A et al.Adipose tissue as a novel target for in vivo gene transfer by adeno-associated viral vectors.Hum Gene Ther. 2006; 17: 921-928Crossref PubMed Scopus (26) Google Scholar) or celastrol.7Zhang FL Jia SQ Zheng SP Ding W Celastrol enhances AAV1-mediated gene expression in mice adipose tissues.Gene Ther. 2011; 18: 128-134Crossref PubMed Scopus (28) Google Scholar In this study, we evaluated the transduction efficiency of a family of newly engineered AAV serotypes.8Charbel Issa P De Silva SR Lipinski DM Singh MS Mouravlev A You Q et al.Assessment of tropism and effectiveness of new primate-derived hybrid recombinant AAV serotypes in the mouse and primate retina.PLoS ONE. 2013; 8: e60361Crossref PubMed Scopus (37) Google Scholar Three newly identified nonhuman primate serotypes: cy5 (cynomolgus macague-variant 5), rh20 (rhesus macaque-variant 20), and rh39 (rhesus macaque-variant 20) have shown greater transduction efficiency than rAAV8 in the brain.9Gao G Vandenberghe LH Alvira MR Lu Y Calcedo R Zhou X et al.Clades of adeno-associated viruses are widely disseminated in human tissues.J Virol. 2004; 78: 6381-6388Crossref PubMed Scopus (775) Google Scholar,10Lawlor PA Bland RJ Mouravlev A Young D During MJ Efficient gene delivery and selective transduction of glial cells in the mammalian brain by AAV serotypes isolated from nonhuman primates.Mol Ther. 2009; 17: 1692-1702Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar To improve transduction efficiency and select desirable tropism, novel hybrid AAV capsid serotypes: Rec1, 2, 3, and 4 were generated by shuffling the fragments of capsid sequences that matched in all three nonhuman primate AAV seroptyes cy5, rh20, and rh39 and AAV8 (ref. 8Charbel Issa P De Silva SR Lipinski DM Singh MS Mouravlev A You Q et al.Assessment of tropism and effectiveness of new primate-derived hybrid recombinant AAV serotypes in the mouse and primate retina.PLoS ONE. 2013; 8: e60361Crossref PubMed Scopus (37) Google Scholar). Rec2 serotype vector showed high transduction efficiency in both brown adipose tissue (BAT) and white adipose tissue (WAT) superior to all the natural serotypes tested including AAV1, AAV8, and AAV9. A proof-of-principle of this improved technology was demonstrated by the Rec2-mediated Cre-loxP knockdown of insulin receptor (IR) in adult mice. To evaluate the efficacy of multiple serotypes of AAV vectors targeting WAT in vivo, we generated pseudotyping vectors in which a transgene cassette flanked by AAV2-inverted repeats (ITR) was packaged into the capsid from another AAV serotype including three natural serotypes: AAV1, AAV8, and AAV9 as well as the four novel serotypes generated by capsid shuffling strategy: Rec1, 2, 3, and 4. Green fluorescent protein (GFP) controlled by a ubiquitous CBA promoter (hybrid cytomegalovirus–chicken β-actin) was used as the reporter. A single dose of AAV vectors (1 × 1010 vector genomes (vg)per injection) was injected to the inguinal WAT (IWAT) of 6-week-old C57BL/6 mice. Four weeks after rAAV injection, GFP expression was examined by immunohistochemistry and western blot. AAV1 and AAV9 led to minimal GFP expression (Figures 1a and 2a). All of the novel engineered serotypes resulted in marked transgene expression (Figures 1a and 2a). In particular, Rec2 showed widespread GFP expression, and transgene expression was more than twofolds higher than AAV8 (Figures 1a and 2a,b). Mizukami et al. reported the in vivo transduction efficiency of AAV serotypes 1, 2, 3, 4, and 5. The highest transduction in IWAT was AAV1 vector in combination with Pluronic F88.6Mizukami H Mimuro J Ogura T Okada T Urabe M Kume A et al.Adipose tissue as a novel target for in vivo gene transfer by adeno-associated viral vectors.Hum Gene Ther. 2006; 17: 921-928Crossref PubMed Scopus (26) Google Scholar The dose in this study was threefold lower than that used in the study by Mizukami et al., and the transduction efficiency was far more superior to the rAAV1 plus Pluronic F88.6Mizukami H Mimuro J Ogura T Okada T Urabe M Kume A et al.Adipose tissue as a novel target for in vivo gene transfer by adeno-associated viral vectors.Hum Gene Ther. 2006; 17: 921-928Crossref PubMed Scopus (26) Google Scholar No transgene expression was found in nearby muscle, kidney (Figure 1b), or brain (data not shown) of Rec2-injected mice. Scarce GFP-positive cells were observed in the liver (Figure 1b) of Rec2-injected mice, while high level of GFP fluorescence was observed in liver of mice injected with AAV1, AAV8, and AAV9 (data not shown) probably because the high infection efficiency of Rec2 to the IWAT prevented the Rec2 vectors to enter the circulation.Figure 2Quantification of transgene expression mediated by seven AAV serotypes. (a) Representative western blot of IWAT. (b) Quantification of western blot. Values are calibrated to GAPDH and expressed as mean ± SEM (n = 6 per group). Bars not connected by same letter are significantly different. (c) GFP fluorescence 10 days after Rec2-GFP injection to retroperitoneal WAT. Image was taken from 90 µm cryosection. Scale bar = 100 µm. AAV, adeno-associated virus; GAPDH, glyceraldehyde-3-phosphate dehydrogenase, GFP, green fluorescent protein; IWAT, inguinal WAT; WAT, white adipose tissue.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To evaluate the transduction efficiency of Rec2 vector to visceral fat, a single dose of Rec2-GFP (1 × 1010 vg per injection) was injected to the retroperitoneal WAT. GFP fluorescence was observed 10 days after Rec2 vector injection (Figure 2c). The transduction efficacy of AAV vectors to BAT has not been examined. We assessed the gene transfer efficiency to BAT by the three natural serotypes and the four novel engineered serotypes. Preliminary data showed that Rec2 transduced BAT more efficiently than IWAT. Therefore, lower dose (1 × 109 vg per injection, bilaterally) was used in the comparison study of the seven serotypes. Similar to IWAT, AAV1 and AAV9 showed the least transgene expression (Figure 3b,c). Rec2 led to the highest transgene expression, at least twofolds of that of AAV8 (Figure 3a–c). Rec2-mediated GFP expression was widespread and more evenly distributed than that was in IWAT possibly due to bilateral injection as well as better tropism to BAT. The dose was three orders lower than that often used for systemic delivery. The low dose of Rec2 (2 × 109 vg per fat pad) was even lower than the dose we often used to transduce hypothalamus via stereotactic surgery (1 × 1010 vg per injection).11Cao L Lin EJ Cahill MC Wang C Liu X During MJ Molecular therapy of obesity and diabetes by a physiological autoregulatory approach.Nat Med. 2009; 15: 447-454Crossref PubMed Scopus (93) Google Scholar,12Cao L Liu X Lin EJ Wang C Choi EY Riban V et al.Environmental and genetic activation of a brain-adipocyte BDNF/leptin axis causes cancer remission and inhibition.Cell. 2010; 142: 52-64Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar Given the much larger size of BAT compared with hypothalamus, Rec2 showed strikingly high transduction efficiency to BAT. Rec2 vector showed the highest transgene expression in both BAT and IWAT among the seven serotypes tested. In addition, Rec2 generally packages higher titer than Rec1, 3, or 4. Therefore, we chose Rec2 to demonstrate the functional efficacy of the AAV-mediated adipose gene transfer. Pseudotype Rec2 vector harboring Cre under the control of CBA promoter was injected to both IWAT pads of 6-week-old female IRlox mice (1 × 1010 vg per injection). High-level GFP expression in AAV vector–injected adipose tissue was associated with inflammation and fibrosis (data not shown). The toxicity was likely due to the transgene instead of vector itself since neither inflammation nor fibrosis was observed in a Rec2 vector harboring expression cassette without a transgene. The Rec2-no transgene vector was used as control in Cre-loxP knockdown study. Rec2-Cre injection had no significant effects on body weight (Figure 4c) while slightly decreased food intake (control: 3.36 ± 0.39 g/day; Cre: 3.02 ± 0.09 g/day; P = 0.041) during the 8 weeks of experiment. No significant changes were observed in insulin tolerance test at fed condition (Figure 4d) or glucose tolerance test at fast (Figure 4e) 4 and 5 weeks after Rec2-Cre injection, respectively. By 8 weeks after Rec2-Cre injection, no significant effects on serum biomarkers were found including insulin, leptin, glucose, cholesterol, and triglyceride (Figure 4f). However, Rec2-Cre decreased IWAT mass by more than 50% (Figure 4a–c), while had no effects on BAT or visceral WAT. Western blot showed that Rec2-Cre knocked down IR protein level by 53% (Figure 4g,h) consistent to the knockdown at mRNA level measured by quantitative RT-PCR (Figure 5b). No changes of IR expression were found in liver, skeletal muscle, or visceral WAT (data not shown). Adipocytes in Rec2-Cre–injected IWAT appeared smaller than that in control (Figure 5a). No increases of apoptosis or cell death were observed in TUNEL (TdT-mediated dUTP-biotin nick end labeling) assay or active caspase-3 immunohistochemistry (data not shown). Gene expression profiling revealed extensive molecular changes in Rec2-Cre–injected IWAT (Figure 5b). Adipokines adiponectin, leptin, and resistin expression were all suppressed. Adipogenic markers Ap2 and Pparg as well as WAT marker Hoxc9 were also decreased significantly.13Kajimura S Seale P Tomaru T Erdjument-Bromage H Cooper MP Ruas JL et al.Regulation of the brown and white fat gene programs through a PRDM16/CtBP transcriptional complex.Genes Dev. 2008; 22: 1397-1409Crossref PubMed Scopus (343) Google Scholar,14Petrovic N Walden TB Shabalina IG Timmons JA Cannon B Nedergaard J Chronic peroxisome proliferator-activated receptor gamma (PPARgamma) activation of epididymally derived white adipocyte cultures reveals a population of thermogenically competent, UCP1-containing adipocytes molecularly distinct from classic brown adipocytes.J Biol Chem. 2010; 285: 7153-7164Crossref PubMed Scopus (1035) Google Scholar Both lipogenic (Fasn) and lipolytic (Cpt1a) markers were reduced together with oxidative markers Cytc and Ppargc1a.15Tiraby C Tavernier G Lefort C Larrouy D Bouillaud F Ricquier D et al.Acquirement of brown fat cell features by human white adipocytes.J Biol Chem. 2003; 278: 33370-33376Crossref PubMed Scopus (380) Google Scholar,16Puigserver P Wu Z Park CW Graves R Wright M Spiegelman BM A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis.Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3102) Google Scholar In addition, angiogenic markers Cd31 and Vegf were also decreased.17Zhang QX Magovern CJ Mack CA Budenbender KT Ko W Rosengart TK Vascular endothelial growth factor is the major angiogenic factor in omentum: mechanism of the omentum-mediated angiogenesis.J Surg Res. 1997; 67: 147-154Abstract Full Text PDF PubMed Scopus (324) Google Scholar In contrast, macrophage marker F4/80 and inflammatory M1 macrophage marker Mgl1 were increased18Barbatelli G Murano I Madsen L Hao Q Jimenez M Kristiansen K et al.The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation.Am J Physiol Endocrinol Metab. 2010; 298: E1244-E1253Crossref PubMed Scopus (566) Google Scholar,19Thompson RJ Doran JF Jackson P Dhillon AP Rode J PGP 9.5–a new marker for vertebrate neurons and neuroendocrine cells.Brain Res. 1983; 278: 224-228Crossref PubMed Scopus (665) Google Scholar (Figure 5b). Immunohistochemistry confirmed the increase of F4/80 in Rec2-Cre–injected IWAT. The extensive molecular changes indicated that the disruption of IR signaling in adult IWAT led to dysfunction of the transduced fat pad, although no overt systemic impact was observed in this relative short-term experiment. Interestingly, the hypothalamus was able to “sense” the knockdown of IR in IWAT (Figure 5c). For example, the neuronal activity marker Fos showed a trend of upregulation in the hypothalamus when IR was knocked down in IWAT.20Nedivi E Hevroni D Naot D Israeli D Citri Y Numerous candidate plasticity-related genes revealed by differential cDNA cloning.Nature. 1993; 363: 718-722Crossref PubMed Scopus (437) Google Scholar Leptin receptor expression was significantly upregulated in the hypothalamus. Brain-derived neurotrophic factor (BDNF) and cocaine-amphetamine–regulated transcript (Cartpt), both anorexigenic,21Rios M Fan G Fekete C Kelly J Bates B Kuehn R et al.Conditional deletion of brain-derived neurotrophic factor in the postnatal brain leads to obesity and hyperactivity.Mol Endocrinol. 2001; 15: 1748-1757Crossref PubMed Scopus (521) Google Scholar,22Xu B Goulding EH Zang K Cepoi D Cone RD Jones KR et al.Brain-derived neurotrophic factor regulates energy balance downstream of melanocortin-4 receptor.Nat Neurosci. 2003; 6: 736-742Crossref PubMed Scopus (702) Google Scholar were upregulated, which might underlie the decreased food intake.Figure 5Phenotypic changes induced by Rec2-Cre–mediated IR knockdown in IWAT. (a) Hematoxylin and eosin staining and F4/80 immunohistochemistry. Scale bar = 50 µm. (b) Gene expression profile of Rec2-injected IWAT (n = 7 per group, *P < 0.05, #P = 0.06). (c) Gene expression profile of hypothalamus (n = 7 per group, *P < 0.05, #P = 0.06). IR, insulin receptor; IWAT, inguinal white adipose tissue.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Rec2-Cre was injected bilaterally to BAT of male IRlox mice at the age of 6 weeks (1 × 109 vg per injection) with empty Rec2 vector as control. No significant difference was found in body weight (Figure 6a) or food intake (control: 4.10 ± 0.19 g/day; Cre: 4.32 ± 0.35 g/day; P = 0.34). Similar to IWAT IR knockdown, no significant effects on insulin tolerance (Figure 6d) or glucose tolerance (Figure 6e) or serum biomarkers (Figure 6f) were observed. Rec2-Cre–injected BAT was 50% smaller than control vector–injected BAT (Figure 6a–c), while no difference in subcutaneous WAT or epididymal WAT mass was found (Figure 6a,c). Western blot showed ∼60% decrease of IR at protein level (Figure 6g,h), while quantitative RT-PCR showed 50% downregulation at mRNA level (Figure 7b). Hematoxylin and eosin staining showed massive reduction of brown adipocytes size with far less lipid droplets (Figure 7a). Rec2-Cre–injected BAT showed marked increase of eosinophilia compared with controls (Figure 7a). These results indicate BAT lipoatrophy, although no apoptosis was found by TUNEL assay or active caspase-3 analysis (data not shown). Cre is reported to have no impact on BAT,23Guerra C Navarro P Valverde AM Arribas M Brüning J Kozak LP et al.Brown adipose tissue-specific insulin receptor knockout shows diabetic phenotype without insulin resistance.J Clin Invest. 2001; 108: 1205-1213Crossref PubMed Scopus (145) Google Scholar and therefore, the phenotypic changes in Rec2-Cre–injected BATs were due to the IR knockdown. The decrease of BAT mass and potential dysfunction had no effect on rectal temperature measured at room temperature (control: 36.2 ± 0.2°C; Cre: 36.8 ± 0.1°C; P = 0.1). Rec2-Cre injection to BAT did not change IR expression in liver or skeletal muscle (Figure 7d) while interestingly upregulated IR expression in visceral WAT (Figure 7d). In contrast to IR knockdown in IWAT (Figure 5b), Rec2-Cre led to less gene expression changes (Figure 7b), although the IR knockdown level was similar. However, greater induction of macrophage markers and inflammation markers were observed in BAT (Figure 7b) with M1 macrophage marker Mcp1 increased by 12-fold and inflammatory marker Saa3 increased by 18-fold.24Sun K Wernstedt Asterholm I Kusminski CM Bueno AC Wang ZV Pollard JW et al.Dichotomous effects of VEGF-A on adipose tissue dysfunction.Proc Natl Acad Sci USA. 2012; 109: 5874-5879Crossref PubMed Scopus (294) Google Scholar The disruption of IR signaling in BAT induced gene expression changes in the hypothalamus (Figure 7c) but showed a different profile compared with IR knockdown in IWAT (Figure 5c). IR expression in hypothalamus showed a trend of upregulation, while melanocortin receptor 4 (Mc4r), a critical pathway in energy balance regulation,25Flier JS Obesity wars: molecular progress confronts an expanding epidemic.Cell. 2004; 116: 337-350Abstract Full Text Full Text PDF PubMed Scopus (962) Google Scholar was downregulated.Figure 7Phenotypic changes induced by Rec2-Cre–mediated IR knockdown in BAT. (a) Hematoxylin and eosin staining of Rec2-injected BAT. Scale bar = 50 µm. (b) Gene expression profile of Rec2-injected BAT (n = 6 per group, *P < 0.05). (c) Gene expression profile of hypothalamus (n = 6 per group, *P < 0.05, #P = 0.06). (d) Insulin receptor expression in liver, muscle, and visceral WAT (n = 6 per group, *P < 0.05). BAT, brown adipose tissue; IR, insulin receptor.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A critical barrier of genetic manipulation of adipose tissue for basic research and translational studies has been the difficulties of transducing adipose tissue with viral vectors such as rAAV. Here, we demonstrate that a newly engineered nonhuman primate–derived hybrid rAAV serotype efficiently transduced both BAT and WAT when directly injected to the fat pad. Rec2 vector showed highest transduction efficacy with lowest off-target transgene expression primarily in liver. Several strategies can be employed to enhance fat-specific expression: using an adipose-specific promoter to drive transgene expression24Sun K Wernstedt Asterholm I Kusminski CM Bueno AC Wang ZV Pollard JW et al.Dichotomous effects of VEGF-A on adipose tissue dysfunction.Proc Natl Acad Sci USA. 2012; 109: 5874-5879Crossref PubMed Scopus (294) Google Scholar,26He W Barak Y Hevener A Olson P Liao D Le J et al.Adipose-specific peroxisome proliferator-activated receptor gamma knockout causes insulin resistance in fat and liver but not in muscle.Proc Natl Acad Sci USA. 2003; 100: 15712-15717Crossref PubMed Scopus (798) Google Scholar, 27Pajvani UB Trujillo ME Combs TP Iyengar P Jelicks L Roth KA et al.Fat apoptosis through targeted activation of caspase 8: a new mouse model of inducible and reversible lipoatrophy.Nat Med. 2005; 11: 797-803Crossref PubMed Scopus (237) Google Scholar, 28Segawa K Matsuda M Fukuhara A Morita K Okuno Y Komuro R et al.Identification of a novel distal enhancer in human adiponectin gene.J Endocrinol. 2009; 200: 107-116Crossref PubMed Scopus (16) Google Scholar; incorporating a microRNA targeting the transgene that is driven by tissue-specific promoters of the tissues intended to avoid11Cao L Lin EJ Cahill MC Wang C Liu X During MJ Molecular therapy of obesity and diabetes by a physiological autoregulatory approach.Nat Med. 2009; 15: 447-454Crossref PubMed Scopus (93) Google Scholar; placing a tissue-specific microRNA target into the 3′ untranslated region of a transgene to mitigate the transgene expression in unintended cells because the specific microRNA is only expressed in the unintended cells such as a liver-specific microRNA-12229Brown BD Venneri MA Zingale A Sergi Sergi L Naldini L Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer.Nat Med. 2006; 12: 585-591Crossref PubMed Scopus (397) Google Scholar, 30Geisler A Jungmann A Kurreck J Poller W Katus HA Vetter R et al.microRNA122-regulated transgene expression increases specificity of cardiac gene transfer upon intravenous delivery of AAV9 vectors.Gene Ther. 2011; 18: 199-209Crossref PubMed Scopus (91) Google Scholar, 31Qiao C Yuan Z Li J He B Zheng H Mayer C et al.Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver.Gene Ther. 2011; 18: 403-410Crossref PubMed Scopus (106) Google Scholar; or the combination of these strategies. Fat pad injection of Rec2 vector showed minimal off-target transgene expression in both the reporter gene experiment using GFP and the functional experiment using the Cre-loxP system. However, restriction of off-target expression of transgene is necessary for systemic gene delivery. Our unpublished data showed that with intravenous infusion, Rec2 showed the greatest transduction of the target tissue cardiac muscle among multiple natural and engineered serotypes while liver also showed high transgene expression. Investigations on targeting adipose tissue via systemic administration of Rec2 are underway. Several hundred human and nonhuman primate isolates of AAV have been discovered.32Gao G Vandenberghe LH Wilson JM New recombinant serotypes of AAV vectors.Curr Gene Ther. 2005; 5: 285-297Crossref PubMed Scopus (417) Google Scholar And the reengineered variants, chimeric, and mutant strains together with the naturally occurring serotypes yielded a broad and versatile AAV vector toolkit for both basic research and clinical application.5Asokan A Schaffer DV Samulski RJ The AAV vector toolkit: poised at the clinical crossroads.Mol Ther. 2012; 20: 699-708Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar Our data show that Rec2 serotype transduces adipose tissue superior to natural serotypes tested. The Rec2 serotype was generated by capsid shuffling of AAV strains isolated from the primate brain. Future studies to isolate AAV stains directly from primate adipose tissue and then to engineer hybrid and mutant strains based on the adipose-derived natural serotypes can further improve tissue tropism and gene transfer efficiency. As a proof-of-efficacy, we used the Rec2 vector to knockdown IR via Cre-loxP recombination in both BAT and WAT. Our data demonstrate that a low-dose injection of Rec2-Cre to fat pads of adult animal led to 50% decrease of IR level and the ensuring loss of fat mass, molecular and morphological changes consistent with impaired adipose function. Fat-specific IR knockout mouse,33Blüher M Michael MD Peroni OD Ueki K Carter N Kahn BB et al.Adipose tissue selective insulin receptor knockout protects against obesity and obesity-related glucose intolerance.Dev Cell. 2002; 3: 25-38Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar BAT-specific IR knockout mouse,23Guerra C Navarro P Valverde AM Arribas M Brüning J Kozak LP et al.Brown adipose tissue-specific insulin receptor knockout shows diabetic phenotype without insulin resistance.J Clin Invest. 2001; 108: 1205-1213Crossref PubMed Scopus (145) Google Scholar and fat-specific IR and insulin-like growth factor-1 receptor double knockout mouse34Boucher J Mori MA Lee KY Smyth G Liew CW Macotela Y et al.Impaired thermogenesis and adipose tissue development in mice with fat-specific disruption of insulin and IGF-1 signalling.Nat Commun. 2012; 3: 902Crossref PubMed Scopus (102) Google Scholar have been generated. These knockout strains have revealed that IR signaling plays a crucial role in the control of fat development. Our data using viral vector–mediated IR knockdown in adult animal suggest that adipose IR signaling is critical to the maintenance of both BAT and WAT. Some phenotypic changes are similar to IR knockout mice. For example, Rec2-Cre injection to BAT led to loss of BAT mass by 50%, which was comparable to the BAT-specific IR knockout mice, whose IR expression in BAT was reduced by 95%.23Guerra C Navarro P Valverde AM Arribas M Brüning J Kozak LP et al.Brown adipose tissue-specific insulin receptor knockout shows diabetic phenotype without insulin resistance.J Clin Invest. 2001; 108: 1205-1213Crossref PubMed Scopus (145) Google Scholar The marked reduction of brown adipocyte size and lipid droplet size as well as increased eosinophilia in Rec2-Cre–injected BAT were consistent to BAT-specific IR knockout mice. BAT-specific IR knockout mice also did not show altered insulin tolerance, although progressive glucose intolerance occurred at the age of 6 months. Long-term experiment using Rec2-Cre might reveal the impact on glucose tolerance that did not change by 8 weeks after viral vector injection. Viral vector–mediated IR knockdown in adipose tissue not only helped to distinguish the role of IR in development from maintenance but also revealed a few changes that were not reported in various fat-specific IR knockout mice such as the strong induction of macrophage markers and the hypothalamic “sensing”" @default.
• W2065821427 created "2016-06-24" @default.
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• W2065821427 date "2014-01-01" @default.
• W2065821427 modified "2023-10-14" @default.
• W2065821427 title "Adipose tissue insulin receptor knockdown via a new primate-derived hybrid recombinant AAV serotype" @default.
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