This work was supported by grants from the NIH: R03 DK089061-01 (VKY); NIH: K08 DK068391 (VKY); the Diabetes and Endocrinology Research Center-(DERC - P30DK079638) at Baylor College of Medicine, a Pilot &amp; Feasibility grant from the DERC (VKY); Juvenile Diabetes Research Foundation: JDRF Award # 5-2006-134 (VKY).

<ol>
	<li>Parks, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+helper-dependent+adenovirus+vector+system:+removal+of+helper+virus+by+Cre-mediated+excision+of+the+viral+packaging+signal.">A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal.</a> Proc. Natl. Acad. Sci. U. S. A. 93, 13565-13570 (1996).</li><li>Kim, I. H., Jozkowicz, A., Piedra, P. A., Oka, K., Chan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifetime+correction+of+genetic+deficiency+in+mice+with+a+single+injection+of+helper-dependent+adenoviral+vector.">Lifetime correction of genetic deficiency in mice with a single injection of helper-dependent adenoviral vector.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 13282-13287 (2001).</li><li>Belalcazar, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+stable+expression+of+human+apolipoprotein+A-I+mediated+by+helper-dependent+adenovirus+gene+transfer+inhibits+atherosclerosis+progression+and+remodels+atherosclerotic+plaques+in+a+mouse+model+of+familial+hypercholesterolemia.">Long-term stable expression of human apolipoprotein A-I mediated by helper-dependent adenovirus gene transfer inhibits atherosclerosis progression and remodels atherosclerotic plaques in a mouse model of familial hypercholesterolemia.</a> Circulation. 107, 2726-2732 (2003).</li><li>Oka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+stable+correction+of+low-density+lipoprotein+receptor-deficient+mice+with+a+helper-dependent+adenoviral+vector+expressing+the+very+low-density+lipoprotein+receptor.">Long-term stable correction of low-density lipoprotein receptor-deficient mice with a helper-dependent adenoviral vector expressing the very low-density lipoprotein receptor.</a> Circulation. 103, 1274-1281 (2001).</li><li>Nomura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-density+lipoprotein+receptor+gene+therapy+using+helper-dependent+adenovirus+produces+long-term+protection+against+atherosclerosis+in+a+mouse+model+of+familial+hypercholesterolemia.">Low-density lipoprotein receptor gene therapy using helper-dependent adenovirus produces long-term protection against atherosclerosis in a mouse model of familial hypercholesterolemia.</a> Gene Ther. 11, 1540-1548 (2004).</li><li>Ng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-efficiency+Cre/loxP-based+system+for+construction+of+adenoviral+vectors.">A high-efficiency Cre/loxP-based system for construction of adenoviral vectors.</a> Hum. Gene Ther. 10, 2667-2672 (1999).</li><li>Palmer, D., Ng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+system+for+helper-dependent+adenoviral+vector+production.">Improved system for helper-dependent adenoviral vector production.</a> Mol. Ther. 8, 846-852 (2003).</li><li>Oka, K., Chan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helper-Dependent+Adenoviral+Vectors.">Helper-Dependent Adenoviral Vectors.</a> Current Protocols in Molecular Biology. , 16.24.1-16.24.23 (2005).</li><li>Yechoor, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenin3+is+sufficient+for+transdetermination+of+hepatic+progenitor+cells+into+neo-islets+in+vivo+but+not+transdifferentiation+of+hepatocytes.">Neurogenin3 is sufficient for transdetermination of hepatic progenitor cells into neo-islets in vivo but not transdifferentiation of hepatocytes.</a> Dev. Cell. 16, 358-373 (2009).</li><li>Yechoor, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Therapy+with+Neurogenin+3+and+Betacellulin+Reverses+Major+Metabolic+Problems+in+Insulin-Deficient+Diabetic+Mice.">Gene Therapy with Neurogenin 3 and Betacellulin Reverses Major Metabolic Problems in Insulin-Deficient Diabetic Mice.</a> Endocrinology. , (2009).</li></ol>

No conflicts of interest declared.

HDAds have been developed to overcome the weakness of early generation Ads and to harness for gene therapy application. However, technical challenges remain. For example, HDAd requires HV for HDAd's packaging and vector amplification is not as efficient as early generation Ads. HV is a first generation Ad and any contamination of HV compromise the effectiveness of HDAd. Therefore, highly efficient transfection and optimal conditions for each serial passage are critical. Another critical parameter for vector production is which passage (P1-P4) should be used for subsequent passage 5 that is directly used as inoculum for suspension cells. To our experience, the best results are obtained by using the passage by which HDAd vector proportion is dramatically increased in the following passage (P3 in Figure 2). The yield of HDAd vectors depends on transgene cassettes. During vector production, both transgenes are expressed because both genes are under ubiquitous promoter. Ngn3 is a transcription factor and Btc is a growth factor, which suggests that HDAd vector expressing transcription factor which may influence cell lineage inhibits vector amplification while that expressing growth hormone helps in vector replication and packaging.
With diabetes assuming epidemic proportions, new approaches to restore b-cell mass are needed. In this report, we describe methods to harness the advantages of HDAd vectors to effect gene transfer of islet lineage-defining transcription factor, Ngn3 along with the islet growth factor, betacellulin to induce islet neogenesis in periportal regions of the liver. To assess the efficacy of this, it is important to choose mice with stable hyperglycemia and ensure that appropriate controls are always included. For this gene transfer experiments, the empty vector treated diabetic mice should always be utilized. In addition, using HDAd-Ngn3 and HDAd-Btc individually treated diabetic mice serves to test the individual contribution of these two genes in islet neogenesis. As our data demonstrates that Ngn3 alone is sufficient to induce islet neogenesis, but the addition of the growth factor, Btc, serves to augment the response leading to robust induction of islet neogenesis. It is also important to test that the vector expression is indeed achieved in the target tissue, the liver and also to demonstrate that the insulin assayed in the plasma of treated mice is not coming from residual islets in the pancreas, by demonstrating the absence of pancreatic islets in the diabetic mice.
In summary, the advantage of the HDAd-vector system for gene transfer lies in its high cloning capacity, efficient transduction and long lasting gene expression in the liver with minimum chronic toxicity as well as its nature of non-integration of vector genome into the host chromosome. The primary limitations are the complex steps involved in its generation and its in vivo application is primarily limited to the liver with the most popular Ad serotype 5. Islet neogenesis can be induced to fully restore plasma insulin and glucose tolerance in diabetic mice by inducing islet neogenesis in the liver by gene transfer of islet lineage-defining transcription factor, Ngn3 along with the islet growth factor, betacellulin. In this report, we show the optimal protocol to generate high quality HDAd-Ngn3 and HDAd-Btc, and demonstrate techniques to induce and assess islet neogenesis in the livers of diabetic mice to reverse hyperglycemia.
Footnote: The viral vectors and the cell lines described here are available from the Vector Production Core Laboratory, Diabetes Research Center, Baylor College of Medicine (<a href="http://www.bcm.edu/mcb/index.cfm?pmid=7731" target="_blank">http://www.bcm.edu/mcb/index.cfm?pmid=7731</a>). Some commercial kits are also available for generating HDAd viruses (e.g. Microbix biosystems Inc.).

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>ProFectionR Mammalian Transfection kit</td>
 <td>Promega</td>
 <td>E1200</td>
 </tr>
 <tr>
 <td>DNeasy Blood &amp; Tissue Kit (50)</td>
 <td>Qiagen</td>
 <td>69504</td>
 </tr>
 <tr>
 <td>PerfeCTa SYBR Green SuperMix, ROX</td>
 <td>Quanta Biosciences</td>
 <td>95055-500</td>
 </tr>
 <tr>
 <td>Sodium deoxycholate</td>
 <td>Sigma</td>
 <td>D6750-25G</td>
 </tr>
 <tr>
 <td>MEM powder</td>
 <td>Invitrogen</td>
 <td>61100087</td>
 </tr>
 <tr>
 <td>Penicillin Streptomycin</td>
 <td>Sigma</td>
 <td>15140122</td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Atlanta Biologicals</td>
 <td>S11150</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Invitrogen</td>
 <td>25030-081</td>
 </tr>
 <tr>
 <td>Hygromycin B</td>
 <td>Sigma</td>
 <td>H0654-1G</td>
 </tr>
 <tr>
 <td>MEM EAGLE JOKLIK</td>
 <td>Sigma</td>
 <td>M0518-10L</td>
 </tr>
 <tr>
 <td>Rnase</td>
 <td>Roche</td>
 <td>10109169001</td>
 </tr>
 <tr>
 <td>DNase I, grade II</td>
 <td>Roche</td>
 <td>10104159001</td>
 </tr>
 <tr>
 <td>Streptozocin</td>
 <td>Sigma</td>
 <td>s0130</td>
 </tr>
 <tr>
 <td>Glass spinner flasks</td>
 <td>Corning</td>
 <td>4500-3L</td>
 </tr>
 <tr>
 <td>Glass spinner flasks</td>
 <td>Corning</td>
 <td>4500-250</td>
 </tr>
 <tr>
 <td>Slide A-lyzer casset</td>
 <td>PIERCE CH</td>
 <td>PI66380</td>
 </tr>
 <tr>
 <td>Tube optiseal poly allomer, 11.2 ml</td>
 <td>Beckman Coulter</td>
 <td>362181</td>
 </tr>
 <tr>
 <td>Cesium chloride 1 kg</td>
 <td>JT4042-2</td>
 <td>VWR</td>
 </tr>
 <tr>
 <td>Beckman LE-80K</td>
 <td>Beckman Coulter</td>
 <td>Optimal LE-80K ultracentrifuge</td>
 </tr>
 <tr>
 <td>Filter</td>
 <td>VWR</td>
 <td>28143-338</td>
 </tr>
 <tr>
 <td>centrifuge tube 500 ml</td>
 <td>Corning</td>
 <td>431123</td>
 </tr>
 <tr>
 <td>tailveiner restrainer</td>
 <td>Braintree scientific , INC</td>
 <td>tv-150</td>
 </tr>
 <tr>
 <td>Insulin, Mouse ELISA</td>
 <td>Mercodia</td>
 <td>10-1247-01</td>
 </tr>
 <tr>
 <td>microvette CB300</td>
 <td>Sarstedt</td>
 <td>16.443.100</td>
 </tr>
 <tr>
 <td>D-glucose</td>
 <td>Sigma</td>
 <td>G8270</td>
 </tr>
 <tr>
 <td>Mouse C-peptide ELISA Kit</td>
 <td>Wako Pure Chemical Industries, Ltd</td>
 <td>#631-07231</td>
 </tr>
 <tr>
 <td>guinea pig anti-insulin antibody</td>
 <td>Abcam</td>
 <td>ab7842</td>
 </tr>
 <tr>
 <td>goat anti-Pdx1 antibody</td>
 <td>gift from Dr. Christopher Wright</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>mouse anti Ngn3 antibody</td>
 <td>Beta Cell Biology Consortium, 
 Univ. of Pennsylvania</td>
 <td>AB2013</td>
 </tr>
 <tr>
 <td>mouse anti Nkx6.1 antibody</td>
 <td>Beta Cell Biology Consortium, 
 Univ. of Pennsylvania</td>
 <td>F64A6B4</td>
 </tr>
 <tr>
 <td>anti- betacellulin antibody</td>
 <td>Cell Sciences</td>
 <td>PAAQ1</td>
 </tr>
 <tr>
 <td>ALT (SGPT) Color Reagent SET.</td>
 <td>Teco Diagnostics</td>
 <td>A526 - 120</td>
 </tr>
 <tr>
 <td>AST/(SGOT), Color Endpoint Reagent Set</td>
 <td>Teco Diagnostics</td>
 <td>A561-120</td>
 </tr>
 </tbody>
</table>
Table 2. Specific Reagents and Equipment.

neo-islet formation in liver of diabetic mice by helper-dependent adenoviral vector-mediated gene transfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.
Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.
In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

Watch this Scientific Journal Video about Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer at JoVE.com

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

neo-islet-formation-liver-diabetic-mice-helper-dependent-adenoviral

Research

JoVE Journal

Medicine

11.7K visualizações.  Baylor College of Medicine. Nós descrevemos a formação de neo-hepática ilhota em STZ (estreptozotocina) induzida ratos diabéticos por transferência de genes de Neurogenin3 (Ngn3) e betacelulina (BTC), utilizando ajudante dependente do vector adenoviral (HDAD) e reversão da hiperglicemia. O nosso método tem vantagens do ajudante dependente de vectores adenovirais com a sua grande eficácia na transdução in vivo e a expressão do gene de longa duração.