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 (http://www.bcm.edu/mcb/index.cfm?pmid=7731
). Some commercial kits are also available for generating HDAd viruses (e.g.
Microbix biosystems Inc.).