Research Article
Pharmaceutics, Drug Delivery and Pharmaceutical Technology
In Vivo Transgene Expression in the Pancreas by the Intraductal Injection of Naked Plasmid DNA

https://doi.org/10.1016/j.xphs.2017.09.021Get rights and content

Abstract

Patients with type I diabetes, which is caused by the destruction of pancreatic islets, now require regular therapeutic injections of insulin. The use of transgene therapy represents an alternate and potent strategy for the treatment of type I diabetes. However, only a limited number of studies regarding in vivo gene delivery targeting the pancreas and islets have been reported. Here, we report on the possibility of in vivo transgene expression in the pancreas by the intraductal injection of naked plasmid DNA (pDNA). Gene expression activities were detected in the pancreas of mice after the injection of naked pDNA encoding luciferase into the common bile duct. We then investigated the effects of injection dose, volume, and speed on gene delivery and determined the optimal conditions for the delivery of pDNA to the pancreas. Exogenous luciferase mRNA was detected in the pancreatic islets by reverse transcription PCR analysis. Moreover, no injury was detected in the liver, the common bile duct, or the pancreas over time after the injection. These findings indicate that the intraductal injection of naked pDNA promises to be a useful technique for in vivo gene delivery targeted to pancreatic tissue and islets.

Introduction

Pancreatic β cells constitute the majority of cells in pancreatic islets. They function to maintain serum glucose levels by secreting insulin in response to fluctuations in serum glucose levels. Patients with type I diabetes, which is caused by islet dysfunction, typically require an external source of insulin. Mutations in genes related to pancreatic β cell function have been associated with certain types of diabetes mellitus.1, 2 These genes are associated with important pancreatic β cell functions, including the production and secretion of insulin. The activation of islets by a transgene represents a potentially viable therapy for many patients who suffer from diabetes mellitus. To achieve such a goal, technology for delivering therapeutic genes to pancreatic islets is needed.

However, our knowledge regarding in vivo gene delivery targeting the pancreas and islets is currently very limited,3, 4, 5, 6 although many studies regarding in vivo gene delivery targeting other tissues including liver have been reported.7, 8, 9, 10 Even though this is a serious situation, Rehman et al.5 reported that the adeno-associated virus (AAV) 8-mediated gene transfer of interleukin-4 to pancreatic β cells prevents the onset of diabetes in nonobese diabetic mice. In this study, the authors succeeded in achieving selective gene expression in pancreatic β cells by the intraperitoneal injection of AAV8 containing a murine insulin promoter. In this case, although selective gene expression in pancreatic β cells was achieved, as evidenced by the finding that AAV8 was distributed throughout the body, toxicity associated with the transfer of AAV to other organs would be unclear. Thus, achieving a safe, simple, and efficient method continues to be a goal, and approaches from various fields are required.

In this study, we first investigated the targeted delivery of pDNA to the pancreas of mice via hydrodynamic injection. Hydrodynamic injection, in which a large volume of naked pDNA is rapidly injected into the tail vein of the mouse, has been frequently used as an efficient in vivo nuclear gene transfer method for delivering naked pDNA to hepatocytes.11, 12, 13 Liu et al.14 reported that this method could also achieve transgene expression in the kidney, spleen, lung, and heart; although these transfection efficiencies were lower than that in the liver. Thus, we hypothesized that hydrodynamic injection might be a candidate for the targeted delivery of a gene to the pancreas.

We next validated the occurrence of in vivo transgene expression in the pancreas by the intraductal injection of naked pDNA. It has been reported that gene delivery by hydrodynamic injection transiently enhances membrane permeability, which would contribute to the introduction of pDNA to the cellular membrane.15 Thus, we expected that the hydrodynamic force caused by the intraductal injection could result in selective targeting to the pancreas. Gene expression activities were measured in the pancreas and other tissues by the intraductal injection of naked pDNA encoding luciferase. We also performed reverse transcription PCR analyses to detect exogenous luciferase mRNA in pancreatic islets. Moreover, injuries to the liver, the common bile duct and the pancreas were evaluated over time after the injection.

Section snippets

Materials

The pcDNA3.1 (+)-luc plasmid was constructed by inserting the firefly luciferase gene (Hind III-Xba I fragment) of the pGL3-Control plasmid (Promega, Madison, WI) into the pcDNA3.1 (+) plasmid (Invitrogen, Carlsbad, CA) pretreated with the same restriction enzymes. The luciferase gene in the pDNA is expressed under the control of the cytomegalovirus promoter. The pDNA was purified using an Endofree Plasmid Giga Kit (Qiagen GmbH, Hilden, Germany). Oligonucleotides for the primers for firefly

Gene Delivery by HTV Injection and Evaluation of Transgene Expression in Various Tissues

We first investigated the targeted delivery of pDNA to the pancreas of mice via hydrodynamic injection. Liu et al.14 reported that this method could also achieve transgene expression in the kidney, spleen, lung, and heart; although the transfection efficiencies in these tissues were lower than that in the liver. Thus, we hypothesized that hydrodynamic injection might be used for the targeted delivery of gene to the pancreas.

In this experiment, 50 μg of a pDNA coding luciferase was administered

Discussion

We report herein on the successful in vivo transgene expression in the pancreas of mice by the intraductal injection of naked pDNA (Fig. 3a). Transgene expression was the highest in the pancreas and much lower in other tissues, including the liver, spleen, kidney, lung, heart, and small intestine (Fig. 3b). These results suggest that the intraductal injection of naked pDNA can be useful for achieving transgene expression in the pancreas. Wang et al.6 reported that the intraductal delivery of

Conclusion

We validated the possibility of the intraductal injection of pDNA for in vivo transgene expression in the pancreas. Our results indicate that gene expression activities were observed in the pancreas by intraductal injection, and the value was the highest in the pancreas, compared with all other tissues. Reverse transcription PCR analysis suggests that the pDNA that was delivered by intraductal injection was localized, not only in the pancreas but also in islets. Moreover, no injuries to the

Acknowledgments

This work was supported founded by the Kowa Life Science Foundation (to Y.Y.), and in part by a grant-in-aid for scientific research (B) from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (MEXT). The authors are grateful to Dr. Jun Yamauchi for her helpful comments. They also thank Oriental Yeast Co., Ltd., for the evaluation of the toxicity (Shiga, Japan) and Dr. Milton Feather for his helpful advice in writing the manuscript.

References (17)

There are more references available in the full text version of this article.

Cited by (5)

  • Designing electrospun fiber platforms for efficient delivery of genetic material and genome editing tools

    2022, Advanced Drug Delivery Reviews
    Citation Excerpt :

    These expression vectors are widely utilized in tissue engineering and regenerative medicine to either stably or transiently transfect mammalian cells and increase the production of mRNA and, subsequently, desirable proteins (Fig. 4) [220]. Due to the excellent stability of plasmids, like circular pDNA that does not possess exposed ends that are more prone to nucleolytic degradation, naked pDNA can be delivered via direct injection [221–223]. Still, a drawback of direct injection of pDNA is that the genetic material must pass through the cell membrane and into the nucleus for expression to occur.

  • Natural hydrogels applied in photodynamic therapy

    2020, Current Medicinal Chemistry

The authors Yuma Yamada and Mai Tabata contributed equally as first author.

Conflicts of interest: The authors declare no conflict of interest associated with this manuscript.

View full text