Pancreatic islet transplantation is a promising treatment for type 1 diabetes mellitus offering improved glycaemic control by restoring insulin production. Improved human pancreatic islet isolation has led to higher islet transplantation success. However, as many as 50% of islets are lost after transplantation due to immune responses and cellular injury, gene therapy presents a novel strategy to protect pancreatic islets for improved survival post-transplantation. To date, most of the vectors used in clinical trials and gene therapy studies have been derived from mammalian viruses such as adeno-associated or retrovirus. However, baculovirus BacMam vectors provide an attractive and safe alternative. Here, a novel BacMam was constructed containing a frameshift mutation within fp25, which results in virus stocks with higher infectious titres. This improved in vitro transduction when compared to control BacMams. Additionally, incorporating a truncated vesicular stomatitis virus G protein increased transduction efficacy and production of EGFP and BCL2 in human kidney (HK-2) and pancreatic islet β cells (EndoC βH3). Lastly, we have shown that our optimized BacMam vector can deliver and express egfp in intact pancreatic islet cells from human cadaveric donors. These results confirm that BacMam vectors are a viable choice for providing delivery of transgenes to pancreatic islet cells.

Autographa californica nucleopolyhedrovirus is a very productive expression vector for recombinant proteins in insect cells. Most vectors are based on the polyhedrin gene promoter, which comprises a TAAG transcription initiation motif flanked by 20 base pairs upstream and 47 base pairs downstream before the native ATG. Many transfer vectors also include a short sequence downstream of the ATG, in which case this sequence is mutated to ATT to abolish translation. However, the ATT sequence, or AUU in the mRNA, is known to be leaky. If a target-coding region is placed in the frame with the AUU, then some products will comprise a chimeric molecule with part of the polyhedrin protein. In this study, we showed that if AUU is placed in the frame with a Strep tag and eGFP coding region, we could identify a protein product with both sequences present. Further work examined if alternative codons in lieu of AUG might reduce translation initiation further. We found that AUA was used slightly more efficiently than AUU, whereas AUC was the least efficient at initiating translation. The use of this latter codon suggested that there might also be a slight improvement of protein yield if this is incorporated into expression vectors.