Transgenic expression and recovery of biologically active recombinant human insulin from Arabidopsis thaliana seeds

The increased incidence of diabetes, coupled with the introduction of alternative delivery methods that rely on higher doses, is expected to result in a substantial escalation in the demand for affordable insulin in the future. Limitations in the capacity and economics of production will make it difficult for current manufacturing technologies to meet this demand. We have developed a novel expression and recovery technology for the economical manufacture of biopharmaceuticals from oilseeds. Using this technology, recombinant human precursor insulin was expressed in transgenic plants. Plant-derived insulin accumulates to significant levels in transgenic seed (0.13% total seed protein) and can be enzymatically treated in vitro to generate a product with a mass identical to that of the predicted product, DesB₃₀-insulin. The biological activity of this product in vivo and in vitro was demonstrated using an insulin tolerance test in mice and phosphorylation assay performed in a mammalian cell culture system, respectively.     

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.     

Expression and recovery of biologically active recombinant Apolipoprotein AIMilano from transgenic safflower (Carthamus tinctorius) seeds

Apolipoprotein AI Milano (ApoAI_Milano) was expressed as a fusion protein in transgenic safflower seeds. High levels of expression corresponding to 7 g of ApoAI_Milano per kilogram of seed have been identified in a line selected for commercialization. The ApoAI_Milano fusion protein was extracted from seed using an oilbody‐based process and matured in vitro prior to final purification. This yielded a Des‐1,2‐ApoAI_Milano product which was confirmed by biochemical characterization including immunoreactivity against ApoAI antibodies, isoelectric point, N‐terminal sequencing and electrospray mass spectrometry. Purified Des‐1,2‐ApoAI_Milano readily associated with dimyristoylphosphatidylcholine in clearance assays comparable to Human ApoAI. Its biological activity was assessed by cholesterol efflux assays using Des‐1,2‐ApoAI_Milano:1‐palmitoyl‐2‐oleoyl phosphatidylcholine complexes in vitro and in vivo. This study has established that high levels of biologically functional ApoAI_Milano can be produced using a plant‐based expression system.