Four in one-Combination therapy using live Lactococcus lactis expressing three therapeutic proteins for the treatment of chronic non-healing wounds.

Diabetes mellitus is one of the major concerns for health care systems, affecting 382 million people worldwide. Among the different complications of diabetes, lower limbs chronic ulceration is a common, severe and costly cause of morbidity. Diabetic foot ulcers are a leading cause of hospitalization in diabetic patients and its rate exceed the ones of congestive heart failure, depression or renal disease. Diabetic non-healing ulcers account for more than 60% of all non-traumatic lower limb amputations and the five-year mortality after amputation is higher than 50%, being equal to several types of advanced cancer. The primary management goals for an existing diabetic foot ulcer are to achieve primary healing as expeditiously as possible and to achieve a reduction of the amputation rate in the patients. Unfortunately, approximately a quarter of patients do not partially or fully respond to the standard of care. Advanced therapies for chronic wounds are existing, however, recent guidelines including the latest reviews and meta-analyses of the scientific and clinical evidence available from current treatment strategies and new therapeutic agents revealed that there is a lack of clinical data and persistent gap of evidence for many of the advanced therapeutic approaches. In addition, no pharmacological wound healing product has gained authority approval for more than 10 years in both US and EU, constituting a highly unmet medical need. In this publication we present data from a live biopharmaceutical product AUP1602-C designed as a single pharmaceutical entity based on the non-pathogenic, food-grade lactic acid bacterium Lactococcus lactis subsp. cremoris that has been genetically engineered to produce human fibroblast growth factor 2,interleukin4 and colony stimulating factor 1. Designed to address different aspects of wound healing (i.e. fibroblast proliferation, angiogenesis and immune cell activation) and currently in phase I clinical study, we show how the combination of the individual components on the wound micro-environment initiates and improves the wound healing in chronic wounds.

Production of HIV-1 integrase fusion protein-carrying lentiviral vectors for gene therapy and protein transduction.

Lentiviral vectors have broad target cell tropism and efficient machinery to integrate transgenes into the host genome. Modification of these vectors by incorporating heterologous proteins into virions has relied mostly on the fusion of proteins into the HIV-1 accessory protein Vpr. Vpr expression can be harmful for cells and its gene has been deleted from third-generation vector production plasmids. We therefore developed a direct integrase fusion protein strategy as an alternative way to package heterologous proteins into vectors. The method was tested by creating two different integrase fusion proteins, IN-p53 and IN-mCherry, cloned into the 3' end of pol in the packaging plasmid. Lentiviral vectors were produced by conventional methods, using the modified packaging plasmids. Vector-incorporated fusion proteins were correctly processed from Gag-Pol, retained the ability to catalyze transgene integration, and showed fusion protein-specific activity by being fluorescent or inducing apoptosis. Functional third-generation lentiviral vectors containing IN-fusion proteins can thus be produced by standard production protocols independent of Vpr expression. Our results suggest that this packaging method is useful for lentiviral vector-mediated protein transduction, such as intranuclear meganuclease, transposon, or zinc finger protein delivery, intracellular imaging of vector particles, and generation of modified lentiviral vectors that contain both toxic and nontoxic IN-fusion proteins.

A 96-well format for a high-throughput baculovirus generation, fast titering and recombinant protein production in insect and mammalian cells.

BACKGROUND: Baculovirus expression vector system (BEVS) has become a standard in recombinant protein production and virus-like particle preparation for numerous applications. FINDINGS: We describe here protocols which adapt baculovirus generation into 96-well format. CONCLUSION: The established methodology allows simple baculovirus generation, fast virus titering within 18 h and efficient recombinant protein production in a high-throughput format. Furthermore, the produced baculovirus vectors are compatible with gene expression in vertebrate cells in vitro and in vivo.