Towards next generation maggot debridement therapy: transgenic Lucilia sericata larvae that produce and secrete a human growth factor

Abstract

Background: Diabetes and its concurrent complications impact a significant proportion of the population of the US and create a large financial burden on the American health care system. FDA-approved maggot debridement therapy (MDT), the application of sterile laboratory-reared Lucilia sericata (green bottle fly) larvae to wounds, is a cost-effective and successful treatment for diabetic foot ulcers and other medical conditions. Human platelet derived growth factor-BB (PDGF-BB) is a secreted dimeric peptide growth factor that binds the PDGF receptor. PDGF-BB stimulates cell proliferation and survival, promotes wound healing, and has been investigated as a possible topical treatment for non-healing wounds. Genetic engineering has allowed for expression and secretion of human growth factors and other proteins in transgenic insects. Here, we present a novel concept in MDT technology that combines the established benefits of MDT with the power of genetic engineering to promote healing. The focus of this study is to create and characterize strains of transgenic L. sericata that express and secrete PDGF-BB at detectable levels in adult hemolymph, whole larval lysate, and maggot excretions/ secretions (ES), with potential for clinical utility in wound healing.

Results: We have engineered and confirmed transgene insertion in several strains of L. sericata that express human PDGF-BB. Using a heat-inducible promoter to control the pdgf-b gene, pdgf-b mRNA was detected via semi-quantitative PCR upon heat shock. PDGF-BB protein was also detectable in larval lysates and adult hemolymph but not larval ES. An alternative, tetracycline-repressible pdgf-b system mediated expression of pdgf-b mRNA when maggots were raised on diet that lacked tetracycline. Further, PDGF-BB protein was readily detected in whole larval lysate as well as larval ES.

Conclusions: Here we show robust, inducible expression and production of human PDGF-BB protein from two conditional expression systems in transgenic L. sericata larvae. The tetracycline-repressible system appears to be the most promising as PDGF-BB protein was detectable in larval ES following induction. Our system could potentially be used to deliver a variety of growth factors and anti-microbial peptides to the wound environment with the aim of enhancing wound healing, thereby improving patient outcome in a cost-effective manner.

Keywords: Diabetic foot ulcer; Excretions/secretions (ES); Growth factor treatment; Lucilia sericata; Maggot debridement therapy (MDT); Platelet-derived growth factor (PDGF); Tetracycline transactivator; Wound healing.

Fig. 1

Heat inducible expression of pdgf-b mRNA in transgenic PD-1 L. sericata. a Schematic of heat-inducible pdgf-b gene construct in a piggyBac transformation vector with a ZsGreen marker gene. b Genomic DNA sequence adjacent to the 5' pBac end in the PD-1 transgenic line. The TTAA insertion site is underlined. c RT-PCR amplification of pdgf-b on total RNA obtained from first instar PD-1 larvae that had been given a heat shock (+HS) or no heat shock (-HS)

Fig. 2

PDGF-BB protein is inducible in transgenic PD-1 L. sericata lysate and hemolymph. a Mean PDGF-BB concentration in wild type (wt) and PD-1 whole larval lysate under control and heat shock conditions. b PDGF-BB concentration in pooled wt or PD-1 adult hemolymph samples after heat shock. Data from two replicate experiments are shown

Fig. 3

tTA-mediated pdgf-b expression in transgenic L. sericata. a Schematic of the DR4 tTA driver and EF-PDGF tTA-regulated effector gene constructs in piggyBac transformation vectors. b Genomic DNA sequence adjacent to 3' pBac for each strain. c DR4#14, EF-PDGF#11, and DR4#14 + EF-PDGF#11 larvae under white light. d Relative expression of pdgf-b mRNA in control effector alone and tTA-driver plus effector larvae. qRT-PCR analysis was performed on RNA isolated from whole larvae and normalized to the 28 s rRNA reference gene

Fig. 4

PDGF-BB protein is detectable in larval lysate and ES of larvae that carry both tTA driver (DR4) and tTA-regulated pdgf-b (EF-PDGF) transgenes. a Mean PDGF-BB concentration in control effector-alone and tTA-driver plus effector larvae. b Mean PDGF-BB concentration in effector-alone and tTA-driver plus effector larval ES