Recombinant plasmid-hepatocyte growth factor(Academy of Military Medical Sciences)

Neuropathic pain (NP) is a refractory disease in the clinic with a tremendous impact on the quality of life of patients. Gene therapy is a potential strategy for the management of NP. In the present study, we examined the analgesic effect and mechanism of hepatocyte growth factor (HGF) in vitro and in vivo.

We examined the proinflammatroy gene changes in lipopolysaccharide (LPS)‐induced microglia BV2 cells with a quantitative real‐time polymerase chain reaction of interleukin (IL)‐1β, IL‐6, tumor necrosis factor (TNF)‐α and inducible nitric oxide synthase (iNOS). Mechanical stimulation tests were performed five times at 5‐min intervals to assess pain thresholds using Von Frey Hair in mice following spared nerve injury (SNI). The glial cell activation of spinal cord was examined by western blotting. Statistical significance was determined by a Tukey's test and a paired t‐test.

We found that recombinant human HGF protein suppressed LPS‐induced BV2 cell activation in vitro, marked by the down‐regulation of IL‐1β, IL‐6, TNF‐α and iNOS expression, as well as decrease of nitric oxide production. Moreover, intrathecal injection of naked plasmid encoding HGF gene (pUDK‐HGF) significantly attenuated SNI‐induced pain behaviors in mice by direct inhibition of spinal cord microglia and astrocyte activation.

The results of the present study indicate that pUDK‐HGF can reduce cytotoxicity products released from activated glial cells, which may provide a promising therapeutic strategy for treating NP.

The demand of a plasmid encoding human hepatocyte growth factor gene (pUDK-HGF) in large quantities at high purity and concentration has increased for gene therapy of critical limb ischemia (CLI) in clinical trials. In this article, we produced pUDK-HGF in compliance with current good manufacturing practices at gram scale. The process included a 50-L batch fermentation, continuous alkaline lysis, and integrated three-step chromatography on Sepharose 6 Fast Flow, PlasmidSelect Xtra, and Source 15Q. The production process has been scaled up to yield 4.24 ± 0.41 g of pharmaceutical pUDK-HGF from 1.0 kg bacterial cell paste and the overall yield reached range from 58.37 to 66.70%. The final pUDK-HGF product exhibited high purity with supercoiled percentage of > 95.8% and undetectable residual RNA, contaminated protein, and bacterial endotoxin. The phase I clinical study indicates that intramuscular injection of pUDK-HGF is safe, well tolerated, and may provide symptomatic relief to CLI patients. These results show that our manufacturing process of pUDK-HGF is efficient in producing pharmaceutical-grade plasmid DNA and is safe for clinical applications.