Epidermal Growth Factor Receptor-targeted Gene Delivery System

Besides surgery, radiotherapy and chemotherapy, gene therapy is potential to be another way for cancer therapy. It may play an important role in preventing cancer from recurrence and metastasis. However, the main issue remains to be resolved is lack of an efficient targeting gene transfer system. Therefore, it is very urgent to develop an efficient targeting gene delivery vector. Since overexpression of EGFR has been frequently found in a wide spectrum of human tumors of epithelial origin, we have attempted to construct efficient EGFR-mediated gene delivery vectors. It will provide new technologies and practical guidance for targeting cancer gene therapy.Section IIdentification of peptide ligands of EGFR by screening phagedisplay peptide libraries[Objective] To obtain candidate peptide ligands of EGFR. [Methods] EGFR proteins purified from A431 cell line or U87 cells stably expressing EGFRvIII were used as targets for screening phage display peptide library. After several rounds of screening, phage clones were sequenced. Phage recovery assay and immunostaining were used to characterize the enriched phage clones; The internalization of the positive phage clones were further analyzed. [Results] This screening yielded an enriched phage encoding an amino acid sequence, YHWYGYTPQNVI, designated as GE11. In phage recovery assay, Phage-GEll bound efficiently to EGFR and this binding can be inhibited by excessive EGF or GE11 peptides. Furthermore, binding of Phage-GEll to EGFR expressing cells SMMC-7721 and NCI-H460 was detected whereas binding was barely detectable in K-562 cells which has no EGFR expression. In addition, Phage-GEll is able to be internalizaed into EGFR positive cells, SMMC-7721. However, no positive phage-GE11 clones were found when using U87-ExERvIII which stablyexpressing extracellular region of EGFRvIII as the panning target. [Conclusion! Phage display technique is plausible to obtain candidate peptide ligand of receptor protein especially when using purified receptor protein as the target; Phage-GEll can specific bind to EGFR and be efficiently internalized by EGFR expressing cells.Section II Characterization of GE11 peptide[Objective] To characterize the biological activities of GE11 peptide.[Methods!: GE11 peptide was labeled with 125I. In vitro binding assay was performed on SMMC-7721 cells or purified EGFR protein; In vivo distribution assay was carried out in athymic mice bearing SMMC-7721 tumor xenografts. MTT assay was performed to measure its biological activity. Internalization assay was performed to analyze its capability of translocation across cell membrane. [ Results ] 125I—GE11 can bind to SMMC-7721 cell and EGFR protein. The binding can be competed by free EGF and GE11 peptides. In addition, GE11 peptide can compete the binding of EGF to EGFR. The dissociation constant (Kd) of GE11 is about 22.28 ±0.4 nM. The radioactivity in tumor was highest among the examined tissues 4 hours after intravenously administration of 125I-GE11. The radioactivities in tumor can be inhibited by several folds when co-injected with excessive free peptide GE11. In MTT assay, GE11 showed about 10% growth stimulating effect on SMMC-7721 cells while EGF showed about 50% of this effect. In the end, FITC labeled GE11 can be internalized into cells overexpressing EGFR; this internalization can be inhibited by free peptide GE11 or EGF. [Conclusion! GE11 is a novel peptide ligand of EGFR. It is potential to be used in EGFR-targeted cancer therapy.Section IIIDevelopment of PEI based nonviral gene delivery system usingGEll as targeting element[Objective] To examine the targeting ability of GEll when it is used in a nonviral gene delivery vector. [Methods] GEL (GEll coupled a linker sequence (GGGGS)3 ) was conjugated to linear PEI (22 kd) with homobifunctional crosslinking agent DSP. The resulted PEI, gPEI was used to condense pGL3 plasmid (encoding luciferase). Gel retardation assay, DNase protection assay and size analysis were used to characterize the DNA polyplexes. In in vitro assay, gPEI/pGL3 and PEI/pGL3 complexes were transducted into SMMO7721 cells. In competitive study, free GEll peptides were co-incubated with the DNA complexes.After 24 hours, the activity of luciferase was measured in relative light units (RLU)/mg protein. In in vivo assay, gPEI/pGL3, PEI/pGL3 complex were injected via tail vein into mice bearing SMMC-7721 tumor xenografts. 24 hours later, intestine, brain, heart, liver, spleen, lung, kidney and tumors were excised. In the competition assay, 100 y g free peptide GEll was co-injected. The luciferase activities were measured. [Results] gPEI was capable of packaging plasmid DNA. When N/P ration is up to 5, most of the DNA was protected against the digestion of DNase I. The size of the gPEI/DNA is about 120 nm when the N/P ratio is equal to 5. Both gPEI/DNA and PEI/DNA polyplexes can transduct genes efficiently into SMMC-7721 cells in vitro. No significant difference was found between these two polyplexes. However, free GEll peptides can inhibit the transduction of gPEI/pGL3 by 3-7 folds while no inhibition was found in PEI/pGL3. In in vivo transduction assay, the luciferase activities in tumors occurred in gPEI/pGL3 polyplexes were 18-fold higher than those occurred in PEI/pGL3 polyplexes and more than ten-fold higher than those activities in the other examined tissues. [Conclusion] PEI based GEll-targeting gene delivery vectors can transfer reporter genes to EGFR overexpressing cells or tumors.