title: Eradication of Human Ovarian Cancer Cells by Transgenic Expression of RecombinantDNASE1, DNASE1L3, DNASE2,andDFFBControlled byEGFRPromoter: Novel Strategy for Targeted Therapy of Cancer.
abstract: BACKGROUND
  Ovarian cancer is the most deadly among all gynecological cancers. Patients undergoing systemic therapies of advanced ovarian cancers suffer from horrendous side effects. Cancer survivors and their offspring suffer from iatrogenic consequences of systemic therapies: genetic mutations. The ultimate goal of our work is development of therapies, which selectively and completely eliminate cancer cells, but do not harm healthy cells. An important consideration for attaining this goal is the fact that ovarian cancer cells over-express EGFR or its mutants, what becomes the factor discriminating them from healthy cells - a potential facilitator of personalized therapy.
  
  
  OBJECTIVE
  The specific aim of this project was threefold: (1) to bioengineer suicide genes' carrying vectors guided by synthetic antibodies for EGFRvIII and EGFR; (2) to genetically engineer DNA constructs for the human, recombinantDNASE1, DNASE1L3, DNASE2,andDFFBcontrolled by theEGFRpromoter; (3) to selectively eradicate ovarian cancer cells by intranuclear targeting of the transgenically expressed recombinant DNases.
  
  
  METHODS
  Synthetic antibodies for EGFR and EGFRvIII were selected from the human library and used to bioengineer biotag-guided transgenes' vectors. Coding sequences for the humanDNASE1, DNASE1L3, DNASE2, DFFBcontrolled by theEGFRpromoter were amplified from the human cDNA and genetically engineered into the plasmid constructs also coding for the fusions with NLS and GFP. The vectors carrying transgenes for the DNases were deliveredin vitrointo human ovarian cancer cells from ascites and cultures.
  
  
  RESULTS
  Synthetic antibody guided vectors delivered the transgenes for the recombinant DNases efficiently into the ovarian cancer cells. Transgenic expression and nuclear targeting of the DNases in those cells resulted in destruction of their genomes and led to their death, as validated by labeling with the molecular death tags. In healthy cells, which did not over-expressEGFR, no changes were recorded.
  
  
  CONCLUSIONS
  Targeted expression of the recombinantDNASE1, DNASE1L3, DNASE2, DFFBin the ovarian cancersin vitroresulted in their complete eradication, but had no effects upon the healthy cells. This novel therapeutic strategy has a potential for streamlining it intoin vivotrials, as personalized, targeted therapy of ovarian and other cancers.
authors.affiliation: PBMEF, San Francisco, CA 94105, USA ; NMRFM, NIH, Madison, WI 53706, USA ; UW, Madison, WI 53706, USA.
authors.forename: Marek
authors.initials: M
authors.lastname: Malecki
authors.affiliation: UW, Madison, WI 53706, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Dahlke
authors.affiliation: UW, Madison, WI 53706, USA.
authors.forename: Melissa
authors.initials: M
authors.lastname: Haig
authors.affiliation: UW, Madison, WI 53706, USA.
authors.forename: Lynn
authors.initials: L
authors.lastname: Wohlwend
authors.affiliation: PBMEF, San Francisco, CA 94105, USA ; SFSU, San Francisco, CA 94132, USA.
authors.forename: Raf
authors.initials: R
authors.lastname: Malecki
keywords: Apoptosis; Cancer of the ovary; DNase; Epidermal growth factor receptor; Nuclear localization signal; Personalized therapy of cancer; Suicide gene therapy of cancer