title: Morpholino-mediated increase in soluble Flt-1 expression results in decreased ocular and tumor neovascularization.
abstract: BACKGROUND
  Angiogenesis is a key process in several ocular disorders and cancers. Soluble Flt-1 is an alternatively spliced form of the Flt-1 gene that retains the ligand-binding domain, but lacks the membrane-spanning and intracellular kinase domains of the full-length membrane bound Flt-1 (mbFlt-1) protein. Thus, sFlt-1 is an endogenous inhibitor of VEGF-A mediated angiogenesis. Synthetic mopholino oligomers directed against splice site targets can modulate splice variant expression. We hypothesize that morpholino-induced upregulation of sFlt-1 will suppress angiogenesis in clinically relevant models of macular degeneration and breast cancer.
  
  
  RESULTS
  In vivo morpholino constructs were designed to target murine exon/intron 13 junction of the Flt-1 transcript denoted VEGFR1_MOe13; standard nonspecific morpholino was used as control. After nucleofection of endothelial and breast adenocarcinoma cell lines, total RNA was extracted and real-time RT-PCR performed for sFlt-1 and mbFlt-1. Intravitreal injections of VEGFR1_MOe13 or control were done in a model of laser-induced choroidal neovascularization and intratumoral injections were performed in MBA-MD-231 xenografts in nude mice. VEGFR1_MOe13 elevated sFlt-1 mRNA expression and suppressed mbFlt-1 mRNA expression in vitro in multiple cellular backgrounds (p<0.001). VEGFR1_MOe13 also elevated sFlt/mbFlt-1 ratio in vivo after laser choroidal injury 5.5 fold (p<0.001) and suppressed laser-induced CNV by 50% (p = 0.0179). This latter effect was reversed by RNAi of sFlt-1, confirming specificity of morpholino activity through up-regulation of sFlt-1. In the xenograft model, VEGFR1_MOe13 regressed tumor volume by 88.9%, increased sFlt-1 mRNA expression, and reduced vascular density by 50% relative to control morpholino treatment (p<0.05).
  
  
  CONCLUSIONS
  Morpholino oligomers targeting the VEGFR1 mRNA exon/intron 13 junction promote production of soluble FLT-1 over membrane bound FLT-1, resulting in suppression of lesional volume in laser induced CNV and breast adenocarcinoma. Thus, morpholino manipulation of alternative splicing offers translational potential for therapy of angiogenic disorders.
authors.affiliation: Department Of Ophthalmology, Moran Eye Center, University of Utah, Salt Lake City, Utah, United States of America.
authors.forename: Leah A
authors.initials: LA
authors.lastname: Owen
authors.forename: Hironori
authors.initials: H
authors.lastname: Uehara
authors.forename: Judd
authors.initials: J
authors.lastname: Cahoon
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.forename: Jacquelyn
authors.initials: J
authors.lastname: Simonis
authors.forename: Balamurali K
authors.initials: BK
authors.lastname: Ambati
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D001943:Breast Neoplasms / Q000098:blood supply / Q000235:genetics / Q000628:therapy; D045744:Cell Line, Tumor; D020256:Choroidal Neovascularization / Q000235:genetics* / Q000517:prevention & control*; D005260:Female; D055785:Gene Knockdown Techniques; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D060172:Morpholinos / Q000235:genetics* / Q000627:therapeutic use*; D009389:Neovascularization, Pathologic / Q000235:genetics* / Q000517:prevention & control*; D034741:RNA, Small Interfering / Q000235:genetics; D012995:Solubility; D014183:Transplantation, Heterologous; D015854:Up-Regulation; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D060172:Morpholinos; D034741:RNA, Small Interfering; C501162:FLT1 protein, human; C501163:Flt1 protein, mouse; D040281:Vascular Endothelial Growth Factor Receptor-1