Department Of Ophthalmology, Moran Eye Center, University of Utah, Salt Lake City, Utah, United States of America.
PLoS One. 2012;7(3):e33576. doi: 10.1371/journal.pone.0033576. Epub 2012 Mar 15.
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.
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).
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.
血管生成是几种眼部疾病和癌症的关键过程。可溶性 Flt-1 是 Flt-1 基因的一种选择性剪接形式，保留了配体结合域，但缺乏全长膜结合 Flt-1（mbFlt-1）蛋白的跨膜和细胞内激酶结构域。因此，sFlt-1 是 VEGF-A 介导的血管生成的内源性抑制剂。针对剪接位点靶标的合成 mopholino 寡核苷酸可调节剪接变体的表达。我们假设，morpholino 诱导的 sFlt-1 上调将抑制黄斑变性和乳腺癌的临床相关模型中的血管生成。
设计了体内 morpholino 构建体以靶向 Flt-1 转录物的鼠外显子/内含子 13 接头，标记为 VEGFR1_MOe13；标准非特异性 morpholino 用作对照。在内皮细胞和乳腺癌腺癌细胞系的核转染后，提取总 RNA 并进行实时 RT-PCR 以检测 sFlt-1 和 mbFlt-1。在激光诱导脉络膜新生血管形成模型中进行 VEGFR1_MOe13 或对照的眼内注射，在裸鼠的 MBA-MD-231 异种移植瘤中进行瘤内注射。VEGFR1_MOe13 在体外多种细胞背景下提高 sFlt-1 mRNA 表达并抑制 mbFlt-1 mRNA 表达（p<0.001）。VEGFR1_MOe13 还使激光脉络膜损伤后 sFlt/mbFlt-1 比值升高 5.5 倍（p<0.001），并抑制激光诱导的 CNV 50%（p=0.0179）。通过 sFlt-1 的 RNAi 逆转了后者的作用，通过上调 sFlt-1 证实了 morpholino 活性的特异性。在异种移植瘤模型中，VEGFR1_MOe13 使肿瘤体积缩小 88.9%，sFlt-1 mRNA 表达增加，并使血管密度降低 50%，与对照 morpholino 治疗相比（p<0.05）。
针对 VEGFR1 mRNA 外显子/内含子 13 接头的 morpholino 寡核苷酸促进可溶性 FLT-1 的产生超过膜结合 FLT-1，从而抑制激光诱导的 CNV 和乳腺癌中的病变体积。因此，剪接的 morpholino 操作为血管生成性疾病的治疗提供了转化潜力。
