Morpholino-Mediated Increase in Soluble Flt-1 Expression Results in Decreased Ocular and Tumor Neovascularization

Abstract

BackgroundAngiogenesis 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.Methods and Findings 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).ConclusionsMorpholino 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.

The corpora lutea proangiogenic state of VEGF system components is turned to antiangiogenic at the later phase of the oestrous cycle in cows

“…The soluble forms of the VEGF receptors bind to the ligand and block their angiogenic action (Kaczmarek et al, 2005). Cancer therapy studies have shown that an increase in the expression of sVEGFR1 (Owen et al, 2012) or sVEGFR2 (Wang et al, 2006;Szentirmai et al, 2008) reduce tumour cell proliferation and increase apoptosis (Szentirmai et al, 2008), which is associated with a reduction in blood flow to the tumour tissues. In the present study, mRNA expression of VEGFR2 on days 4 and 6 (CL formation) of the cycle was at least eightfold higher than that Figure 2 Changes in the ratio of mRNA expression for angiogenic (VEGF120a, VEGFR1 and VEGFR2) and antiangiogenic (VEGF120b, sVEGFR1 and sVEGFR2) VEGF system components in the corpus luteum during the oestrous cycle.…”

Targeting RNA splicing for disease therapy

“…In some cases redirection of splicing to an aberrant isoform that is not normally generated from a gene transcript can be an effective means to eliminate a detrimental RNA isoform that is implicated in disease (Table 1, detrimental gene expression). Some of the more advanced studies, showing promise in animal models, include ASOs targeting TNFRSF1B 112 for the treatment of inflammatory disease, and FLT1 92 as a cancer therapy.…”

Vivo-Morpholinos Induced Transient Knockdown of Physical Activity Related Proteins

“…To date there are only twenty-eight studies using Vivo-morpholinos [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44] and all have reported at least 50% knockdown of the target gene with no adverse side effects. Fourteen of these studies used a mouse model [17], [18], [19], [20], [21], [24], [31], [32], [34], [35], [40], [41], [42], [43] with the remaining studies using rats [36], [37], newts [29], chicken embryos [27], fish [15], [16], [22], [23], [25], [26] tadpoles [30], [38], or frogs [28].…”