No abstract

Antisense morpholino oligonucleotides (MO) have been used successfully in zebrafish and Xenopus to knock down gene function by gene-specific inhibition of mRNA translation (Ekker, 2000). In addition to their ability to block cytosolic processes, MO can enter the nucleus (Partridge et al., 1996) and have been shown to be effective inhibitors of pre-mRNA splicing in mammalian tissue-culture cell lines (Schmajuk et al., 1999). We show here that MO efficiently block pre-mRNA splicing in zebrafish embryos. Splice-blocking MO have the advantages that the efficacy of gene knockdown can be quantified without the use of antibodies, and that they specifically target zygotic, and not maternal, transcripts.We targeted the fgf8 gene (Furthauer et al., 1997;Reifers et al., 1998) with splice-blocking MO. An ENU induced mutation in fgf8, acerebellar (ace; referred to here as fgf8 it282 ), has previously been described (Reifers et al., 1998). fgf8 it282 is a splice donor mutation that results in the production of an aberrantly spliced mRNA (Reifers et al., 1998). Hence, blocking the same splicing event using MO should result in a phenotype similar to fgf8 it282 .We determined the fgf8 exon/intron structure and designed two 25-mer MO complementary to the exon 2 and exon 3 splice donor sites (designated E2I2 and E3I3, respectively; Fig. 1a). Both MO span the exon/intron junction, including the most conserved residues of the splice donor consensus sequence (Fig. 1 legend).We first asked whether splice site-targeted MO can alter splicing of fgf8 mRNA. Using reverse transcriptase polymerase chain reaction, we found that injection of the E3I3 MO into early zebrafish embryos results in the production of two aberrantly spliced messages we term variant 1 and 2 (Fig. 1a, b). Sequence analysis of the corresponding cDNAs revealed that variant 1 results from excluding exon 3, whereas variant 2 results from the use of a cryptic splice donor located 32 bases 5Ј of the normal exon 3 splice donor (Fig. 1c). Use of the cryptic splice donor creates a premature termination codon at the novel splice junction (Fig. 1c). Additionally, we found that injected splice site-targeted MO only alter the structure of zygotically expressed, but not maternally supplied, mRNA, as expected (Fig. 2d). Thus, E3I3 can

The vertebrate inner ear develops from the otic placode, an ectodermal thickening that forms adjacent to the presumptive hindbrain. Previous studies have suggested that competent ectodermal cells respond to signals from adjacent tissues to form the placode. Members of the Fgf family of growth factors and the Dlx family of transcription factors have been implicated in this signal-response pathway. We show that compromising Fgf3 and Fgf8 signaling blocks ear development; only a few scattered otic cells form. Removal of dlx3b, dlx4b and sox9a genes together also blocks ear development, although a few residual cells form an otic epithelium. These cells fail to form if sox9b function is also blocked. Combined loss of Fgf signaling and the three transcription factor genes, dlx3b,dlx4b and sox9a, also completely eliminates all indications of otic cells. Expression of sox9a but not dlx3b, dlx4b or sox9b requires Fgf3 and Fgf8. Our results provide evidence for Fgf3-and Fgf8-dependent and -independent genetic pathways for otic specification and support the notion that Fgf3 and Fgf8 function to induce both the otic placode and the epithelial organization of the otic vesicle.

We have developed a new transporter structure that provides effective delivery of Morpholino antisense oligomers into a wide variety of tissues in living mice. This transporter comprises a dendritic structure assembled around a triazine core which serves to position eight guanidinium head groups in a conformation effective to penetrate cell membranes. This transporter structure is conjugated to a Morpholino oligomer to form a delivery-enabled product referred to as a Vivo-Morpholino. Vivo-Morpholinos are shown to effectively enter and function within cultured cells in the presence of 100% serum using a rigorous positive test system based on correction of a defined splicing error in a pre-messenger RNA. In addition, Vivo-Morpholinos are demonstrated to enter into a wide variety of tissues in a similar positive test system in transgenic mice, as evidenced by correction of the targeted splicing error in all tissues assessed, including near-complete splice correction in the small intestine, colon, stomach, liver kidney, and a number of muscles. Finally, Vivo-Morpholinos, which target the exon-skipping of exon 23 harboring a premature termination codon in the mdx mouse model, effectively restore the reading frame of dystrophin and restore expression of a functional dystrophin protein.

Steric-block antisense oligonucleotides (AONs) are able to target RNAs for destruction and splicing alteration. Reading frame restoration of the dystrophin transcript can be achieved by AON-mediated exon skipping in the dystrophic mdx mouse model. However, simple, unmodified AONs exhibit inefficient delivery systemically, leading to dystrophin induction with high variability in skeletal muscles and barely detectable in cardiac muscle. Here, we examined a Morpholino oligomer conjugated with a dendrimeric octaguanidine (Vivo-Morpholino) and demonstrated that the delivery moiety significantly improved dystrophin production in both skeletal and cardiac muscles in mdx mice in vivo. Single intravenous (IV) injections of 6 mg/kg Vivo-MorpholinoE23 (Vivo-ME23) generated dystrophin expression in skeletal muscles at the levels higher than the injection of 300 mg/kg unmodified ME23. Repeated injections at biweekly intervals achieved near 100% of fibers expressing dystrophin in skeletal muscles bodywide without eliciting a detectable immune response. Dystrophin protein was restored to approximately 50 and 10% of normal levels in skeletal and cardiac muscles, respectively. Vivo-Morpholinos showed no signs of toxicity with the effective dosages and regime, thus offering realistic prospects for the treatment of a majority of Duchenne muscular dystrophy (DMD) patients and many other diseases by targeting RNAs.