Delivery is key: lessons learnt from developing splice-switching antisense therapies.

The use of splice-switching antisense therapy is highly promising, with a wealth of pre-clinical data and numerous clinical trials ongoing. Nevertheless, its potential to treat a variety of disorders has yet to be realized. The main obstacle impeding the clinical translation of this approach is the relatively poor delivery of antisense oligonucleotides to target tissues after systemic delivery. We are a group of researchers closely involved in the development of these therapies and would like to communicate our discussions concerning the validity of standard methodologies currently used in their pre-clinical development, the gaps in current knowledge and the pertinent challenges facing the field. We therefore make recommendations in order to focus future research efforts and facilitate a wider application of therapeutic antisense oligonucleotides.

Distinct patterns of heparan sulphate in pancreatic islets suggest novel roles in paracrine islet regulation.

Heparan sulphate proteoglycans (HSPGs) exist in pancreatic beta cells, and HS seems to modulate important interactions in the islet microenvironment. However, the intra-islet structures of HS in health or altered glucose homeostasis are currently unknown. Here we show that distinct spatial distribution of HS motifs is present in islets in the adult, that intra-islet HS motifs are mostly conserved between rodents and humans, and that HS is abundant in glucagon producing islet alpha cells. In beta cells HS is characterised by 2-O, 6-O and N-sulphated moieties, whereas HS in alpha cells is N-acetylated, N-, and 2-O sulphated and low in 6-O groups. Differential expression of three HS modifying genes in alpha and beta cells was observed and may account for the different HS patterns. Furthermore, we found that FGF1 and FGF2 were present in alpha cells, whereas functional FGFRs exist in beta cells, but not in the alpha cell line aTC1-6, or in primary alpha cells in islets. FGF1 induced signalling was dependent on 2-O, and 6-O HS sulphation in beta cells, and HS desulphation reduced beta cell proliferation and potentiated oxidant induced apoptosis. In leptin resistant animals and in islets from streptozotocin treated rats there was a reduction in alpha cell HS expression. These data demonstrate the distinct HS expression patterns in alpha and beta islet cells and propose a novel role for alpha cells as a source of paracrine FGF ligands to neighbouring beta cells with specific cell-associated HS domains mediating the activation and diffusion of paracrine ligands.

Development of therapeutic splice-switching oligonucleotides.

Synthetic splice-switching oligonucleotides (SSOs) target nuclear pre-mRNA molecules to change exon splicing and generate an alternative protein isoform. Clinical trials with two competitive SSO drugs are underway to treat Duchenne muscular dystrophy (DMD). Beyond DMD, many additional therapeutic applications are possible, with some in phase 1 clinical trials or advanced preclinical evaluation. Here, we present an overview of the central factors involved in developing therapeutic SSOs for the treatment of diseases. The selection of susceptible pre-mRNA target sequences, as well as the design and chemical modification of SSOs to increase SSO stability and effectiveness, are key initial considerations. Identification of effective SSO target sequences is still largely empirical and published guidelines are not a universal guarantee for success. Specifically, exon-targeted SSOs, which are successful in modifying dystrophin splicing, can be ineffective for splice-switching in other contexts. Chemical modifications, importantly, are associated with certain characteristic toxicities, which need to be addressed as target diseases require chronic treatment with SSOs. Moreover, SSO delivery in adequate quantities to the nucleus of target cells without toxicity can prove difficult. Last, the means by which these SSOs are administered needs to be acceptable to the patient. Engineering an efficient therapeutic SSO, therefore, necessarily entails a compromise between desirable qualities and effectiveness. Here, we describe how the application of optimal solutions may differ from case to case.

Pathogenetic mechanisms of amyloid A amyloidosis.

Systemic amyloid A (AA) amyloidosis is a serious complication of chronic inflammation. Serum AA protein (SAA), an acute phase plasma protein, is deposited extracellularly as insoluble amyloid fibrils that damage tissue structure and function. Clinical AA amyloidosis is typically preceded by many years of active inflammation before presenting, most commonly with renal involvement. Using dose-dependent, doxycycline-inducible transgenic expression of SAA in mice, we show that AA amyloid deposition can occur independently of inflammation and that the time before amyloid deposition is determined by the circulating SAA concentration. High level SAA expression induced amyloidosis in all mice after a short, slightly variable delay. SAA was rapidly incorporated into amyloid, acutely reducing circulating SAA concentrations by up to 90%. Prolonged modest SAA overexpression occasionally produced amyloidosis after long delays and primed most mice for explosive amyloidosis when SAA production subsequently increased. Endogenous priming and bulk amyloid deposition are thus separable events, each sensitive to plasma SAA concentration. Amyloid deposits slowly regressed with restoration of normal SAA production after doxycycline withdrawal. Reinduction of SAA overproduction revealed that, following amyloid regression, all mice were primed, especially for rapid glomerular amyloid deposition leading to renal failure, closely resembling the rapid onset of renal failure in clinical AA amyloidosis following acute exacerbation of inflammation. Clinical AA amyloidosis rarely involves the heart, but amyloidotic SAA transgenic mice consistently had minor cardiac amyloid deposits, enabling us to extend to the heart the demonstrable efficacy of our unique antibody therapy for elimination of visceral amyloid.

Exon skipping of hepatic APOB pre-mRNA with splice-switching oligonucleotides reduces LDL cholesterol in vivo.

Familial hypercholesterolemia (FH) is a genetic disorder characterized by extremely high levels of plasma low-density lipoprotein (LDL), due to defective LDL receptor-apolipoprotein B (APOB) binding. Current therapies such as statins or LDL apheresis for homozygous FH are insufficiently efficacious at lowering LDL cholesterol or are expensive. Treatments that target APOB100, the structural protein of LDL particles, are potential therapies for FH. We have developed a series of APOB-directed splice-switching oligonucleotides (SSOs) that cause the expression of APOB87, a truncated isoform of APOB100. APOB87, like similarly truncated isoforms expressed in patients with a different condition, familial hypobetalipoproteinemia, lowers LDL cholesterol by inhibiting very low-density lipoprotein (VLDL) assembly and increasing LDL clearance. We demonstrate that these "APO-skip " SSOs induce high levels of exon skipping and expression of the APOB87 isoform, but do not substantially inhibit APOB48 expression in cell lines. A single injection of an optimized APO-skip SSO into mice transgenic for human APOB resulted in abundant exon skipping that persists for >6 days. Weekly treatments generated a sustained reduction in LDL cholesterol levels of 34-51% in these mice, superior to pravastatin in a head-to-head comparison. These results validate APO-skip SSOs as a candidate therapy for FH.

Antisense-mediated exon-skipping to induce gene knockdown.

Exon-skipping antisense oligonucleotides (ASOs) can be used to knockdown the expression of an undesired gene or specific gene isoform. This chapter discusses the potential therapeutic applications of the technique and provides a sample protocol for inducing exon-skipping in Apolipoprotein B in vitro, as well as a protocol for quantifying exon-skipping using real-time PCR.

Oligonucleotide-mediated gene editing is underestimated in cells expressing mutated green fluorescent protein and is positively associated with target protein expression.

BACKGROUND: Single-stranded DNA oligonucleotides (ssODNs) can introduce small, specific sequence alterations into genomes. Potential applications include creating disease-associated mutations in cell lines or animals, functional studies of single nucleotide polymorphisms and, ultimately, clinical therapy by correcting genetic point mutations. Here, we report feasibility studies into realizing this potential by targeting a reporter gene, mutated enhanced green fluorescent protein (mEGFP). METHODS: Three mammalian cell lines, CHO, HEK293T and HepG2, expressing multiple copies of mEGFP were transfected with a 27-mer ssODN capable of restoring fluorescence. Successful cell correction was quantified by flow cytometry. RESULTS: Gene editing in each isogenic cell line, as measured by percentage of green cells, correlated tightly with target protein levels, and thus gene expression. In the total population, 2.5% of CHO-mEGFP cells were successfully edited, although, remarkably, in the highest decile producing mEGFP protein, over 20% of the cells had restored green fluorescence. Gene-edited clones initially selected for green fluorescence lost EGFP expression during cell passaging, which partly reflected G2-phase cycle arrest and perhaps eventual cell death. The major cause, however, was epigenetic down-regulation; incubation with sodium butyrate or 5-aza-2'-deoxycytidine reactivated fluorescent EGFP expression and hence established that the repaired genotype was stable. CONCLUSIONS: Our data establish that ssODN-mediated gene editing is underestimated in cultured mammalian cells expressing nonfluorescent mutated EGFP, because of variable expression of this mEGFP target gene in the cell population. This conclusion was endorsed by studies in HEK293T-mEGFP and HepG2-mEGFP cells. We infer that oligonucleotide-directed editing of endogenous genes is feasible, particularly for those that are transcriptionally active.

Use of internally nuclease-protected single-strand DNA oligonucleotides and silencing of the mismatch repair protein, MSH2, enhances the replication of corrected cells following gene editing.

BACKGROUND: Gene editing is potentially a powerful technology for introducing genetic changes by using short single-stranded DNA oligonucleotides (ssODNs). However, their efficiency is reduced by the mismatch repair system, especially MSH2, which may suppress gene editing, although findings vary depending on readout and type of oligonucleotide used. Additionally, successfully edited cells are reported to arrest at the S- or G2-phase. In the present study, we evaluate whether a novel ssODN design and down-regulation of MSH2 expression allows the isolation of replicating gene-edited cells. METHODS: Cultured Chinese hamster ovary cells expressing mutated enhanced green fluorescent protein were targeted with ssODNs of varying design, all capable of restoring fluorescence, which allows the monitoring of correction events by flow cytometry. Converted cells were isolated by cell sorting and grown to determine colony formation efficiencies. MSH2 expression was suppressed with small interfering RNA and the cell cycle distribution of cells transfected with ssODN was quantified by flow cytometry, following propidium iodide or DRAQ5 staining. RESULTS: Although efficiency was higher using ssODN end-protected with phosphorothioate, the potential of edited cells to form colonies was lower than those targeted with unmodified ssODN. We established that ssODN transfection itself perturbs the cell cycle and that MSH2 gene silencing increases correction efficiency. In both cases, however, the effect was dependent on the positioning of the protected nucleotides. Importantly, when internally protected ssODN was used in combination with MSH2 suppression, a higher proportion of G1-phase corrected cells was observed 48-64 h after transfection. CONCLUSIONS: Use of internally protected ssODN and downregulating cellular MSH2 activity may facilitate isolation of viable, actively replicating gene-edited cells.

The presence of valine at residue 129 in human prion protein accelerates amyloid formation.

The polymorphism at residue 129 of the human PRNP gene modulates disease susceptibility and the clinico-pathological phenotypes in human transmissible spongiform encephalopathies. The molecular mechanisms by which the effect of this polymorphism are mediated remain unclear. It has been shown that the folding, dynamics and stability of the physiological, alpha-helix-rich form of recombinant PrP are not affected by codon 129 polymorphism. Consistent with this, we have recently shown that the kinetics of amyloid formation do not differ between protein containing methionine at codon 129 and valine at codon 129 when the reaction is initiated from the alpha-monomeric PrP(C)-like state. In contrast, we have shown that the misfolding pathway leading to the formation of beta-sheet-rich, soluble oligomer was favoured by the presence of methionine, compared with valine, at position 129. In the present work, we examine the effect of this polymorphism on the kinetics of an alternative misfolding pathway, that of amyloid formation using partially folded PrP allelomorphs. We show that the valine 129 allelomorph forms amyloids with a considerably shorter lag phase than the methionine 129 allelomorph both under spontaneous conditions and when seeded with pre-formed amyloid fibres. Taken together, our studies demonstrate that the effect of the codon 129 polymorphism depends on the specific misfolding pathway and on the initial conformation of the protein. The inverse propensities of the two allelomorphs to misfold in vitro through the alternative oligomeric and amyloidogenic pathways could explain some aspects of prion diseases linked to this polymorphism such as age at onset and disease incubation time.

Methionine 129 variant of human prion protein oligomerizes more rapidly than the valine 129 variant: implications for disease susceptibility to Creutzfeldt-Jakob disease.

The human PrP gene (PRNP) has two common alleles that encode either methionine or valine at codon 129. This polymorphism modulates disease susceptibility and phenotype of human transmissible spongiform encyphalopathies, but the molecular mechanism by which these effects are mediated remains unclear. Here, we compared the misfolding pathway that leads to the formation of beta-sheet-rich oligomeric isoforms of the methionine 129 variant of PrP to that of the valine 129 variant. We provide evidence for differences in the folding behavior between the two variants at the early stages of oligomer formation. We show that Met(129) has a higher propensity to form beta-sheet-rich oligomers, whereas Val(129) has a higher tendency to fold into alpha-helical-rich monomers. An equimolar mixture of both variants displayed an intermidate folding behavior. We show that the oligomers of both variants are initially a mixture of alpha- and beta-rich conformers that evolve with time to an increasingly homogeneous beta-rich form. This maturation process, which involves no further change in proteinase K resistance, occurs more rapidly in the Met(129) form than the Val(129) form. Although the involvement of such beta-rich oligomers in prion pathogenesis is speculative, the misfolding behavior could, in part, explain the higher susceptibility of individuals that are methionine homozygote to both sporadic and variant Creutzfeldt-Jakob disease.

Multigene RNA vector based on coronavirus transcription.

Coronavirus genomes are the largest known autonomously replicating RNAs with a size of ca. 30 kb. They are of positive polarity and are translated to produce the viral proteins needed for the assembly of an active replicase-transcriptase complex. In addition to replicating the genomic RNA, a key feature of this complex is a unique transcription process that results in the synthesis of a nested set of six to eight subgenomic mRNAs. These subgenomic mRNAs are produced in constant but nonequimolar amounts and, in general, each is translated to produce a single protein. To take advantage of these features, we have developed a multigene expression vector based on human coronavirus 229E. We have constructed a prototype RNA vector containing the 5' and 3' ends of the human coronavirus genome, the entire human coronavirus replicase gene, and three reporter genes (i.e., the chloramphenicol acetyltransferase [CAT] gene, the firefly luciferase [LUC] gene, and the green fluorescent protein [GFP] gene). Each reporter gene is located downstream of a human coronavirus transcription-associated sequence, which is required for the synthesis of individual subgenomic mRNAs. The transfection of vector RNA and human coronavirus nucleocapsid protein mRNA into BHK-21 cells resulted in the expression of the CAT, LUC, and GFP reporter proteins. Sequence analysis confirmed the synthesis of coronavirus-specific mRNAs encoding CAT, LUC, and GFP. In addition, we have shown that human coronavirus-based vector RNA can be packaged into virus-like particles that, in turn, can be used to transduce immature and mature human dendritic cells. In summary, we describe a new class of eukaryotic, multigene expression vectors that are based on the human coronavirus 229E and have the ability to transduce human dendritic cells.

Characterization of 2'-fluoro-RNA aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion.

We have isolated artificial ligands or aptamers for infectious prions in order to investigate conformational aspects of prion pathogenesis. The aptamers are 2'-fluoro-modified RNA produced by in vitro selection from a large, randomized library. One of these ligands (aptamer SAF-93) had more than 10-fold higher affinity for PrPSc than for recombinant PrPC and inhibited the accumulation of PrPres in near physiological cell-free conversion assay. To understand the molecular basis of these properties and to distinguish specific from non-specific aptamer-PrP interactions, we studied deletion mutants of bovine PrP in denatured, alpha-helix-rich and beta-sheet-rich forms. We provide evidence that, like scrapie-associated fibrils (SAF), the beta-oligomer of PrP bound to SAF-93 with at least 10-fold higher affinity than did the alpha-form. This differential affinity could be explained by the existence of two binding sites within the PrP molecule. Site 1 lies within residues 23-110 in the unstructured N terminus and is a nonspecific RNA binding site found in all forms of PrP. The region between residue 90 and 110 forms a hinge region that is occluded in the alpha-rich form of PrP but becomes exposed in the denatured form of PrP. Site 2 lies in the region C-terminal of residue 110. This site is beta-sheet conformation-specific and is not recognized by control RNAs. Taken together, these data provide for the first time a specific ligand for a disease conformation-associated site in a region of PrP critical for conformational conversion. This aptamer could provide tools for the further analysis of the processes of PrP misfolding during prion disease and leads for the development of diagnostic and therapeutic approaches to TSEs.