Stargardt disease-associated in-frame ABCA4 exon 17 skipping results in significant ABCA4 function.

BACKGROUND: ABCA4, the gene implicated in Stargardt disease (STGD1), contains 50 exons, of which 17 contain multiples of three nucleotides. The impact of in-frame exon skipping is yet to be determined. Antisense oligonucleotides (AONs) have been investigated in Usher syndrome-associated genes to induce skipping of in-frame exons carrying severe variants and mitigate their disease-linked effect. Upon the identification of a STGD1 proband carrying a novel exon 17 canonical splice site variant, the activity of ABCA4 lacking 22 amino acids encoded by exon 17 was examined, followed by design of AONs able to induce exon 17 skipping. METHODS: A STGD1 proband was compound heterozygous for the splice variant c.2653+1G>A, that was predicted to result in in-frame skipping of exon 17, and a null variant [c.735T>G, p.(Tyr245*)]. Clinical characteristics of this proband were studied using multi-modal imaging and complete ophthalmological examination. The aberrant splicing of c.2653+1G>A was investigated in vitro in HEK293T cells with wild-type and mutant midigenes. The residual activity of the mutant ABCA4 protein lacking Asp864-Gly885 encoded by exon 17 was analyzed with all-trans-retinal-activated ATPase activity assay, along with its subcellular localization. To induce exon 17 skipping, the effect of 40 AONs was examined in vitro in WT WERI-Rb-1 cells and 3D human retinal organoids. RESULTS: Late onset STGD1 in the proband suggests that c.2653+1G>A does not have a fully deleterious effect. The in vitro splice assay confirmed that this variant leads to ABCA4 transcripts without exon 17. ABCA4 Asp864_Gly863del was stable and retained 58% all-trans-retinal-activated ATPase activity compared to WT ABCA4. This sequence is located in an unstructured linker region between transmembrane domain 6 and nucleotide-binding domain-1 of ABCA4. AONs were designed to possibly reduce pathogenicity of severe variants harbored in exon 17. The best AON achieved 59% of exon 17 skipping in retinal organoids. CONCLUSIONS: Exon 17 deletion in ABCA4 does not result in the absence of protein activity and does not cause a severe STGD1 phenotype when in trans with a null allele. By applying AONs, the effect of severe variants in exon 17 can potentially be ameliorated by exon skipping, thus generating partial ABCA4 activity in STGD1 patients.

Intravitreal antisense oligonucleotide sepofarsen in Leber congenital amaurosis type 10: a phase 1b/2 trial.

CEP290-associated Leber congenital amaurosis type 10 (LCA10) is a retinal disease resulting in childhood blindness. Sepofarsen is an RNA antisense oligonucleotide targeting the c.2991+1655A>G variant in the CEP290 gene to treat LCA10. In this open-label, phase 1b/2 ( NCT03140969 ), 12-month, multicenter, multiple-dose, dose-escalation trial, six adult patients and five pediatric patients received ≤4 doses of intravitreal sepofarsen into the worse-seeing eye. The primary objective was to evaluate sepofarsen safety and tolerability via the frequency and severity of ocular adverse events (AEs); secondary objectives were to evaluate pharmacokinetics and efficacy via changes in functional outcomes. Six patients received sepofarsen 160 µg/80 µg, and five patients received sepofarsen 320 µg/160 µg. Ten of 11 (90.9%) patients developed ocular AEs in the treated eye (5/6 with 160 µg/80 µg; 5/5 with 320 µg/160 µg) versus one of 11 (9.1%) in the untreated eye; most were mild in severity and dose dependent. Eight patients developed cataracts, of which six (75.0%) were categorized as serious (2/3 with 160 µg/80 µg; 4/5 with 320 µg/160 µg), as lens replacement was required. As the 160-µg/80-µg group showed a better benefit-risk profile, higher doses were discontinued or not initiated. Statistically significant improvements in visual acuity and retinal sensitivity were reported (post hoc analysis). The manageable safety profile and improvements reported in this trial support the continuation of sepofarsen development.

QR-313, an Antisense Oligonucleotide, Shows Therapeutic Efficacy for Treatment of Dominant and Recessive Dystrophic Epidermolysis Bullosa: A Preclinical Study.

Dystrophic epidermolysis bullosa (DEB) is a blistering skin disease caused by mutations in the gene COL7A1 encoding collagen VII. DEB can be inherited as recessive DEB (RDEB) or dominant DEB (DDEB) and is associated with a high wound burden. Perpetual cycles of wounding and healing drive fibrosis in DDEB and RDEB, as well as the formation of a tumor-permissive microenvironment. Prolonging wound-free episodes by improving the quality of wound healing would therefore confer substantial benefit for individuals with DEB. The collagenous domain of collagen VII is encoded by 82 in-frame exons, which makes splice-modulation therapies attractive for DEB. Indeed, antisense oligonucleotide-based exon skipping has shown promise for RDEB. However, the suitability of antisense oligonucleotides for treatment of DDEB remains unexplored. Here, we developed QR-313, a clinically applicable, potent antisense oligonucleotide specifically targeting exon 73. We show the feasibility of topical delivery of QR-313 in a carbomer-composed gel for treatment of wounds to restore collagen VII abundance in human RDEB skin. Our data reveal that QR-313 also shows direct benefit for DDEB caused by exon 73 mutations. Thus, the same topically applied therapeutic could be used to improve the wound healing quality in RDEB and DDEB.

Evaluation of eluforsen, a novel RNA oligonucleotide for restoration of CFTR function in in vitro and murine models of p.Phe508del cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the epithelial chloride channel CF transmembrane conductance regulator (CFTR) protein. The most common mutation is a deletion of three nucleotides leading to the loss of phenylalanine at position 508 (p.Phe508del) in the protein. This study evaluates eluforsen, a novel, single-stranded, 33-nucleotide antisense oligonucleotide designed to restore CFTR function, in in vitro and in vivo models of p.Phe508del CF. The aims of the study were to demonstrate cellular uptake of eluforsen, and its efficacy in functional restoration of p.Phe508del-CFTR both in vitro and in vivo. In vitro, the effect of eluforsen was investigated in human CF pancreatic adenocarcinoma cells and human bronchial epithelial cells. Two mouse models were used to evaluate eluforsen in vivo. In vitro, eluforsen improved chloride efflux in CF pancreatic adenocarcinoma cell cultures and increased short-circuit current in primary human bronchial epithelial cells, both indicating restoration of CFTR function. In vivo, eluforsen was taken up by airway epithelium following oro-tracheal administration in mice, resulting in systemic exposure of eluforsen. In female F508del-CFTR mice, eluforsen significantly increased CFTR-mediated saliva secretion (used as a measure of CFTR function, equivalent to the sweat test in humans). Similarly, intranasal administration of eluforsen significantly improved nasal potential difference (NPD), and therefore CFTR conductance, in two CF mouse models. These findings indicate that eluforsen improved CFTR function in cell and animal models of p.Phe508del-CFTR-mediated CF and supported further development of eluforsen in human clinical trials, where eluforsen has also been shown to improve CFTR activity as measured by NPD.

Effect of an intravitreal antisense oligonucleotide on vision in Leber congenital amaurosis due to a photoreceptor cilium defect.

Photoreceptor ciliopathies constitute the most common molecular mechanism of the childhood blindness Leber congenital amaurosis. Ten patients with Leber congenital amaurosis carrying the c.2991+1655A>G allele in the ciliopathy gene centrosomal protein 290 (CEP290) were treated (ClinicalTrials.gov no. NCT03140969 ) with intravitreal injections of an antisense oligonucleotide to restore correct splicing. There were no serious adverse events, and vision improved at 3 months. The visual acuity of one exceptional responder improved from light perception to 20/400.

Vaccine-induced effector-memory CD8+ T cell responses predict therapeutic efficacy against tumors.

CD8(+) T cells have the potential to attack and eradicate cancer cells. The efficacy of therapeutic vaccines against cancer, however, lacks defined immune correlates of tumor eradication after (therapeutic) vaccination based on features of Ag-specific T cell responses. In this study, we examined CD8(+) T cell responses elicited by various peptide and TLR agonist-based vaccine formulations in nontumor settings and show that the formation of CD62L(-)KLRG1(+) effector-memory CD8(+) T cells producing the effector cytokines IFN-γ and TNF predicts the degree of therapeutic efficacy of these vaccines against established s.c. tumors. Thus, characteristics of vaccine-induced CD8(+) T cell responses instill a predictive determinant for the efficacy of vaccines during tumor therapy.

Systemic administration of PRO051 in Duchenne's muscular dystrophy.

BACKGROUND: Local intramuscular administration of the antisense oligonucleotide PRO051 in patients with Duchenne's muscular dystrophy with relevant mutations was previously reported to induce the skipping of exon 51 during pre-messenger RNA splicing of the dystrophin gene and to facilitate new dystrophin expression in muscle-fiber membranes. The present phase 1-2a study aimed to assess the safety, pharmacokinetics, and molecular and clinical effects of systemically administered PRO051. METHODS: We administered weekly abdominal subcutaneous injections of PRO051 for 5 weeks in 12 patients, with each of four possible doses (0.5, 2.0, 4.0, and 6.0 mg per kilogram of body weight) given to 3 patients. Changes in RNA splicing and protein levels in the tibialis anterior muscle were assessed at two time points. All patients subsequently entered a 12-week open-label extension phase, during which they all received PRO051 at a dose of 6.0 mg per kilogram per week. Safety, pharmacokinetics, serum creatine kinase levels, and muscle strength and function were assessed. RESULTS: The most common adverse events were irritation at the administration site and, during the extension phase, mild and variable proteinuria and increased urinary α(1)-microglobulin levels; there were no serious adverse events. The mean terminal half-life of PRO051 in the circulation was 29 days. PRO051 induced detectable, specific exon-51 skipping at doses of 2.0 mg or more per kilogram. New dystrophin expression was observed between approximately 60% and 100% of muscle fibers in 10 of the 12 patients, as measured on post-treatment biopsy, which increased in a dose-dependent manner to up to 15.6% of the expression in healthy muscle. After the 12-week extension phase, there was a mean (±SD) improvement of 35.2±28.7 m (from the baseline of 384±121 m) on the 6-minute walk test. CONCLUSIONS: Systemically administered PRO051 showed dose-dependent molecular efficacy in patients with Duchenne's muscular dystrophy, with a modest improvement in the 6-minute walk test after 12 weeks of extended treatment. (Funded by Prosensa Therapeutics; Netherlands National Trial Register number, NTR1241.).

Local dystrophin restoration with antisense oligonucleotide PRO051.

BACKGROUND: Duchenne's muscular dystrophy is associated with severe, progressive muscle weakness and typically leads to death between the ages of 20 and 35 years. By inducing specific exon skipping during messenger RNA (mRNA) splicing, antisense compounds were recently shown to correct the open reading frame of the DMD gene and thus to restore dystrophin expression in vitro and in animal models in vivo. We explored the safety, adverse-event profile, and local dystrophin-restoring effect of a single, intramuscular dose of an antisense oligonucleotide, PRO051, in patients with this disease. METHODS: Four patients, who were selected on the basis of their mutational status, muscle condition, and positive exon-skipping response to PRO051 in vitro, received a dose of 0.8 mg of PRO051 injected into the tibialis anterior muscle. A biopsy was performed 28 days later. Safety measures, composition of mRNA, and dystrophin expression were assessed. RESULTS: PRO051 injection was not associated with clinically apparent adverse events. Each patient showed specific skipping of exon 51 and sarcolemmal dystrophin in 64 to 97% of myofibers. The amount of dystrophin in total protein extracts ranged from 3 to 12% of that found in the control specimen and from 17 to 35% of that of the control specimen in the quantitative ratio of dystrophin to laminin alpha2. CONCLUSIONS: Intramuscular injection of antisense oligonucleotide PRO051 induced dystrophin synthesis in four patients with Duchenne's muscular dystrophy who had suitable mutations, suggesting that further studies might be feasible.

Synthesis and in vitro evaluation of PNA-peptide-DETA conjugates as potential cell penetrating artificial ribonucleases.

We report the synthesis of novel artificial ribonucleases with potentially improved cellular uptake. The design of trifunctional conjugates 1a and 1b is based on the specific RNA-recognizing properties of PNA, the RNA-cleaving abilities of diethylenetriamine (DETA), and the peptide (KFF)(3)K for potential uptake into E. coli. The conjugates were assembled in a convergent synthetic route involving native chemical ligation of a PNA, containing an N-terminal cysteine, with the C-terminal thioester of the cell-penetrating (KFF)(3)K peptide to give 12a and 12b. These hybrids contained a free cysteine side-chain, which was further functionalized with an RNA-hydrolyzing diethylenetriamine (DETA) moiety. The trifunctional conjugates (1a, 1b) were evaluated for RNA-cleaving properties in vitro and showed efficient degradation of the target RNA at two major cleavage sites. It was also established that the cleavage efficiency strongly depended on the type of spacer connecting the PNA and the peptide.