In vitro antisense therapeutics for a deep intronic mutation causing Neurofibromatosis type 2

Abstract

Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder affecting about 1:33 000 newborns, mainly characterized by the development of tumors of the nervous system and ocular abnormalities. Around 85% of germline NF2 mutations are point mutations. Among them, ∼25% affect splicing and are associated with a variable disease severity. In the context of our NF2 Multidisciplinary Clinics, we have identified a patient fulfilling clinical criteria for the disease and exhibiting a severe phenotype. The patient carries a deep intronic mutation (g. 74409T>A, NG_009057.1) that produces the insertion of a cryptic exon of 167pb in the mature mRNA between exons 13 and 14, resulting in a truncated merlin protein (p.Pro482Profs*39). A mutation-specific antisense phosphorodiamidate morpholino oligomer was designed and used in vitro to effectively restore normal NF2 splicing in patient-derived primary fibroblasts. In addition, merlin protein levels were greatly recovered after morpholino treatment, decreasing patient's fibroblasts in vitro proliferation capacity and restoring cytoeskeleton organization. To our knowledge, this is the first NF2 case caused by a deep intronic mutation in which an in vitro antisense therapeutic approximation has been tested. These results open the possibility of using this approach in vivo for this type of mutation causing NF2.

Figure 1

Deep intronic NF2 mutation description. (a) Analysis of patient fibroblasts showed a proportion of NF2 transcripts containing the inclusion of a cryptic exon (NF2 CEI) compared with the WT NF2 mRNA (NF2 WT) (upper panel). Forward sequence of cryptic exon inclusion is shown (bottom panel). (b) Schematic representation of the identified NF2 deep intronic mutation and MS-PMO location. Constitutive and cryptic exons are represented by dark and light gray boxes, respectively. The boundaries of the cryptic inserted exon are shown in uppercase; flanking intronic sequences are shown in lowercase. MS-PMO sequence is underlined. Mutated nucleotide is shown in bold and the nucleotide change is indicated by an arrow.

Figure 2

Correcting the effect of the deep intronic NF2 mutation on splicing by PMO treatment. (a) Correction of aberrant splicing by MS-PMO treatment of patient's primary fibroblasts. The treatment was performed using 5, 10 and 20 μℳ PMO concentrations for 24 h. The percentage of the cryptic exon-containing transcripts (NF2 CEI) in relation to the total (NF2 WT+NF2 CEI) is plotted in the Y axes of the graph. Mean±SD is depicted in the bar, representing at least three independent experiments. The corresponding agarose gel electrophoresis is shown below the graph. (b) Representative western blot showing the restoration of merlin protein expression in MS-PMO-treated patient's primary fibroblasts. Patient and control fibroblasts were treated with morpholinos. MS-PMO and CTL-PMO (20 μℳ) were used to treat patient's fibroblast for 24, 48 and 72 h. MS-PMO was used to treat control fibroblast using the same conditions. For comparison purposes, a control sample consisting in patient fibroblasts treated with PMO delivery vehicle alone was added for the 24 h time point. Asterisks highlight merlin recovery. (c) Quantification of merlin expression in NF2-mutant fibroblast cultures after either MS-PMO or CTL-PMO (5 μℳ) treatment for 24, 48 and 72 h. In the Y axis, it is plotted the proportion of merlin expression after MS-PMO treatment in relation to merlin expression after CTL-PMO treatment. All merlin values were previously normalized with actin levels. Mean±SD is depicted in the bar, representing at least three independent experiments. (*) Denotes statistical significance using the Mann–Whitney U-test (P>0.0001). (d) Merlin expression level restoration decrease the proliferation capacity of patient's fibroblasts, whereas not interfering in the proliferation capacity of control fibroblasts. Mean±SD is depicted in the bar, representing at least three independent experiments. (*) Denotes statistical significance using the T-test (P>0.05). (e) Cytoskeletal abnormalities of patient's derived fibroblasts were restored after MS-PMO treatment. NF2-mutated fibroblasts treated with CTL-PMO (left panels) showed poorly organized cytoskeleton when compared with the same fibroblast cultures treated with MS-PMO (right panels) at 5 μℳ PMO concentration for 48 h. Scale bar: 75 μm. Lower panels display a magnification of a detail of the cellular membrane. Microvilli structures are indicated by arrows.