Efficient bypass of mutations in dysferlin deficient patient cells by antisense-induced exon skipping

Abstract

Mutations in DYSF encoding dysferlin cause primary dysferlinopathies, autosomal recessive diseases that mainly present clinically as Limb Girdle Muscular Dystrophy type 2B and Miyoshi myopathy. More than 350 different sequence variants have been reported in DYSF. Like dystrophin, the size of the dysferlin mRNA is above the limited packaging size of AAV vectors. Alternative strategies to AAV gene transfer in muscle cells must then be addressed for patients. A gene therapy approach for Duchenne muscular dystrophy was recently developed, based on exon-skipping strategy. Numerous sequences are recognized by splicing protein complexes and, when specifically blocked by antisense oligoucleotides (AON), the corresponding exon is skipped. We hypothesized that this approach could be useful for patients affected with dysferlinopathies. To confirm this assumption, exon 32 was selected as a prioritary target for exon skipping strategy. This option was initially driven by the report from Sinnreich and colleagues of a patient with a very mild and late-onset phenotype associated to a natural skipping of exon 32. Three different antisense oligonucleotides were tested in myoblasts generated from control and patient MyoD transduced fibroblasts, either as oligonucleotides or after incorporation into lentiviral vectors. These approaches led to a high efficiency of exon 32 skipping. Therefore, these results seem promising, and could be applied to several other exons in the DYSF gene. Patients carrying mutations in exons whose the in-frame suppression has been proven to have no major consequences on the protein function, might benefit of exon-skipping based gene correction.

Proteasomal Inhibition Restores Biological Function of Mis-sense Mutated Dysferlin in Patient-derived Muscle Cells

“…Currently, no causal pharmacological treatment is available for patients affected by the progressive and debilitating muscular dystrophies caused by dysferlin deficiency. Based on clinical observations of dysferlinopathy patients with internally truncated dysferlin molecules and mild phenotype (26,27), exon-skipping strategies have been developed (28,29), analogous to strategies currently tested in patients with dystrophinopathies (30,31). Other experimental treatment possibilities include the generation of small dysferlin molecules suitable for adeno-associated virus (AAV)-mediated gene delivery (27), or expression of dysferlin coding fragments which recombine after dual adeno-associated virus-mediated gene transfer to generate one transcript able to produce the full-length protein (32).…”

Full‐length dysferlin expression driven by engineered human dystrophic blood derived CD133+ stem cells

“…encode the C-terminal part of the protein, which has a fundamental role in anchoring dysferlin at the membrane and cannot be eliminated without impairing protein function [20]. Here, we developed a strategy based on delivery of full-length dysferlin by lentiviral vector to allow dysferlin expression in both patients 1 and 2.…”

“…2C). Unfortunately, the same approach could not be used to create a D51-55 EGFP dysferlin, mimicking the skipping of the mutation of patient 2, because this block of exons encodes two crucial elements, the 3′ UTR sequence and the transmembrane domain of the protein [20].…”

“…We next developed a strategy based on complete dysferlin delivery by lentiviral vector. Importantly, this approach represented a valid alternative to exon skipping technology for the treatment of patient 2, for whom we could not remove the exons located in the C-terminal part of the protein (such as exons 51-55) because their removal would impair dysferlin protein expression and function [20]. Moreover, other regions required skipping of three or more exons to restore a correct ORF, a result hardly obtainable with a classical exon skipping strategy, as we showed in patient 1.…”

“…Unfortunately, it is only applicable for proteins in which part of the amino acid sequence can be deleted without important deleterious impact on their overall function. In the dysferlin gene, it may be possible to skip some exons in the functionally redundant C2 domains, such as exon 32 [18,19], but not others, such as exons 53 and 54 that encode the unique terminal transmembrane domain [20]. Other techniques complementary to the exon skipping approach include the use of adeno-associated viruses (AAVs) to carry wild-type cDNA of specific mutated genes and perform gene replacement [21,22] and/or the use of gene miniaturizing and transplicing approaches to overcome the packaging limits of AAVs [23,24].…”