Meganucleases can restore the reading frame of a mutated dystrophin

Abstract

Mutations in Duchenne muscular dystrophy (DMD) are either inducing a nonsense codon or a frameshift. Meganucleases (MGNs) can be engineered to induce double-strand breaks (DSBs) at specific DNA sequences. These breaks are repaired by homologous recombination or by non-homologous end joining (NHEJ), which results in insertions or deletions (indels) of a few base pairs. To verify whether MGNs could be used to restore the normal reading frame of a dystrophin gene with a frameshift mutation, we inserted in a plasmid coding for the dog m-dystrophin sequences containing a MGN target. The number of base pairs in these inserted sequences changed the reading frame. One of these modified target m-dystrophin plasmids and an appropriate MGN were then transfected in 293FT cells. The MGN induced micro-deletion or micro-insertion in the m-dystrophin that restored dystrophin expression. MGNs also restored m-dystrophin expression in myoblasts in vitro and in muscle fibers in vivo. The mutation of the targeted m-dystrophin was confirmed by PCR amplification followed by digestion with the Surveyor enzyme and by cloning and sequencing of the amplicons. These experiments are thus a proof of principle that MGNs that are adequately engineered to target appropriate sequences in the human dystrophin gene should be able to restore the normal reading frame of that gene in DMD patients with an out-of-frame deletion. New MGNs engineered to target a sequence including or near nonsense mutation could also be used to delete it.

Targeting Sarcoplasmic Reticulum Calcium ATPase by Gene Therapy

“…Similarly, zinc finger protein transcription factors that specifically activate or repress virtually any gene can be engineered. Meganucleases and zinc finger proteins have been used to target dystrophin mutations and vascular endothelial growth factor (VEGF) expression by gene transfer (Chapdelaine et al, 2010). With the ever-expanding genetic mutations in dilated and hypertrophic cardiomyopathies (Konno et al, 2010), these nucleases can be used in the future to repair DNA in the affected organs by gene therapy.…”

On a Break with the X: The Role of Repair of Double-Stranded DNA Breaks in X-Linked Disease

“…thus, by using nheJ on pre-cut sequences containing mutations disrupting the reading frame, it could be expected that micro-insertions or micro-deletions would be generated at the joining site, which may effectively restore the reading frame though at the expense of several improperly added nucleotides or, alternatively, risking a loss of one or more amino acid residues. As of now this has been demonstrated in vitro with out-of-frame mutated dystrophin gene (21,84). It is also believed that specific nuclease activities coupled with subsequent nheJ could be used for targeting sequences containing a nonsense mutation and deleting them, or introducing more than one DSB in sites flanking an inserted or translocated sequence, causing its excision (21,49).…”

“…As of now this has been demonstrated in vitro with out-of-frame mutated dystrophin gene (21,84). It is also believed that specific nuclease activities coupled with subsequent nheJ could be used for targeting sequences containing a nonsense mutation and deleting them, or introducing more than one DSB in sites flanking an inserted or translocated sequence, causing its excision (21,49).…”

Duchenne Muscular Dystrophy: Therapeutic Approaches to Restore Dystrophin

“…During this process often small deletions or insertions occur, which can restore the reading frame. Proof of principle for this approach has been recently demonstrated in vitro and after local delivery in vivo, albeit at low levels (Chapdelaine et al, 2010). The challenge of this approach will be the delivery of the meganucleases and the limited recognition of target sequences of meganucleases.…”