Abstract
Therapeutic strategies for rare diseases based on exon skipping are aimed at mediating the elimination of mutated exons and restoring the reading frame of the affected protein. We explored the capability of polypurine reverse-Hoogsteen hairpins (PPRHs) to cause exon skipping in NB6 cells carrying a duplication of exon 2 of the DHFR gene that causes a frameshift abolishing DHFR activity. Methods: Different editing PPRHs were designed and transfected in NB6 cells followed by incubation in a DHFR-selective medium lacking hypoxanthine and thymidine. Surviving colonies were analyzed by DNA sequencing, RT-PCR, Western blotting and DHFR enzymatic activity. Results: Transfection of editing PPRHs originated colonies in the DHFR-selective medium. DNA sequencing results proved that the DHFR sequence in all these colonies corresponded to the wildtype sequence with just one copy of exon 2. In the edited colonies, the skipping of the additional exon was confirmed at the mRNA level, the DHFR protein was restored, and it showed high levels of DHFR activity. Conclusions: Editing-PPRHs are able to cause exon skipping at the DNA level and could be applied as a possible therapeutic tool for rare diseases.
Highlights
Rare diseases are the result of point mutations in gene sequences that can alter the reading frame of the encoded protein or produce a premature stop codon
We described the ability of the polypurine reverse-Hoogsteen hairpins (PPRHs) technologgyy to causee exonn skippppiinngg at the DNA level in a cell line model bearinngg ann eexxttrraa ccooppyy ooff DDHHFFRR eexxoonn 22 tthhaatt lleeaaddss ttoo aa frameshhiifftt
In our conditions, these spontaneous revertants from NB6 cells were not able to neither express the DHFR protein nor display its enzymatic activity (Figures 6 and 7). This spontaneous deletion was associated with defective processing of DHFR pre-mRNA in NB6 cells, as shown by the levels of intron 1 retention in the corresponding mRNA molecules (Figures 4 and 5)
Summary
Rare diseases are the result of point mutations in gene sequences that can alter the reading frame of the encoded protein or produce a premature stop codon. A therapeutic strategy for DMD based on exon skipping has been developed, in which antisense oligonucleotides (aODNs) are used to mediate the elimination of the mutated exon, restoring the reading frame of the protein. In these conditions, the resulting synthesis of the dystrophin protein mimics the BMD phenotype [4,5,6,7,8,9]. Each PPRH core was extended at its 5’ end by a sequence tail homologous to 20 nt upstream and/or 20 nt downstream of the PstI restriction site present in the original DHFR minigene pDCH1P (Table 1 and Figure 2), which corresponded to the insertion site of the additional sequence including the extra exon 2. Depend5i’nGgGoTnAGthGeClCoCcGaCtiCoGnGoCfTtGhCeAPGPRH target with respect to the sequence toLbDeSHedpEit3eI1dU, DthPestIeditinCgCtCaTilTwGCaCsCaArrTaGnCgCeCdGtCoGhGaTve the same sequenEcxeonan d orientation as the strand of the DNA tToTTbTeTrGeGcAoCmCbAiAnGedA.GGTAAGGAT As a negative control, sequence TUTDTTPAsGtIGcAoArTrGesGpAoGnAdAinCgCAoGnGly3’ to the extension tail, 20 nt upstream and 20 nt UdDowPsnt1stream of5t’hGeGPTsAtGI rGeCsCtrCicGtCioCnGGsiCteTGpCreAsGent in the originalNDoHneFR minigene, but without a PPRH core aCttCaCchTTeGdC, CwCaAsTuGsCeCdCGinCGthGeTt3r’ansfection experiments
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