Aberrant Splicing in Transgenes Containing Introns, Exons, and V5 Epitopes: Lessons from Developing an FSHD Mouse Model Expressing a D4Z4 Repeat with Flanking Genomic Sequences

Abstract

The DUX4 gene, encoded within D4Z4 repeats on human chromosome 4q35, has recently emerged as a key factor in the pathogenic mechanisms underlying Facioscapulohumeral muscular dystrophy (FSHD). This recognition prompted development of animal models expressing the DUX4 open reading frame (ORF) alone or embedded within D4Z4 repeats. In the first published model, we used adeno-associated viral vectors (AAV) and strong viral control elements (CMV promoter, SV40 poly A) to demonstrate that the DUX4 cDNA caused dose-dependent toxicity in mouse muscles. As a follow-up, we designed a second generation of DUX4-expressing AAV vectors to more faithfully genocopy the FSHD-permissive D4Z4 repeat region located at 4q35. This new vector (called AAV.D4Z4.V5.pLAM) contained the D4Z4/DUX4 promoter region, a V5 epitope-tagged DUX4 ORF, and the natural 3’ untranslated region (pLAM) harboring two small introns, DUX4 exons 2 and 3, and the non-canonical poly A signal required for stabilizing DUX4 mRNA in FSHD. AAV.D4Z4.V5.pLAM failed to recapitulate the robust pathology of our first generation vectors following delivery to mouse muscle. We found that the DUX4.V5 junction sequence created an unexpected splice donor in the pre-mRNA that was preferentially utilized to remove the V5 coding sequence and DUX4 stop codon, yielding non-functional DUX4 protein with 55 additional residues on its carboxyl-terminus. Importantly, we further found that aberrant splicing could occur in any expression construct containing a functional splice acceptor and sequences resembling minimal splice donors. Our findings represent an interesting case study with respect to AAV.D4Z4.V5.pLAM, but more broadly serve as a note of caution for designing constructs containing V5 epitope tags and/or transgenes with downstream introns and exons.

Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in FaciosScapuloHumeral Muscular Dystrophy (FSHD)

“…For its use in the AO evaluation the AAV had to be modified in order to include the DUX4 3’ UTR target, but the co-insertion of a DNA sequence encoding a V5 epitope tag at the end of the DUX4 ORF generated artefactual splicing [84]. After muscle injection the repaired AAV vector did express DUX4-V5 protein that caused a local myopathy.…”

“…Two mice were injected in the Tibialis anterior (TA) with rAAV-D4Z4/pLAM virus (described in [84]) and pLAM3A vivo-PMO (Figure 2) directed against the DUX4 mRNA. For negative controls we similarly injected two mice with rAAV-D4Z4/pLAM and a vivo-PMO targeting human beta-globin mRNA.…”

“…The mice were sacrificed 10 days later, their TAs collected, and total RNAs were extracted with Trizol. DUX4 mRNAs were amplified by reverse transcription (RT) followed by a nested PCR (primer sequences in [84]). An electrophoresis of the RT-PCR products on agarose gel yielded a band at the expected 450-bp length for the TAs injected with control vivo-PMO (Figure 6).…”

“…Various chemical moieties have been added to PMOs in order to facilitate their membrane crossing and cell uptake (for examples, [88,89]) and some of these structures could be optimized for FSHD muscles. As well in the transgenic mouse with inducible PITX1 expression [83] and in a local myopathy induced by AAV-DUX4 injection [84] AOs with the vivo-morpholino chemistry could reduce the target mRNA levels while PMOs were not efficient. Further progress in DUX4-targeted therapeutic approaches for FSHD will also have to deal with DUX4 normal functions in non-muscle tissues such as testis and MSC.…”

AAV-mediated follistatin gene therapy improves functional outcomes in the TIC-DUX4 mouse model of FSHD

“…defect that rendered the DUX4 gene nontoxic (47). This necessitated eliminating the first lines, repairing the targeting construct, rederiving new ES cells, and generating another set of animals, at which time we had to seek additional funding to support characterization.…”