Mouse Dux is myotoxic and shares partial functional homology with its human paralog DUX4

D4Z4 repeats are present in at least 11 different mammalian species, including humans and mice. Each repeat contains an open reading frame encoding a double homeodomain (DUX) family transcription factor. Aberrant expression of the D4Z4 ORF called DUX4 is associated with the pathogenesis of Facioscapulohumeral muscular dystrophy (FSHD). DUX4 is toxic to numerous cell types of different species, and over-expression caused dysmorphism and developmental arrest in frogs and zebrafish, embryonic lethality in transgenic mice, and lesions in mouse muscle. Because DUX4 is a primate-specific gene, questions have been raised about the biological relevance of over-expressing it in non-primate models, as DUX4 toxicity could be related to non-specific cellular stress induced by over-expressing a DUX family transcription factor in organisms that did not co-evolve its regulated transcriptional networks. We assessed toxic phenotypes of DUX family genes, including DUX4, DUX1, DUX5, DUXA, DUX4-s, Dux-bl and mouse Dux. We found that DUX proteins were not universally toxic, and only the mouse Dux gene caused similar toxic phenotypes as human DUX4. Using RNA-seq, we found that 80% of genes upregulated by Dux were similarly increased in DUX4-expressing cells. Moreover, 43% of Dux-responsive genes contained ChIP-seq binding sites for both Dux and DUX4, and both proteins had similar consensus binding site sequences. These results suggested DUX4 and Dux may regulate some common pathways, and despite diverging from a common progenitor under different selective pressures for millions of years, the two genes maintain partial functional homology

Pre-clinical Safety and Off-Target Studies to Support Translation of AAV-Mediated RNAi Therapy for FSHD

RNAi emerged as a prospective molecular therapy nearly 15 years ago. Since then, two major RNAi platforms have been under development: oligonucleotides and gene therapy. Oligonucleotide-based approaches have seen more advancement, with some promising therapies that may soon reach market. In contrast, vector-based approaches for RNAi therapy have remained largely in the pre-clinical realm, with limited clinical safety and efficacy data to date. We are developing a gene therapy approach to treat the autosomal-dominant disorder facioscapulohumeral muscular dystrophy. Our strategy involves silencing the myotoxic gene DUX4 using adeno-associated viral vectors to deliver targeted microRNA expression cassettes (miDUX4s). We previously demonstrated proof of concept for this approach in mice, and we are now taking additional steps here to assess safety issues related to miDUX4 overexpression and sequence-specific off-target silencing. In this study, we describe improvements in vector design and expansion of our miDUX4 sequence repertoire and report differential toxicity elicited by two miDUX4 sequences, of which one was toxic and the other was not. This study provides important data to help advance our goal of translating RNAi gene therapy for facioscapulohumeral muscular dystrophy

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

<div><p>The <i>DUX4</i> 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 <i>DUX4</i> 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 <i>DUX4</i> cDNA caused dose-dependent toxicity in mouse muscles. As a follow-up, we designed a second generation of <i>DUX4</i>-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 <i>DUX4</i> ORF, and the natural 3’ untranslated region (pLAM) harboring two small introns, <i>DUX4</i> exons 2 and 3, and the non-canonical poly A signal required for stabilizing <i>DUX4</i> 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 <i>DUX4</i> 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.</p></div

Mis-splicing is not unique to DUX4.V5 cDNAs.

<p>(A) The myotilin cDNA fused to V5 creates a functional splice donor that was fused to splice acceptors resident in the DUX4 3’ UTR and that of an unrelated gene, MDM2. In both circumstances, sequence chromatograms showed that the V5 tag was deleted, similar to the event that occurred in the original DUX4.V5 transgene arising from the AAV D4Z4.V5.pLAM vector. (B) The humanized renilla luciferase cDNA contained 5 predicted splice donor sites. When attached to the DUX4 3’ region (exon 1 UTR, intron 1, exon 2, intron 2, exon 3), one site (SD5; boxed in red) was utilized and fused to DUX4 exon 2. PCR of 3’ RACE products produced the SD5 transcript (1,268 bp) and some smaller products on an ethidium bromide stained gel. Only the 1,268 bp band contained Renilla luciferase sequences, and chromatograms confirmed the SD5-mediated mis-splicing event. This construct showed significantly reduced luciferase enzyme activity in vitro (indicated by **, p<0.0001; ANOVA with Tukey’s multiple comparison test, n = 3 replicates). Mutating the SD5 T nucleotide (boxed) destroyed the SD5 splice donor and restored luciferase activity to above normal levels (indicated by #, p = 0.0076; ANOVA with Tukey’s multiple comparison test, n = 3 replicates). (C) Comparison of splice donor sequences identified in this study to the consensus splice donor site.</p

Schematic of chromosome 4, D4Z4, and DUX4-expressing AAV vectors.

<p>A: A representation of the telomeric region of the chromosome 4 long arm (4q35). Drawing is not to scale. The 4q35 subtelomere harbors polymorphic, 3.3 kb D4Z4 repeat arrays, as well as other genes, some of which are indicated. This region is normally embedded in repressive heterochromatin. Contraction of the D4Z4 repeat array (in FSHD1) or mutations in SMCHD1 (in FSHD2) leads to epigenetic changes in the 4q35 region, and subsequently permits transcription of the DUX4 gene. An “FSHD permissive” haplotype creates a polyA signal in the pLAM region located downstream of the array. DUX4 transcripts initiated in the last D4Z4 unit extend to this signal and are stabilized by a polyA tail, thereby allowing the mRNA to be translated into the toxic, pro-apoptotic DUX4 protein. B: Two different AAV vectors were engineered to express DUX4. The first generation vector utilized a CMV promoter and SV40 polyA signal. The DUX4 ORF was tagged at the 3’ end with sequences encoding a V5 tag, thereby producing a full-length DUX4 protein containing a carboxy-terminal V5 epitope fusion. ITR, AAV2 inverted terminal repeats. The second generation AAV.D4Z4.V5 vector essentially recapitulates the terminal D4Z4 repeat and pLAM sequences isolated from an FSHD patient, but engineered to express DUX4 with a carboxy-terminal V5 epitope fusion.</p

AAV.D4Z4.V5 vectors are non-toxic compared to AAV.CMV.DUX4.V5.

<p>H&E stained cryosections of tibialis anterior muscles isolated from C57BL/6 mice, 2 weeks after injection with 3x10¹⁰ particles of the indicated AAV6 vectors, or saline. Top panels (A) show low power images, and (B) are high power images of the same sections. Arrows point out examples of myofibers with centrally located nuclei, which are a histological indication that muscles were damaged and subsequently repaired. Note that AAV.DUX4.V5 vectors cause widespread muscle damage and regeneration, while AAV.D4Z4.V5 and saline did not.</p

Repaired AAV.D4Z4.V5.pLAM-2.0 vector produces full-length DUX4.V5.

<p>(A) Mutation of the DUX4-V5 splice donor from GGT to GGG (lowercase red letters) maintained the V5 glycine residue but destroyed the invariant T required for splicing. Note the yellow boxed area containing linker sequences joining the V5 epitope to the natural DUX4 exon 1 untranslated region. The red uppercase G (indicated by arrow) at the beginning of the linker indicates a second aberrant splice site utilized instead of the natural DUX4 exon 1 splice donor, only in the repaired sequence. BsaAI restriction sites are indicated. (B) Schematic of 3’ RACE/BsaAI restriction digestion assay to identify mis-spliced DUX4-V5 products. The BsaAI site is located in the V5 tag. Removal of this sequence by mis-splicing creates a BsaAI-resistant 3’ RACE product of 383 bp, evident by gel electrophoresis (C, original V5). The repaired vector incorporated the V5 tag and its resident BsaAI, making this product susceptible to digestion by the enzyme. The expected full-length and BsaAI-digested products were empirically smaller following electrophoresis (C, mutated V5). (D) Sequence chromatogram of the mutated DUX4.V5 transcript revealed that full-length DUX4.V5 was produced, but a second splice donor sequence was encountered and preferentially utilized instead of the natural DUX4 exon 1 donor. Arrow points to the G residue indicated in the linker sequence of (A).</p

The DUX4-55aa protein expressed from mis-spliced DUX4 V5 mRNA does not activate apoptosis in vitro.

<p>(A) Western blot confirms expression of DUX4.V5, DUX4-55aa, and DUX4.HOX1.V5 constructs in HEK293 cells transfected two days earlier. The DUX4-55aa construct lacked a V5 tag, and proteins were therefore detected using rabbit anti-DUX4 primary antibodies followed by HRP-coupled goat-anti-rabbit secondary antibodies. DUX4-55aa is ~6 kDa larger than full-length DUX4, which migrates at ~52 kDa. (B) DUX4-55aa protein does not activate apoptosis in vitro. HEK293 cells were transfected with 200 ng of the indicated CMV expression plasmids and plated simultaneously on 96-well plates. Caspase-3/7 activity was measured 48 hours later using a fluorescent plate reader. High caspase-3/7 activity in CMV.DUX4.V5-transfected HEK293 cells indicated that the DUX4.V5 protein caused apoptotic cell death, as previously reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118813#pone.0118813.ref008" target="_blank">8</a>]. The non-toxic DUX4.HOX1.V5 protein, which was engineered to lack DNA binding function, failed to induce apoptosis, as expected [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118813#pone.0118813.ref008" target="_blank">8</a>]. Likewise, CMV.DUX4-55aa did not activate caspase-3/7. *, indicates significant difference from DUX4.V5-treated cells. Data represent means +/- sem, averaged from 6 separate samples performed from two independent experiments. UNT, untransfected.</p

The V5 epitope tag coding sequence caused mis-splicing of DUX4 mRNA.

<p>A: <i>Top</i> shows the exon-intron structure of the DUX4 gene arising from the distal D4Z4 unit, and flanking pLAM region. Exons and introns are indicated but not to scale. The asterisk (*) represents the normal DUX4 stop codon. The final 11 nucleotides of exon 1 are untranslated, as are those in exons 2 and 3 (the latter is part of the pLAM sequence). In an FSHD permissive genotype, the DUX4 polyA signal is located in exon 3. <i>Bottom</i> shows the same locus but with the V5 coding sequences inserted. A new stop codon was placed downstream of the V5 tag, followed by 40 nucleotides of linker sequences (N40). The lower case “g” indicated by an arrow and red text, is the aberrant splice site created by the DUX4-V5 fusion coding sequence. B: AAV.D4Z4.V5 vectors express DUX4.V5 mRNA. A previously reported nested PCR/3’ RACE strategy [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118813#pone.0118813.ref024" target="_blank">24</a>] was used to amplify 3’ end of the DUX4 transcript from RNAs harvested from AAV.D4Z4.V5-injected C57BL/6 TA muscles. A ~450 bp band was amplified from muscles that received AAV.D4Z4.V5 but not AAV.GFP controls. The size of this band corresponded to the expected product of the reverse transcribed and PCR-amplified DUX4 3’ end if introns 1 and 2 were spliced out. C: Sequence chromatogram of cloned 3’ RACE products identifying the mis-splicing event. The arrow points out the same g nucleotide indicated in panel A. D: The V5 epitope, N40 linker sequences, and intron 1 are spliced out of the D4Z4.V5 transcript. E: This mis-splicing event created a new missense DUX4 protein containing an additional 55 amino acids (DUX4-55aa), until an in-frame stop codon occurred in exon 3.</p