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. 2012 Feb 21;20(6):1212–1221. doi: 10.1038/mt.2012.26

Engineering Multiple U7snRNA Constructs to Induce Single and Multiexon-skipping for Duchenne Muscular Dystrophy

Aurélie Goyenvalle 1,2,*, Jordan Wright 1, Arran Babbs 1, Vivienne Wilkins 1, Luis Garcia 2, Kay E Davies 1,*
PMCID: PMC3369406  PMID: 22354379

Abstract

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD but still faces personalized medicine challenges as different mutations found in DMD patients require skipping of different exons. However, 70% of DMD patients harbor dystrophin gene deletions in a mutation-rich area or “hot-spot” in the central genomic region. In this study, we have developed 11 different U7 small-nuclear RNA, to shuttle antisense sequences designed to mask key elements involved in the splicing of exons 45 to 55. We demonstrate that these constructs induce efficient exon skipping both in vitro in DMD patients' myoblasts and in vivo in human DMD (hDMD) mice and that they can be combined into a single vector to achieve a multi skipping of at least 3 exons. These very encouraging results provide proof of principle that efficient multiexon-skipping can be achieved using adeno-associated viral (AAV) vectors encoding multiple U7 small-nuclear RNAs (U7snRNAs), offering therefore very promising tools for clinical treatment of DMD.

Introduction

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. The majority of mutations causing DMD disrupts the open-reading frame and gives rise to prematurely truncated proteins, thus leading to progressive muscle wasting. One of the most promising strategies aims to convert an out-of-frame mutation into an in-frame mutation, which would give rise to internally deleted, but still functional dystrophin1,2 (Figure 1a). This can be achieved by using antisense oligonucleotides (AO) that interfere with splice sites or regulatory elements within the exon and thus induce the skipping of specific exons at the pre-mRNA level.3,4 The applicability of AO therapy has now been demonstrated in phase I/II clinical trials with two different chemistries of AO targeting the human dystrophin exon 51 in DMD patients.5,6 More recently, systemic treatment of DMD patients with AOs was reported to induce dose-dependent exon-skipping efficacy leading to detectable amount of dystrophin protein in skeletal muscles of patients treated with 2 mg or more per kg.7,8

Figure 1.

Figure 1

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Antisense-mediated exon-skipping rationale for Duchenne muscular dystrophy (DMD). (a) Patients with Duchenne muscular dystrophy have mutations which disrupt the open-reading frame of the dystrophin pre-mRNA. In this example, exon 50 is deleted, creating an out-of-frame mRNA and leading to the synthesis of a truncated non-functional or unstable dystrophin (left panel). A modified U7 small-nuclear RNA (U7snRNA) construct directed against exon 51 can induce effective skipping of exon 51 and restore the open-reading frame, therefore generating an internally deleted but partly functional dystrophin (right panel). (b) Multiexon-skipping rationale for DMD. The optimal skipping of exons 45–55 leading to the del45–55 artificial dystrophin could transform the DMD phenotype into the asymptomatic or mild Becker muscular dystrophy (BMD) phenotype. This multiple-exon skipping could theoretically rescue up to 63% of DMD patients with a deletion.16

Alternatively, the antisense sequence can be delivered to cells using viral vectors carrying a gene from which the antisense sequence can be transcribed, such as a modified U7 small-nuclear RNA (U7snRNA) gene.9,10 U7snRNA is normally involved in histone pre-mRNA 3′-end processing, but can be converted into a versatile tool for splicing modulation by a small change in the binding site for Sm/Lsm proteins.11 The antisense sequence embedded into a small-nuclear ribonucleoprotein particle is therefore protected from degradation and accumulates in the nucleus where splicing occurs. We have previously demonstrated that appropriately modified U7snRNA induces efficient exon-skipping of the dystrophin pre-mRNA in both the mdx mouse where it results in the sustained correction of muscular dystrophy12 and in DMD patients' myoblasts where it restores dystrophin expression to near normal level.13 The long-term restoration of dystrophin achieved using adeno-associated viral (AAV) vectors represents a strong advantage of this approach over synthetic AOs by eliminating the need for repeated injections.

Considering the diversity of mutations among DMD patients, the translation of this strategy to human will require specific tools adapted to different human dystrophin exons. However, 70% of DMD patients harbor dystrophin gene deletions in a mutation-rich area or “hot-spot” in the central genomic region (exons 45–55).1,14,15

In this study, we have thus designed 11 specific U7snRNA targeting every exon between exons 45 and 55 of the human dystrophin gene. Each construct has been inserted into lentiviral vectors for in vitro analysis in myoblasts from DMD patients. Following transduction of these cells with lentiviral vectors encoding the various U7 constructs, specific skipping of the targeted exon was confirmed by reverse transcription-PCR (RT-PCR) and dystrophin restoration was assessed by western blot when appropriate DMD genotypes were available. In parallel, we demonstrated and quantified the efficacy of these constructs in vivo in transgenic mice carrying the entire human DMD locus (hDMD mice) after intramuscular injection of AAV vectors encoding the U7snRNAs. This work therefore reports a number of modified U7snRNA able to induce efficient exon skipping that would suggest them worthy of clinical evaluation.

We also show here that these U7snRNA constructs can be combined into a single AAV vector and achieve an efficient multiskipping of at least 3 exons in vivo in the hDMD mice. These very encouraging results provide proof of principle that efficient multiexon-skipping can be achieved using AAV vectors encoding multiple U7snRNAs. Since the skipping of an entire stretch of exons such as exons 45 to 55 has been suggested to be applicable to up to 63%,16 this vectorized approach combining multiple U7snRNAs offers very promising tools for clinical treatment of DMD (Figure 1b).

Results

Rationale and design of U7snRNA constructs targeting 11 exons between 45 and 55

The clinical applicability of the exon skipping approach will require specific tools adapted to different human dystrophin exons. Considering that a large proportion of DMD patients harbor dystrophin gene deletions in the central genomic region,1,14,15 and would be eligible for a multiexon skipping of exons 45 to 5516 (Figure 1b), we have developed modified U7snRNAs targeting each of the 11 exons between 45 and 55 inclusive. The U7snRNA gene was engineered into a versatile tool for splicing modulation as previously described 12 and specific antisense sequence to the targeted exon, reported to induce exon skipping as AOs,17,18 were inserted. For most exons, the two best antisense sequences described to induce the most efficient exon skipping were chosen to be inserted into the modified U7snRNA (Figure 2a). Table 1 recapitulates all antisense sequences used. These antisense sequences target exon-internal elements believed to be exon splicing enhancers,17 except for exon 47 for which the only efficient sequence described was targeting the acceptor splice site. Each of the modified U7snRNA was cloned separately into lentiviral vector constructs (Figure 2b) and viral particles were subsequently produced as previously described13 to transduce human myoblasts.

Figure 2.

Figure 2

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Scheme depicting the different U7 small-nuclear RNA (U7snRNA) constructs and the lentiviral vector used in this work. (a) Representation of the different U7snRNAs and their target on the dystrophin pre-mRNA. Antisense sequences are represented by rectangular boxes. Most of the U7snRNA constructs target two exon-internal sequences believed to be exonic splicing enhancer (ESE) (represented by black and white boxes) and previously reported as targets for antisense oligonucleotides (see Table 1 for details). The U7snRNA directed against exon 47 targets the acceptor splice site of this exon (represented by hatched boxes). (b) Schematic map of the U7snRNA lentiviral vectors. The two long-terminal repeats (LTRs) enclose the encapsidation signal (ψ), the Rev-responsive element (RRE), the central polypurine tract (cPPT), the woodchuck hepatitis virus responsive element (WPRE), and the U7snRNA cassette inversely inserted. The U7snRNA cassette is constituted of the engineered U7snRNA sequence (gray box) carrying different antisense sequences, placed under the control of its natural U7 promoter (hatched box) and 3′ downstream elements (open box).

Table 1. Antisense sequences inserted into U7snRNA constructs.

graphic file with name mt201226t1.jpg

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In vitro evaluation of the U7snRNA constructs in human myoblasts

We first assessed the efficacy of the different constructs on normal dystrophin pre-mRNA. Immortalized human normal myoblasts19 were transduced with the different lentiviral vectors with previously described optimized transduction conditions inducing >95% efficiency.13 Following transduction, cells were amplified for further analysis. Total RNA was extracted from the transduced cells and analyzed by nested RT-PCR using primers surrounding the targeted exons (sequences available upon request). Figure 3a reports the exon-skipping analysis representative of results obtained from at least three independent transductions. For most exons, we detect a band corresponding to the skipped product at the expected size, which was confirmed by sequence analysis (data not shown). However, exon-skipping efficiency varies between exons, and whilst good levels of skipping were obtained for exons 45, 46, 48, 49, 50, 51, 54, and 55, only a very faint band was detected in the case of exon 47 and no skipping at all for exons 52 and 53. These variations in efficacy were not due to differences in transduction levels since quantitative PCR (qPCR) performed on genomic DNA extracted from the different transduced myoblast populations revealed a very similar copy number of lentiviral genomes per cell (data not shown).

Figure 3.

Figure 3

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Comparison of exon-skipping levels in human wild-type or Duchenne muscular dystrophy (DMD) patients myoblasts transduced by different lentiviral vectors. (a) Normal human myoblasts were transduced with lentiviral vectors encoding different U7 small-nuclear RNA (U7snRNA) constructs targeting exons 45–55 and corresponding exon skipping is assessed by nested reverse transcription-PCR (RT-PCR). In each case, the Ctl lane corresponds to untreated normal myoblasts. (b) DMD myoblasts carrying the Δ49–50 deletion were transduced with lentiviral vectors encoding the U7snRNA constructs targeting exons 45, 46, 47, 48, 51, 52, 53, 54, and 55. Constructs targeting exons 49 and 50 were tested on DMD myoblasts carrying an exon 52 deletion. In each case the exon skipping was assessed by nested RT-PCR. Note that additional bands between the nonskipped and skipped products are visible in some analyses. This is due to heteroduplex formation and has been described previously.53

We also transduced immortalized DMD myoblasts from a DMD patient carrying a deletion of exons 49 and 50 with the lentiviral vectors encoding the different U7 constructs except for the ones targeting exons 49 and 50, for which we used DMD myoblasts carrying a nonsense mutation in exon 52. Analysis of transduced DMD myoblasts by RT-PCR revealed an apparent higher level of skipping for some exons compared to the level observed in normal myoblasts (Figure 3b), which has been previously described.13 Exons 52 and 53 skipping could be detected on a DMD background, and exon 47 skipping levels appeared a little bit higher, although 47 is an in-frame exon. Remarkably, in some cases like exons 46, 48, 51, and 55, the skipping was nearly complete and the band corresponding to the unskipped product was almost no longer detectable.

In order to check that such exon-skipping led to restoration of dystrophin, we evaluated the levels of dystrophin protein in appropriate DMD genotypes by western blot. Limited by the availability of skippable DMD myoblasts, we only investigated the skipping of exon 46 on primary myoblasts carrying a deletion of exon 45 and the skipping of exon 51 on immortalised myoblasts Δ49–50. As shown in Figure 4, the level of exon skipping was maximal in both cases, inducing complete skipping of exons 46 and 51, respectively (Figure 4a and b). Protein samples were isolated from the same transduced DMD myoblasts after differentiation into myotubes and analyzed by western blotting. In both cases, dystrophin expression was detected in transduced cells, and levels of restoration were particularly high in myoblasts Δ49–50 treated with U7dtex51 construct, where dystrophin expression reaches a level comparable with that detected in normal myoblasts (Figure 4c and d).

Figure 4.

Figure 4

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Specific exon skipping in Duchenne muscular dystrophy (DMD) myoblasts and subsequent dystrophin rescue. (a) Primary DMD myoblasts carrying a Δ45 deletion were transduced with a lentiviral vector encoding a specific U7 small-nuclear RNA (U7snRNA) construct targeting exon 46. Exon 46 skipping was assessed by nested reverse transcription-PCR (RT-PCR) and a fragment of the expected size was detected. (b) Immortalized DMD myoblasts carrying a Δ49–50 deletion were transduced with lentiviral vectors encoding a specific U7snRNA construct targeting exon 51. Exon 51 skipping was assessed by nested RT-PCR and a band of the expected size was detected. Additional bands due to heteroduplex formation and activation of a cryptic splice site within the exon 51 (previously described in ref. 53) are visible in same analyses. (c,d) Western blot probed with dystrophin antibody (top gels) and α-actinin antibody (bottom gels).

In vivo validation of the U7snRNA constructs in hDMD mice

The hDMD mouse offers the possibility to evaluate in vivo the efficiency of constructs specifically engineered to target the human dystrophin gene and therefore represents an excellent preclinical model. In order to validate the effectiveness of the U7snRNA-mediated exon-skipping approach in vivo, we cloned the different engineered U7snRNA constructs into an AAV2-based vector (Figure 5a) and subsequently produced AAV1 pseudotyped vectors (AAV2/1) encoding the various U7snRNA constructs. All 11 AAV vectors were separately injected into the tibialis anterior (TA) muscles of hDMD mice and RNA from treated muscles were isolated 4 weeks after the injection. RT-PCR results presented in Figure 5b demonstrate an efficient exon skipping for each of the 11 exons confirming the efficacy of this approach in vivo. Interestingly, levels of exon skipping appeared higher in vivo than in human myoblasts following lentiviral transduction and most U7snRNA constructs seem to induce almost complete exon skipping (exons 46, 47, 48, 51, 54, and 55). In order to quantify more accurately the levels of each exon skipping, we developed Taqman assays for qPCR analysis of skipping for each of the 11 exons (Figure 5c). The percentage of exon skipping was quantified by comparing the relative abundance of skipped transcripts to unskipped transcripts. Only a low level of background skipping was observed in control mice, although we never observed any skipped product by nested RT-PCR. We therefore subtracted the average background from a minimum of two control mice from the percentage skipping in each of the treated mice. In all cases a substantial level of exon skipping was observed by qPCR, though the skipping efficiencies appeared lower than those indicated by nested PCR analyses. The minimum level of skipping quantified by qPCR was at or above ~20%, confirming the efficacy of all the U7 constructs in vivo. We considered these results to be encouraging as, in some cases, exon-skipping of the in-frame native transcript of hDMD mice would disrupt the open-reading frame, rendering the skipped molecule subject to nonsense-mediated decay (NMD) and thus decreasing the apparent efficiency of skipping.

Figure 5.

Figure 5

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Adeno-associated virus (AAV)-U7 small-nuclear RNA (U7snRNA) mediated exon skipping in vivo in human Duchenne muscular dystrophy (hDMD) mice induced by AAV vectors encoding engineered U7snRNA. (a) Top: Structure of the AAV vectors encoding the different U7snRNA cassettes. The U7snRNA cassette is inserted between two AAV2 inverted terminal repeats (ITR) and is constituted of the engineered U7snRNA sequence (gray box) carrying different antisense sequences (bottom), placed under the control of its natural U7 promoter (hatched box) and 3′ downstream elements (open box). (b) AAV2/1 vectors encoding different engineered U7snRNA were injected in the tibialis anterior of adult hDMD mice. Four weeks after the injection, muscles were harvested and analyzed by nested reverse transcription-PCR (RT-PCR) for skipping of the exon of interest. (c) Quantitative PCR (qPCR) analysis of exon skipping. Taqman qPCR was performed on cDNA from treated and control mice to calculate the relative abundance of skipped to unskipped transcripts. The error bars represent the standard deviation from the mean value of exon skipping from a minimum of three treated mice.

AAV-mediated multiexon-skipping potential in hDMD mice

Although the development of efficient U7snRNA constructs targeting each of these 11 exons represents a promising advance in itself and offers potential tools for clinical application relevant to a large proportion of patients, we wanted to investigate the possibility of multiple exon skipping. Targeting multiple exons by combining several constructs into a single vector could indeed increase the number of eligible patients and decrease the number of constructs that would need clinical evaluation. We have therefore introduced sequentially three U7snRNA constructs targeting exons 45, 46, and 47, respectively into the same AAV vector (Figure 6a). Following production, these vectors containing 1, 2, or 3 U7snRNA constructs were injected into the TA muscles of hDMD mice. Exon-skipping efficacy was assessed by RT-PCR on total RNA isolated from treated muscles 4 weeks after the injection. Figure 6b illustrates the representative skipping obtained from at least three injected muscle in each case. The major product detected in each lane corresponds to the expected skipping of 1, 2, or 3 exons, as confirmed by sequence analysis (Supplementary Figure S1). Interestingly, the skipping of exons 45 and 46 or 45, 46, and 47 simultaneously seems almost as efficient as the skipping of exon 46 alone, which confirms the feasibility of multiexon skipping in vivo and suggests a great therapeutic potential for DMD.

Figure 6.

Figure 6

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Multiexon skipping in vivo in human Duchenne muscular dystrophy (hDMD) mice induced by adeno-associated viral (AAV) vectors encoding engineered U7 small-nuclear RNAs (U7snRNAs) targeting exons 45 to 47. (a) Top: Structure of the AAV vectors encoding the different U7snRNA cassettes. The multiple U7snRNA cassettes are inserted adjacent to each other between two AAV2 inverted terminal repeats (ITR). Each of them is constituted of the engineered U7snRNA sequence (gray box) carrying antisense sequences as previously described (bottom), placed under the control of their natural U7 promoter (hatched box) and 3′ downstream elements (open box). (b) AAV2/1 vectors encoding a single U7snRNA, two U7snRNAs or three U7snRNAs were injected in the tibialis anterior of adult hDMD mice. Four weeks after the injection, muscles were harvested and analyzed by reverse transcription-PCR (RT-PCR) for exon skipping.

Discussion

Antisense-mediated exon-skipping as a therapeutic strategy for DMD has developed from a plausible notion in the mid-1990's to the stage where it holds realistic prospects of therapeutic benefits. Early clinical trials have demonstrated promising results after both local and systemic administration. However, some substantial scientific and regulatory barriers remain, especially regarding the personalized medicine nature of such an approach since it is mutation specific. There is a large variety of mutations causing DMD and most patients have unique mutations.20 The clinical applicability of the exon-skipping approach requires different antisense sequences adapted to each subset of patients. In this study, we have developed 11 different U7snRNA constructs targeting each exon between 45 and 55, which correspond to a mutation hot spot in the DMD gene. We first demonstrated the efficiency of these constructs in vitro in human myoblasts following lentiviral transduction. Whilst few U7 constructs (i.e., U7-ex47, U7ex52, and U7ex53) were hardly inducing any exon skipping in normal myoblasts, we were able to detect an efficient skipping for all exons in DMD myoblasts. This difference may be explained by the NMD pathway acting upon exon-skipped dystrophin mRNA. For instance, in normal myoblasts, where the native reading frame is intact, the application of exon skipping may induce the production of downstream stop codons within the mRNA (depending on the exon being skipped), thus leading to its degradation. This would result in less skipped mRNA being observed. In contrast, the native dystrophin mRNA in DMD myoblasts already encodes a premature stop codon and may therefore already be subject to NMD. The application of exon skipping to this mRNA would in some cases restore the reading frame allowing this internally truncated mRNA to escape degradation by NMD. This would lead to increased levels of exon-skipped mRNA over the native, full-length mRNA. In cases where exon skipping would not restore the reading frame, both native and exon-skipped mRNAs could be subject to NMD with no bias against either type of molecule.

Western blots were performed on treated DMD myoblasts with appropriate genotypes and whilst levels of exon skipping were almost complete for both exons 46 and 51, levels of dystrophin restoration appeared higher in myoblasts Δ49–50 treated with U7dtex51. This may be due to the presence of fibroblasts in the primary culture of Δ45 cells, which despite being transduced would not express dystrophin, therefore resulting in lower levels of dystrophin across the entire culture albeit similar levels of α-actinin. This would not occur in the immortalized myoblasts Δ49–50, where the cell population is more homogenous and mostly composed of myoblasts.

We also demonstrated in this study the efficacy of these 11 U7snRNA constructs in vivo in a mouse model transgenic for the entire human dystrophin locus following intramuscular injection of AAV vectors encoding the various U7snRNAs. Interestingly, exon skipping levels detected by nested RT-PCR in vivo appeared higher than in vitro in normal myoblasts, although the target dystrophin pre-mRNA is in frame in both cases. We have previously observed a similar difference between in vitro and in vivo efficacies in the mdx mouse,12 where levels of exon 23 skipping where higher following AAV injection than lentiviral transduction of mdx myoblasts. This difference may be related to the high efficiency of AAV-mediated gene transfer into mature skeletal muscle and the resulting high copy number of transgene into the myofibers.

Although nested RT-PCR is widely used to analyze exon-skipping, it does not provide an accurate quantification of exon-skipping and has been shown to overestimate skipping levels when compared with absolute quantification using a digital array.21 We therefore developed an alternative method of quantification using Taqman assays specific to skipped and unskipped templates. Comparative ΔCt analysis was used to compare the relative abundance of exon-skipped template to its respective unskipped, native form. Selected assays were performed in parallel with standard curve analyses using known concentrations of plasmids containing exon skipped or unskipped templates, and results were found not to differ from those of the comparative ΔCt analyses, confirming the accuracy of the method (data not shown). qPCR data clearly indicate lower levels of exon-skipping than those observed by nested RT-PCR, although to an extent both methods correlate in determining the most efficient constructs. Exons 46, 48, 54, and 55 all displayed highly efficient skipping (close to 100%) by nested PCR, and accordingly these exons were also identified as the most efficiently skipped by qPCR, though levels varied between 55 and 95%. The skipping levels of other exons, although less efficient than the aforementioned examples, were always quantified as being at least ~20% efficient and therefore demonstrated the efficacy of our constructs in vivo. Although it would have been interesting to determine how exon-skipping levels ascertained by qPCR in vivo compare with those seen in vitro, we found that qPCR analysis of complementary DNA (cDNA) derived from human myoblasts was problematic due to the very low levels of dystrophin mRNA present.

Since mono exon skipping of each of these 11 exons would be applicable to over 44% of DMD patients,22 these 11 U7snRNA constructs, shown to be efficient both in vitro and in vivo, represent promising tools for future clinical application. However, the number of eligible patients dramatically increases when considering double and multiexon skipping, reaching up to 83% of all DMD mutations.22 Of particular interest, the multiskipping of exons 45–55 in the hot spot mutation region has been suggested as worthwhile for investigation.16 Not only would this multiskipping be applicable to 63% of patients, but it would also create a deletion associated with a mild phenotype.23 This is of crucial importance considering it is not yet clear how functional each of the resulting internally deleted dystrophins will be. A few studies of small recombinant dystrophin fragments have examined the stability of non-native junctions expected from exon skipping approaches for DMD and highlighted the potential for decreased stability or protein aggregation when non-native repeat junctions are created in the dystrophin protein.24,25 More recently, biophysical analyses of recombinant dystrophin and utrophin constructs demonstrated that minidystrophin functionality may be compromised by the presence of non-native protein junctions that result in protein misfolding, instability, and aggregation.26 These studies confirm the importance of protein stability as a new factor to consider in the design of therapeutic vectors for the treatment of DMD.

For most targeted exons, each U7snRNA construct is composed of a combination of two antisense sequences (except for exon 47). This double-targeting strategy within one exon has been suggested in previous studies using the U7snRNA gene as a shuttle for antisense sequences10,27 and generally induces better skipping efficacy. Although this could evoke some concerns regarding off-target effects by doubling the target sites, the length of each individual sequence is on average at least 19 nucleotide, which constitutes sufficient sequence information to uniquely target a selected gene transcript. For clinical application, the specificity of each selected antisense sequence needs to be confirmed as with AOs against exon 51 used in recent clinical trials, which were shown to be highly sequence-specific (ref. 28 and http://www.prosensa.eu).

While double exon-skipping has been achieved in vitro and in vivo using AOs,29,30 skipping more exons and especially up to 11 can be very challenging with this technique.31 This can be intuitively explained by the difficulty of delivering all the different AOs to the same cell or myofiber. The use of viral vectors on the other hand, offers the advantage of transporting the different antisense constructs to the same nucleus, making the viral approach a much more appealing method to achieve multiexon-skipping. In order to test this hypothesis, we introduced up to three U7snRNA constructs into the same AAV vector and demonstrated efficient double and triple exon skipping in vivo. Interestingly the skipping efficiency does not seem to decrease much when targeting one or two additional exons, which is very encouraging for future larger exon-skipping attempts. While it would be feasible to utilize qPCR analysis to quantify levels of multiexon skipping in hDMD mice, we refrained from using it for this purpose due to the considerable number of Taqman assays required to quantify an increasing number of simultaneous exon skips. Moreover, qPCR results obtained on mono exon skipping confirmed that high level of skipping observed by nested RT-PCR correlated with substantial levels of skipping by qPCR. Nonetheless, qPCR may prove to be a valuable tool in the future when quantifying block skipping of exons 45–55.

Current AO-mediated exon-skipping approaches, despite showing encouraging results, are still facing some delivery issues with very variable restoration levels amongst muscles, as well as the hurdle of readministration that could potentially lead to toxicity problem due to the accumulation of AOs (see ref. 32 for review). The use of AAV vectors to induce exon-skipping may circumvent some of these obstacles as these vectors enable extremely stable in vivo expression in the vast majority of the musculature. The vectors introduce multiple extrachromosomal copies of the transgene in cells and display a variety of tissue tropisms, depending on the type of capsid used in the packaging system. Vectors packaged with AAV1, 6, 8, and 9 capsids are of particular interest for gene transfer into muscle33 and represent very efficient tools for the delivery of U7snRNA, as we previously demonstrated in the mdx mouse.12

Results from recent studies in larger animal models and in early human trials highlight immunological complications associated with viral vector-mediated gene transfer as the major barrier to clinical success. While AAV vectors do not elicit a cellular immune response in mice, multiple labs have now observed that AAVs 1, 2, 6, and 8 can elicit an immune response in the dog model for DMD,34,35,36,37 in monkeys38 and in humans.39,40,41,42,43 However, studies in dystrophic dogs also showed that this T-cell response could be blocked with transient immunosuppression,44 which has also been applied with success in nonhuman primates.38,45

There is also some concern that dystrophin itself may be immunogenic in dystrophin deficient patients. In the human trial of dystrophin gene transfer by Mendell and colleagues,46 intramuscular injections of rAAV2-minidystrophin resulted in robust minidystrophin-specific T-cell activity and none of the six patients injected displayed any detectable exogenous dystrophin. The authors proposed that dystrophin epitopes from revertant dystrophin fibres could prime the T-cell response. However, recent clinical trials evaluating AO-mediated exon-skipping have reported substantial levels of dystrophin expression reaching up to 97% of the myofibers in the biopsy after intramuscular injection,5 suggesting the absence of T-cell response against these fibers. This difference raises the hypothesis that the immune response observed in the AAV-minidystrophin trial might have been primed by the transduction of antigen-presenting cells. Differing from a conventional gene therapy approach where the transgene expression is driven by a ubiquitous promoter, the AAV-U7snRNA system induces dystrophin expression only in cells naturally expressing the dystrophin mRNA, and not in cells involved in the immune response for example. This advantage reduces the chances of activating the immune response through transduction of antigen-presenting cells. The AAV-U7snRNA approach may therefore circumvent this T-cell activation and represents the best of both worlds, combining the efficacy of a viral gene transfer system with the specificity of the exon-skipping strategy.

In conclusion, we developed efficient U7snRNA constructs targeting 11 different exons which could on their own be relevant to a large proportion of DMD patients. Furthermore, we demonstrated the feasibility of mutiexon-skipping in vivo by combining multiple U7snRNA constructs into a single AAV vector. This study therefore offers promising tools for the clinical evaluation of the U7snRNA-mediated exon skipping approach.

Materials and Methods

U7snRNA constructs design. The different U7snRNA constructs specific to each exon between 45 and 55 were engineered from the previously described U7smOPT-SD23/BP22 (modified murine U7snRNA gene).12 Antisense sequences targeting the mouse exon 23 were replaced by antisense sequences targeting the different exons of dystrophin mRNA, previously reported to induce exon skipping as AOs.17,18 Sequences inserted into U7snRNA constructs are reported in Table 1. The choice of targeting two sequences when designing the U7snRNA constructs was directed by the previous observation that combination of antisense sequences in U7snRNA seems to increase its efficiency.27 The resulting U7snRNA fragments were then introduced either in a lentiviral vector construct for further lentiviral production or into an AAV vector construct for AAV production. For the multiexon skipping vectors, each modified U7snRNA was inserted sequentially into the AAV backbone plasmid.

Viral vector production. All the lentiviral vectors were based on the pRRL-cPPT-hPGK-eGFP-WPRE constructs47 where the hPGK-GFP cassette was removed and replaced with the U7snRNA construct. For a large tropism, lentiviral vectors were pseudotyped with the vesicular stomatosis virus-G protein, and were generated by transfection into 293T cells of a packaging construct, pCMVΔR8.74, a plasmid producing the vesicular stomatosis virus-G envelope (pMD.G) and the vector itself as previously described.48 Viral titers (infectious particles) were determined by transduction of 105 NIH3T3 cells with serial dilutions of the vector preparation in a 12-well plate. Seventy-two hours later, genomic DNA from transduced cells was extracted using a genomic DNA purification kit (Qiagen, Crawley, UK). The infectious particles titer (infectious particle/ml) was determined by quantitative real-time PCR as described elsewhere.49

For subsequent AAV vector production, the different U7snRNA fragments were introduced at the XbaI site of the pSMD2 AAV2 vector.50 AAV2/1 pseudotyped vectors were prepared by cotransfection in 293 cells of pAAV2-U7snRNA, pXX6 encoding adenovirus helper functions and pAAV1pITRCO2 that contains the AAV2 rep and AAV1 cap genes. Vector particles were purified on Iodixanol gradients from cell lysates obtained 48 hours after transfection and titers were measured by quantitative real-time PCR.51

Cell culture and lentiviral transduction. Human myoblasts (courtesy of V. Mouly, Institut de Myologie, Paris, France) have been immortalized as described in ref. 19. Immortalized myoblasts (normal and DMD) were grown in DMEM medium (Invitrogen, UK) supplemented with 20% fetal bovine serum (Invitrogen, Paisley, UK), 100 U/ml penicillin and 100 U/ml streptomycin. For lentiviral transduction, 1 × 105 myoblasts were seeded in each well of a 12-well plate. After 24 hours, cells were incubated with 106–107 infectious particle of lentiviral vector in a total volume of 500 µl of their respective medium mentioned above. After 4 hours of incubation, medium was replaced. Cells were subsequently amplified for further analysis (RNA and protein analysis). All analysis was repeated on cells amplified from at least three independent transductions for each U7 construct. In all cases, copy number of integrated lentiviral vector has been measured by quantitative real-time PCR and shown to be directly comparable.

RNA isolation and RT-PCR analysis. Total RNA was isolated from the human myoblasts using the RNeasy kit according to the manufacturer's instructions (Qiagen). Aliquots of 200 ng of total RNA were used for RT-PCR analysis using the Access RT-PCR System (Promega, Southampton, UK) in a 50-µl reaction using the external primers surrounding the exon of interest (primer sequence list available upon request). The cDNA synthesis was carried out at 45 °C for 45 minutes, directly followed by the primary PCR of 30 cycles of 94 °C (30 seconds), 58 °C (1 minute), and 72 °C (2 minutes). Two microlitres of these reactions were then reamplified in nested PCRs by 25 cycles of 94 °C (30 seconds), 58 °C (1 minute), and 72 °C (2 minutes) using the internal primers surrounding the exon of interest. For in vivo experiments in hDMD mice, total RNA was isolated from the injected muscles using TRIzol reagent according to the manufacturer's instructions (Invitrogen). 200 ng of total RNA were used for RT-PCR analysis using the Access RT-PCR System (Promega) in a 50-µl reaction using the external primers. The cDNA synthesis was carried out at 45 °C for 45 minutes, directly followed by the primary PCR of 30 cycles of 94 °C (40 seconds), 60 °C (40 seconds), and 72 °C (40 seconds). Two microlitres of these reactions were then reamplified in nested PCRs by 25 cycles of 94 °C (40 seconds), 60 °C (40 seconds), and 72 °C (40 seconds) using the internal primers. PCR products were analyzed on 2% agarose gels.

qRT-PCR analysis. RNA was isolated from mouse tissue as described above. Contaminating DNA was removed from the RNA preparations using the Turbo DNA-free system (Ambion, Huntingdon, UK). One microgram aliquots of DNase-treated RNA were then subjected to RT using the First-Strand Synthesis System (Invitrogen) with random hexamers according to the manufacturer's instructions. qPCR was performed using Taqman assays that were designed against exon-skipped or unskipped templates using the Custom Assay Design Tool (Applied Biosystems, Warrington, UK, primer/probe sequences available upon request). When possible, existing inventoried Taqman assays were utilized. All dystrophin Taqman probes used incorporated a FAM fluorophore and NFQ-MGB quencher. An 18S Taqman assay (VIC/TAMRA) was used as an endogenous control of cDNA input (Applied Biosystems; 4310893E). Ten nanogram of cDNA was used as input per reaction and assays were carried out in singleplex. Assays were performed on a fast cycle using an Applied Biosystems StepOne Plus Thermocycler and all data were analyzed by the comparative Ct method using the associated StepOne analytical software. Δ-Ct values of skipped and unskipped assays within a given sample were used to calculate the relative abundance of templates and this was then expressed as percentage skipping.

Western blot analysis. Protein extracts were obtained from pelleted myoblasts treated with Newcastle buffer (3.8% SDS, 75 mmol/l Tris–HCl pH 6.7, 4 mol/l urea, 10% β-mercaptoethanol, 10% glycerol, 0.001% bromophenol blue). To determine the total protein amount in samples, the bicinchoninic acid protein assay kit and protocol was used (Perbio Science, Northumberland, UK). Samples were denatured at 95 °C for 5 minutes before 50 µg of protein was loaded in a 5% polyacrylamide gel with a 4% stacking gel. Gels were electrophoresed for 4–5 hours at 100 V and blotted to a polyvinylidene fluoride membrane overnight at 40 V. Blots were blocked for 1 hour with 10% nonfat milk in phosphate-buffered saline–Tween (PBST) buffer. Dystrophin and α-actinin proteins were detected by probing the membrane with 1:100 dilution of NCL-DYS1 primary antibody (monoclonal antibody to dystrophin R8 repeat; NovoCastra, Newcastle upon Tyne, UK) and 1:200 dilution of α-actinin primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA), respectively. An incubation with a mouse horseradish peroxidase-conjugated secondary antibody (1:2,000) or goat horseradish peroxidase-conjugated secondary antibody (1:160,000) allowed visualization using ECL Analysis System (GE Healthcare, Chalfont St Giles, UK).

Transgenic human DMD mice injection. Transgenic human DMD mice were imported from the Leiden University Medical Center.28,52 All animal experiments were performed according to the guidelines and protocols approved by the Home Office. Adult hDMD mice (between 6 and 8 weeks of age) were anesthetized under isofluorane and injected with 1011 vg of AAV2/1 vector, encoding the different U7snRNA constructs, into the TA muscles. Mice were sacrificed 4 weeks after the injection and TA muscles were isolated and snap frozen in liquid nitrogen-cooled isopentane.

SUPPLEMENTARY MATERIAL Figure S1. Sequence analysis of the unskipped and skipped products.

Acknowledgments

We are grateful to Vincent Mouly (Institut de Myologie, Paris, France) for providing the immortalized myoblasts used in this study. We also thank Annemieke Aarstma-Rus and Johan T. den Dunnen (Leiden University Medical Center, the Netherlands) for providing the transgenic human DMD mice. This work was supported by the UK Medical Research Council, the Muscular Dystrophy Campaign, the Muscular Dystrophy Association USA, the Association Monegasque contre les myopathies and the Duchenne Parent project France.

Supplementary Material

Figure S1.

Sequence analysis of the unskipped and skipped products.

Click here for additional data file. (66.2MB, tiff)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Sequence analysis of the unskipped and skipped products.

Click here for additional data file. (66.2MB, tiff)

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