Triple Trans-Splicing Adeno-Associated Virus Vectors Capable of Transferring the Coding Sequence for Full-Length Dystrophin Protein into Dystrophic Mice

Abstract

Recombinant adeno-associated virus (rAAV) vectors have been shown to permit very efficient widespread transgene expression in skeletal muscle after systemic delivery, making these increasingly attractive as vectors for Duchenne muscular dystrophy (DMD) gene therapy. DMD is a severe muscle-wasting disorder caused by DMD gene mutations leading to complete loss of dystrophin protein. One of the major issues associated with delivery of the DMD gene, as a therapeutic approach for DMD, is its large open reading frame (ORF; 11.1 kb). A series of truncated microdystrophin cDNAs (delivered via a single AAV) and minidystrophin cDNAs (delivered via dual-AAV trans-spliced/overlapping reconstitution) have thus been extensively tested in DMD animal models. However, critical rod and hinge domains of dystrophin required for interaction with components of the dystrophin-associated protein complex, such as neuronal nitric oxide synthase, syntrophin, and dystrobrevin, are missing; these dystrophin domains may still need to be incorporated to increase dystrophin functionality and stabilize membrane rigidity. Full-length DMD gene delivery using AAV vectors remains elusive because of the limited single-AAV packaging capacity (4.7 kb). Here we developed a novel method for the delivery of the full-length DMD coding sequence to skeletal muscles in dystrophic mdx mice using a triple-AAV trans-splicing vector system. We report for the first time that three independent AAV vectors carrying “in tandem” sequential exonic parts of the human DMD coding sequence enable the expression of the full-length protein as a result of trans-splicing events cojoining three vectors via their inverted terminal repeat sequences. This method of triple-AAV-mediated trans-splicing could be applicable to the delivery of any large therapeutic gene (≥11 kb ORF) into postmitotic tissues (muscles or neurons) for the treatment of various inherited metabolic and genetic diseases.

Introduction

Adeno-associated virus (AAV) vectors provide one of the most efficient delivery vehicles for gene transfer. Currently, at least 11 primate AAV serotypes have been described that demonstrate diverse tissue tropisms. Many serotypes, including AAV 1, 2, 6, 8, and 9, have now been demonstrated to efficiently transduce into skeletal muscles after systemic injection (Zincarelli et al., 2008). Importantly for application for gene therapies, recombinant AAV6 (rAAV6) and in particular rAAV8 and 9 vectors have been shown to transduce both cardiac and skeletal muscle at high efficiencies after systemic gene transfer, allowing the potential for body-wide gene transfer (Gregorevic et al., 2004; Wang et al., 2005; Foster et al., 2008; Zincarelli et al., 2008). A number of studies have also demonstrated very stable gene expression: over 8 years in canine models after systemic injection of rAAV2 (Niemeyer et al., 2009) and 6 years in nonhuman primate models after intramuscular injection of rAAV2/1 (Rivera et al., 2005). These factors, along with the apparent lack of immunogenicity to both transgene and virus in animal models, have made rAAV vectors very promising facilitators of genetic delivery in gene therapy. However, one of the main drawbacks of using AAV in gene therapy applications is limited packaging capacity (Grieger and Samulski, 2005; Lai et al., 2010; Wu et al., 2010). Recombinant AAVs utilizing the cis-acting 145 bp inverted terminal repeats (ITRs) to flank vector transgene cassettes provide a packaging capacity of up to 4.7 kb for foreign DNA. In muscle, molecular assembly of the AAV vector genomes occurs through intermolecular recombination between two ITRs, leading to genome concatemerization (Yang et al., 1999). This aspect of AAV vector biology has led to attempts to develop trans-splicing or recombination-based overlapping vector approaches for the transfer of larger genes. Trans-splicing or recombination-based overlapping AAV vector approaches have increased the gene delivery capacity by up to twofold (Lai et al., 2005; Li et al., 2008). Two independent AAV vectors each carrying parts of a transgene with appropriate splice signals or with overlapping sequence elements are independently packaged in AAV vectors. Each AAV vector encoding either the 5′ or the 3′ expression cassette can then be targeted to infect single postmitotic cells simultaneously. One full-length mRNA is produced by head-to-tail intermolecular recombination between two independent genomes, transcription, and subsequent splicing across the ITR junction in coinfected cells.

Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder caused by mutations in the DMD gene. Despite an expansive wealth of research after the discovery of the DMD gene 25 years ago, there is still no curative treatment for DMD. Out-of-frame mutations of the DMD gene lead to the complete loss of the dystrophin protein and severe muscle damage in DMD patients. In the context of gene therapy for DMD, AAV vectors have been used for microdystrophin (single-AAV cassettes) or minidystrophin (dual trans-splicing or overlapping methods) gene transfer approaches to restore dystrophin expression with great muscle improvement. The microdystrophin gene transfer using an AAV vector seems very efficient in protecting muscle rigidity and restoring the muscle function significantly without muscle necrosis (Foster et al., 2008; Banks et al., 2009; Koo et al., 2011a,b). Attempts to transfer the minidystrophin genes by trans-splicing AAV vector approaches (Lai et al., 2005; Zhang et al., 2013) or protein trans-splicing (Li et al., 2008) have also been successfully demonstrated. However, there is no currently available trans-splicing AAV vector-mediated gene delivery system documented to deliver the full-length 11.1 kb DMD coding sequence or any other large ≥8 kb open-reading-frame (ORF) transgene to this effect. Such a delivery system would be required for a universal gene therapy approach for DMD, in principle, across all DMD patient groups.

In this study, we demonstrate a triple trans-splicing approach for the transfer of the ∼11.1 kb full-length ORF of the human DMD gene to overcome the limited AAV packaging capacity. Three independent AAV vectors were designed to deliver three exonic sections of a human DMD coding sequence, which, if introduced simultaneously into the same cells in vitro or in vivo, are able to produce full-length dystrophin protein by intermolecular recombination “in tandem” and cojoining between the three vector genomes. With further development, the triple trans-splicing vector system has the potential to compensate for dystrophin loss of function by facilitating a highly efficient ∼11.1 kb full-length DMD gene/ORF transfer into skeletal muscles. This novel approach could be also applicable for the transfer of other large genes as a therapy for a range of other diseases.

Materials and Methods

Construction of triple trans-splicing AAV ITR-based vector plasmids carrying split DMD coding sequence

cDNA encoding DMD exons 1–25 from the pCihdys plasmid was cloned between the StuI and AleI cutting site in hybrid pAAV2:5ITR-part of dys1. This vector carried the CAG promoter, HA-tag sequence, part of the DMD gene (from 5′ end of exon 1 to StuI site, from AleI to 3′ end of exon 25), and a synthetic splicing donor signal. cDNA encoding DMD exons 26–54 from the pCihdys plasmid was cloned between the Bsu361I and BstXI cutting site into pAAV5:2ITR-part of dys2. This vector carried a synthetic splicing acceptor signal, part of the DMD gene (from 5′ end of exon 26 to Bsu351I site, from BstXI to 3′ end of exon 54) and a synthetic splicing donor signal. cDNA encoding DMD exons 55–79 from the pCihdys plasmid was cloned between the XhoI and BlpI enzyme site into pAAV2:2ITR-part of dys3. This vector carried a synthetic splicing acceptor signal, part of the DMD gene (from 5′ end of exon 55 to XhoI site, from BlpI to 3′ end of exon 79), enhanced green fluorescent protein (eGFP), and a poly A signal. The woodchuck hepatitis posttranscriptional regulatory element (WPRE) was inserted between the ClaI enzyme sites after the eGFP gene. The vectors were de novo synthesized (GeneART). Details of the vector sequence are available upon request.

Production, purification, and characterization of AAV2/5 trans-splicing viral vectors

To produce AAV vectors, all triple-AAV vectors were pseudotyped in AAV5 capsid. For vector1-dys1 production, HEK293T cells were transfected with pAAV2:5ITR-dys1, pAAV2–5, and pAAVhelpercap5. For vector2-dys2 vector, HEK293T cells were transfected with pAAV5:2ITR-dys2, pAAV2–5, and pAAVhelpercap5 plasmids. For vector3-dys3, HEK293T cells were transfected with pAAV2:2ITR-dys3 and pAAVhelpercap5 plasmids. The pAAVhelpercap5 encoding rep2/cap5 and the pAAV2–5 vector encoding the Rep5 protein were sufficient to allow virus propagation. HEK293T cells were cultured in roller bottles in Dulbecco's modified Eagle's medium, supplemented with 10% (v/v) fetal bovine serum, and incubated at 37°C and 5% CO₂. Reagents for cell culture were purchased from Invitrogen. Recombinant pseudotyped AAV vector stocks were generated using calcium phosphate coprecipitation and triple-transfection with plasmids at a molar ratio of 1:1:1 in HEK293T cells. After 72 hr of incubation, cells were lysed and particles were purified by iodixanol (Sigma-Aldrich) step-gradient ultracentrifugation. The number of vector genomes was determined by dot blot hybridization.

In vivo gene delivery

AAV was administered to 8-week-old young adult male mdx mice anesthetized with 2%–4% isoflurane, and the mice were injected intramuscularly with cocktails of vector1-dys1, vector2-dys2, and vector3-dys3 in 40 μl of physiological saline. As a positive control for histological analysis, wild-type C57BL/10 mice were purchased from Harlan. As a negative control, mdx mice were injected with 40 μl of physiological saline only. C57BL/10ScSn-Dmdmdx (mdx) mice were bred in-house and maintained in minimal-disease facilities (Royal Holloway, University of London) with food and water ad libitum. In vivo experiments were conducted under statutory Home Office recommendation; regulatory, ethics, and licensing procedures; and the Animals (Scientific Procedures) Act 1986.

Analysis of total RNA

About 30 mg of cryosections of tibialis anterior (TA) tissue was homogenized using tungsten carbide beads (3 mm; Qiagen) and a TissueLyser (Qiagen). RNA was harvested by the RNeasy Fibrous Tissue Mini Kit (Qiagen), and nested reverse transcription–polymerase chain reaction (RT-PCR) was performed to examine trans-splicing across the different AAV vectors using the GeneScript RT-PCR system kit (GeneSys) for the first round and 2× PCR Mastermix (GeneSys) for the second round. Human-specific primers were designed, and specificity was confirmed on control human and mdx RNA. For analysis of trans-splicing between vector1 and vector2, first-round human-specific primers were designed to exons 23 (vector1) and 27 (vector2), and human-specific second-round primers were designed to exons 23 (vector1) and 26 (vector2). For confirmation of correct splicing between vector2 and vector3, seminested RT-PCR was performed using human-specific first-round primers to exon 53 (vector2) and over exon 54/55 boundary (vector2/vector3 junction), and human-specific second-round primers to exon 54 (vector2) and over exon 54/55 boundary (vector2/vector3 junction). Products were separated on 2.5% agarose gel in Tris-borate/EDTA buffer, and Hyper Ladder V (Bioline) was used as a marker. Sequencing of the exon 23–26 amplicons and exon 54–54/55 amplicons was performed by MWG Eurofins. Sequences were aligned against the respective human and mouse sequences by Vector NTI software (Invitrogen). Details of primers and RT-PCR protocols used in this study are available upon request.

To confirm the presence of transcript corresponding to vector1/vector2/vector3 trans-splicing, cDNA was generated from 1 μg RNA from treated samples by RevertAid H Minus First Strand cDNA synthesis Kit (ThermoScientific) with a human-specific primer designed to exon 77. The cDNA was then subjected to seminested PCR using human-specific primers to exons 23 (vector1) and 57 (vector3) in the first round, and exons 23 (vector1) and 56 (vector3) in the second round by LongAmp Taq DNA polymerase (New England Biolabs). Specificity of primers used for reverse transcription and PCR was tested on control human and mouse RNA. Human DMD cDNA was included as a positive control in the PCRs.

Histological analysis

TA muscles of mice were excised from tendon to tendon, weighed, and rapidly frozen in liquid nitrogen-cooled isopentane. To assess muscle pathology, 10 μm cryosections of muscles were prepared and fixed in cold acetone. For immunohistochemistry, muscle sections were stained by dystrophin antibodies for dys1 (sc-15376; a rabbit polyclonal antibody corresponding to amino acids 801–1100; Santa Cruz Biotechnology) and dys3 (P6; a rabbit polyclonal antibody corresponding to amino acids 2814–3028, a gift from Prof. Glenn Morris, Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, UK). An antibody against the eGFP protein (ab6556; rabbit polyclonal; Abcam) was used. The signal was visualized by Alexafluor 568-conjugated anti-rabbit IgG (Invitrogen).

Results

Generation of a triple trans-splicing AAV vector system to deliver the full-length DMD coding sequence

The 11.1 kb of full-length DMD coding sequence was split into three pieces and packaged into AAV vectors (AAV vectors 1–3). Optimal gene-splitting sites in the DMD gene were designed to be out of frame to avoid expression of one part of the DMD gene (after circular monomers formation) as delivered by a single AAV vector (Fig. 1). Two gene-splitting sites in the 11.1 kb full-length DMD gene were defined at the junction of exon 25 and exon 26 and at the junction of exon 54 and exon 55 to obtain high consensus splicing values (CV) between exon/intron/exon junctions. Score of CV was calculated according to nucleotide percentages at splice junctions in primates (Shapiro and Senapathy, 1987). AAV vector1 contained exon 1 to exon 25 of the DMD gene (dys1), followed by a splice donor consensus sequence at the 3′ end of exon 25. Expression of this part of the DMD gene was controlled by the strong ubiquitous CAG promoter. An optimal Kozak sequence and HA tag was incorporated at the 5′ end of exon 1 of the DMD gene. The size of the transgene cassette between AAV-ITRs for vector1 was 4.3 kb. To overcome the limitation of random recombination between vector genomes, a nonhomologous ITR hybrid vector system was developed with head-to-tail recombination of ITRs derived from AAV serotypes 2 and 5, respectively. The vector1 transgene cassette was packaged by hybrid ITR2 and ITR5 derived from AAV serotype 2 and 5 at the 5′ and 3′ ends, respectively. AAV vector2 harbored exons 26–54 of the DMD gene (dys2) flanked by splice acceptor and splice donor sequences at 5′ and 3′ ends, respectively. The size of the transgene cassette between AAV-ITRs for vector2 was 4.7 kb. This was flanked by hybrid ITR5 and ITR2 derived from AAV serotypes 5 and 2 at 5′ and 3′ ends, respectively. Finally, AAV vector3 contained exons 55–79 of the DMD gene (dys3) with a splice acceptor sequences incorporated to the 5′ end of exon 55. An eGFP gene was fused at the 3′ end of exon 79 to allow visualization of the fused dystrophin protein expression, under direct fluorescence microscopy. Moreover, the stop codon at the 3′ end of dys3 and start codon at the 5′ end of eGFP were removed to ensure the concurrent expression of dystrophin and eGFP and to avoid eGFP expression without dystrophin expression. Ahead of the polyA signal, WPRE was incorporated to increase mRNA stability and avoid the rapid degradation of the full-length recombinant dystrophin mRNA. This vector harbored the homologous ITR2 sequences as derived from AAV serotype 2 to lead to intermolecular recombination formation between 3′ ITR2 in vector2 and 5′ ITR2 in vector3, as well as 3′ ITR2 in vector3 and 5′ ITR2 in vector1. Thus, compared with vector1 and vector2, vector3 having flanking-compatible ITRs was more prone to deliver circular monomer formations. The size of the transgene cassette between AAV-ITRs for vector3 was 4.1 kb. ITR-mediated intermolecular recombination of the three AAV vectors after splicing of the three parts of the resultant DMD pre-mRNA transcript “in tandem” should result in transduction of the full-length human dystrophin protein in a triple-AAV-transduced muscle cell.

FIG. 1.

Schematic diagram of triple trans-splicing AAV vector approaches for overcoming the size limitation of gene delivery. For the triple-vector trans-splicing approach, 11.1 kb full-length DMD ORF can be carried from three independent sets of trans-splicing AAV vectors. Vector1-dys1 carries the CAG promoter, a DMD cDNA encoding exons 1–25 with an N-terminal HA-tag, and a splice donor signal at the 3′ end of DMD exon 25. Vector2-dys2 contains DMD exons 26–54 flanked by splice acceptor and donor signals at the 5′ end and the 3′ end. Vector3-dys3 contains a DMD exons 55–79 fused to eGFP, the splice acceptor signal at the 5′ end of exon 55, the WPRE, and the SV40 poly A signal. AAV, adeno-associated virus; DMD, Duchenne muscular dystrophy; eGFP, enhanced green fluorescent protein; ORF, open reading frame; SA, splicing acceptor; SD, splicing donor; seq, sequence; WPRE, woodchuck hepatitis posttranscriptional regulatory element.

To optimize the splicing efficiency of the trans-splicing AAV vector, the endogenous introns at the junction of exon 25 and exon 26 and at the junction of exon 54 and exon 55 were replaced with an optimized synthetic intron that had the highest CV at the 5′ and 3′ splice sites at exon/intron junctions of DMD pre-mRNA (Fig. 2). The nucleotides were optimized in the synthetic intron to have the highest splicing value so as to enhance splicing efficiency. The synthetic intron was split into two pieces, and the 5′ end of the intron contained a splicing donor signal, derived from the first intron in the human-globulin gene, while the 3′ end of the intron contained a splicing acceptor signal, derived from the human immunoglobulin heavy chain gene. The splicing acceptor signal incorporated two branch point sites. The splicing signals from synthetic introns can obtain 100% CV at AG of the 3′ end of the exon and 96.56% CV at G of the 5′ end of the exon.

FIG. 2.

The highly efficient synthetic intron acts as a splicing donor and as a splicing acceptor in the reconstituted AAV genome. The synthetic intron splicing signal was inserted between exon junctions. The splicing donor from the first intron in the human β-globin gene was used, which gives a 100% consensus splicing value when it is incorporated to AG at the 3′ end of the exon. The splicing acceptor derived from the human immunoglobulin heavy chain gene renders a 96.56% consensus splicing value with G at the 5′ end of the exon.

Evaluation of triple trans-splicing efficacy by transcript/amplicon analysis

To evaluate the transduction efficiency of triple trans-splicing vectors, three viral vectors were packaged into AAV serotype 5 capsids and injected simultaneously into TA muscles of 8-week-old male mdx mice (n=4). TA muscles of mdx mice were injected with 1×10¹⁰ vector genomes each of three AAV2/5 vectors carrying the three parts of DMD coding sequences (dys1, dys2, and dys3, respectively), and TA muscles were harvested 8 weeks after intramuscular injection. As a negative control, TA muscles of age-matched mdx mice were injected with physiological saline (n=4) and harvested at the same time point.

To assess the presence of trans-spliced full-length human DMD mRNA, seminested RT-PCR was performed to amplify over the junction between exon 25 and exon 26 and between exon 54 and exon 55 of human DMD coding sequences. We used a human-specific primer set to specifically delineate between the relatively rare events of false-positive mouse dystrophin revertant fibers and formation of human triple trans-spliced transcripts. RNA harvested from normal human primary myoblast and that from mdx muscle tissue were included as positive and negative controls, respectively. The amplicons from exon 23 within vector1-dys1 to exon 26 within vector2-dys2 were successfully detected in three out of the four mdx TA muscles (samples 1, 2, and 4) injected with triple trans-splicing AAV vectors (Fig. 3a). This strongly suggested that there was a successful trans-splicing event between the two split human DMD coding sequences derived from two independent AAV vectors, leading to trans-spliced human DMD mRNA product expression. The amplicon from exon 54 within vector2-dys2 to exon 54/55 boundary across the vector2-dys2/vector3-dys3 boundary was also detectable in one out of four mdx TA muscles (sample 2) (Fig. 4a); this TA also showed successful trans-splicing between vector1 and vector2 (Fig. 3a), demonstrating that full-length human DMD mRNA was detectable through two consequential trans-splicing events using triple trans-splicing vector system. These amplicons were further analyzed by sequencing to ascertain species specificity of the human DMD mRNA (Figs. 3b and 4b). The amplicons from exon 23 and exon 26 and from exon 54 and exon 54/55 boundary were human DMD specific and showed complete homology to the human DMD sequence (ensemble ID ENSG00000198947) (Figs. 3c and 4c). To ensure that our results were not biased on the basis of RNA quality, we performed RT-PCR using mouse 18s primers to confirm that the RNA was of equally good quality in all tissue samples (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/hum).

FIG. 3.

Vector1/vector2 RT-PCR analysis of RNA harvested from TA muscle of mdx after intramuscular injection of triple trans-splicing AAV2/5 vectors. (a) Nested RT-PCR analysis was performed using human-specific primers to amplify a sequence of 510 bp between exons 23 and 26 in the second round of PCR. (1) Untreated mdx TA muscle; (2) saline-treated mdx TA muscle; (3) TTS-AAV-treated mdx TA muscle (sample 1); (4) TTS-AAV-treated mdx TA muscle (sample 2); (5) TTS-AAV-treated mdx TA muscle (sample 3); (6) TTS-AAV-treated mdx TA muscle (sample 4); (7) no transcript control; (8) human muscle cell RNA. (b) The amplicons were sequence-analyzed, and (c) aligned against human dystrophin using Vector NTI software. RT-PCR, reverse transcription–polymerase chain reaction; TA, tibialis anterior.

FIG. 4.

Vector2/vector3 RT-PCR analysis of RNA harvested from TA muscle of mdx after intramuscular injection of triple trans-splicing AAV2/5 vectors. (a) Seminested RT-PCR analysis was performed using human-specific primers to amplify between exon 54 and the exon 54/55 boundary. (1) Untreated mdx TA muscle; (2) saline-treated mdx TA muscle; (3) TTS-AAV-treated mdx TA muscle (sample 1); (4) TTS-AAV-treated mdx TA muscle (sample 2); (5) TTS-AAV-treated mdx TA muscle (sample 3); (6) TTS-AAV-treated mdx TA muscle (sample 4); (7) human muscle cell RNA; (8) no transcript control. (b) The amplicons were sequence-analyzed, and (c) aligned against human dystrophin using Vector NTI software.

To demonstrate the presence of transcripts corresponding to splicing between all three vectors, cDNA was generated using a human-specific primer to exon 77 and then subjected to seminested PCR using LongAmp Taq DNA polymerase with human-specific primers to exon 23 (vector1) and exon 57 (vector3) in the first round and to exon 23 (vector1) and exon 56 (vector3) in the second round. An amplicon of the correct size (5199 bp) was evident in one sample out of the four treated mdx TA muscles (sample 2) (Fig. 5), which corresponded with the sample that showed successful trans-splicing between vector1 and vector2, and also vector2 and vector3.

FIG. 5.

Analysis of cDNA derived from TA muscle of mdx after intramuscular injection of triple trans-splicing AAV2/5 vectors for vector1/vector2/vector3 triple trans-splicing of RNA. Complementary cDNA was generated using a human-specific primer to exon 77. Using LongAmp Taq DNA polymerase, the cDNA was subjected to seminested PCR using human-specific primers to amplify between exon 23 and exon 56. cDNA generated from human primary cells using the same human-specific primer to exon 77 (human primary) and DNA from a plasmid carrying full-length DMD (human pCihdys) were included as positive controls. (1) Saline-treated mdx TA muscle; (2) TTS-AAV-treated mdx TA muscle (sample 1); (3) TTS-AAV-treated mdx TA muscle (sample 4); (4) TTS-AAV-treated mdx TA muscle (sample 2); (5) human muscle cell cDNA; (6) human pCihdys plasmid.

Taken collectively, analysis of the RNA showed that although it was of low efficiency, triple trans-splicing between the three vectors was occurring and led to full-length human DMD mRNA in treated mdx mice.

The full-length human dystrophin protein was expressed in mdx skeletal muscles

To assess the level of the trans-spliced full-length human dystrophin protein at the sarcolemma, TA muscles were cross-sectioned and stained using dystrophin antibodies: H-300 antibody against the N-terminus (NT) domain of dystrophin (corresponding to amino acids 801–1100), and P6 antibody against the C-terminus (CT) domain of dystrophin (corresponding to amino acids 2814–3028). Staining was also performed using eGFP antibodies against the eGFP that was fused to the 3′ end of the CT domain of dys3. The level of dystrophin expression was evaluated by counting dystrophin-positive fibers and total fibers in treated mdx muscles, and the percentage of dystrophin-positive fibers was compared with muscles injected with saline. The dystrophin expression was elevated in mdx TA muscle injected with AAV2/5-trans-splicing vectors carrying the three exonic parts of the DMD coding sequence, displaying 5.3- and 2.4-fold increases in the level of NT- and CT-dystrophin-positive fibers, respectively, compared with the level of revertant fibers in mdx control TA muscles injected with saline (Fig. 6). This TA muscle (sample 2) showed trans-splicing event between vector1 and vector2, as well as vector2 and vector3 by the transcript/amplicon analysis (Figs. 4 and 5). The discrepancy in the dystrophin level change between the NT- and CT-domain of dystrophin was caused by difference in the number of revertant fibers recognized by antibodies to different domains of dystrophin. Very few revertant fibers were observed using NT-dys-recognizing antibodies in saline-injected mdx TA control muscles, whereas an increasing number of revertant fibers were observed by CT-dys-recognizing antibodies in serial sections (Fig. 6). There was no difference in the percentage of revertant fibers between saline-injected and untreated mdx muscles. This implied that the increased dystrophin expression in the triple trans-spliced vector-treated muscles relative to the saline-injected muscles was not caused by muscle de/regeneration induced by muscle damage from injection itself, but was caused by trans-spliced dystrophin protein expression.

FIG. 6.

Expression of the dystrophin-eGFP fusion protein in TA muscles of mdx after intramuscular injection of triple trans-splicing AAV2/5 vectors. TA muscles of 8-week-old mdx mice were injected with 1×10¹⁰ vector genomes of each of AAV vector1-dys1, vector2-dys2, and vector3-dys3, simultaneously. After 8 weeks, TA muscle (sample 2) was recovered and three serial sections were stained using H-300 antibody against the NT domain of dystrophin, P6 antibody against the CT domain of dystrophin, and eGFP antibody against the eGFP protein that was fused in the 3′ end of the CT domain of dystrophin. Negative control TA muscles from age-matched mdx were injected with a same volume of physiological saline. TA muscles from wild-type C57BL/10 mice were used as a positive control. Asterisks indicate the same fibers in three serial sections for orientation. CT, C-terminal; NT, N-terminal; tts-AAV; triple trans-splicing AAV.

Expression of the eGFP protein was observed at the sarcolemma of triple trans-spliced AAV-treated muscle in 4.1% of the total fibers, whereas saline-injected mdx TA muscles did not show any eGFP-expressing fibers (Fig. 6). There was no eGFP expression in the sarcoplasm except for a very few muscle fibers (less than 3 fibers/section), since eGFP protein was fused to the CT domain of dystrophin. Therefore, eGFP and the full-length human dystrophin protein were expressed as a single molecule with sequential localization at the sarcolemma.

Discussion

Several groups have successfully transferred genes with ORFs larger than 4.5 kb, such as a 6.3 kb Becker-type human minidystrophin transgene by dual trans-splicing or overlapping AAV approaches (Ghosh et al., 2006; Li et al., 2008; Zhang et al., 2013). It has also been reported that a systemically delivered trans-splicing AAV can transduce transgenes into skeletal and heart muscles of mdx mice (Ghosh et al., 2009). Despite a report suggesting that a packaging capacity of up to 8.9 kb transgene can be accommodated via an AAV serotype 5 vector (Allocca et al., 2008), other studies suggest that a transgene larger than 6 kb is not fully packaged by the AAV5 capsid, and that partially packaged larger transgenes of AAV are preferentially degraded by the proteasome (Lai et al., 2010; Wu et al., 2010). There has been no AAV vector system yet described that can potentially transfer the full-length DMD coding sequence of ∼11.1 kb.

This study has focused on the early stage development/conceptualization of a novel triple trans-splicing AAV vector system capable of transferring the full-length 11.1 kb human DMD ORF sequence. To our knowledge, this is the first report on the use of a triple AAV vector system for the transfer of a large gene. A prerequisite of this AAV-TTS system to generate the correct full-length human dystrophin/eGFP fusion is that all three AAV vectors have to be correctly aligned/orientated in a 1-2-3 (or 2-3-1 or 3-1-2) circular concatamer hierarchy/orientation and should transduce the same single muscle cell/fiber, resulting in the expression of a full dystrophin-eGFP fusion protein. This constitutes a ∼25% (3/12) frequency event probability (and that is if all three vectors end up in the same cell) from all the possible theoretical combinations of either single [1, 2, 3 (as circular monomers)], dual [1–2, 1–3, 2–3 (circular dimers)], or reversed engineered event [1-3-2, 2-1-3, 3-2-1 (circular concatamers)], which theoretically would not necessarily result in right patterns of expression.

Trans-splicing approaches have great potential for the transfer of large therapeutic genes into tissues, exploiting the natural serotype and species/tissue-specific tropism of AAV vectors and their low immunogenicity. In this study, three independent vectors containing three parts of human DMD ORF “in tandem” were generated for the transfer of full-length human DMD coding sequences into muscle cells. In principle, when the three AAV vectors are simultaneously infected into a single muscle cell, the intrinsic AAV vector biology can reconstitute and generate full-length human DMD mRNA through ITR intermolecular recombination/concatamerization and cojoining. After splicing of DMD mRNA, a full-length 427 kDa dystrophin protein can be obtained through utilization of the translation machinery of muscle cells. However, one of the limitations of the AAV trans-splicing system is that the transduction efficiency of the system is lower than that of a single intact AAV vector. To optimize the splicing efficiency, each piece of the DMD coding sequence was linked to synthetic introns at the junctions of the splice site, with the aim to boost mRNA production. It has also been reported that trans-splicing vectors carrying a synthetic intron had improved transduction efficiency of AAV relative to endogenous intron-based vectors (Lai et al., 2006). Another main hurdle of the trans-splicing AAV vector to overcome is the limitation of the hetero-oligomerization of vector genomes, and directionality of the recombination between independent vector genomes. Formation of circular monomers of the single vectors through self-circularization without recombination with another vector genome of AAV is possible. This can cause expression of broken pieces of unstable mRNA, leading to rapid degradation. Moreover, the double-D-ITR junctions between two fused ITRs can prevent the accumulation of spliced mRNA, and it may result in reduced expression of the gene (Xu et al., 2004). The difference in the nucleotide sequence between ITRs derived by AAV2 and AAV5 is higher compared with other serotypes of AAV (serotypes 1, 3, 4, 6, and 7). It has been reported that the ITR2:ITR5 hybrid vector can decrease the frequency of circularization of the genomes (Yan et al., 2007). This leads to enhanced reconstruction of the gene compared with homologous ITR2:ITR2 and ITR5:ITR5 vectors, not only after coinfection of Hela and primary fetal fibroblast, but also in vivo in mouse skeletal muscle, liver, and heart (Yan et al., 2005, 2007). It may also provide more efficient directional recombination because of increased abundance of linear-form genomes. To increase the directional recombination between intermolecular genomes of triple trans-splicing AAV vectors, the hybrid AAV vector1-dys1 and AAV vector2-dys2 were flanked by ITR2:ITR5 and ITR5:ITR2 head-to-tail ITR configuration, respectively. However it was not feasible under the current system to avoid ITR2-mediated homologous circular monomer formation of AAV vector3 alongside the required intermolecular recombination formation between 3′ ITR2 in vector2 and 5′ ITR2 in vector 3, as well as 3′ ITR2 in vector3 and 5′ ITR2 in vector1.

We demonstrate that the full-length dystrophin-fused eGFP protein was successfully expressed at the sarcolemma of the skeletal muscles, although at low levels. In theory, this would not have occurred without “in tandem” trans-splicing of the three parts of the DMD coding sequence carried by triple AAV vectors, since AAV-dys3-eGFP does not harbor a promoter to drive its expression. Since the stop codon on exon 79 has been removed in dys3, it is possible that an alternative less efficient stop codon before the exon 54/exon 55 boundary acts to prevent full-length dystrophin expression. The discrepancy in the expression level of dystrophin and eGFP protein is caused by a marked difference in the sensitivity of the antibodies used in detection.

A possible explanation for the very few sarcoplasmic eGFP-positive fibers is that an alternative start codon in the eGFP gene could be used to produce the eGFP protein without the dystrophin protein and sequentially would be localized in the sarcoplasm. The probability of random integration of the eGFP transgene is very low and in any case would result in endocellular patterns of localization, since it would not be fused with the sarcolemal dystrophin protein. However, we cannot exclude the possibility that the 5′ ITR of vector3 can act as a weak promoter.

Very few fibers expressing both NT- and CT-dystrophin but not eGFP were observed that would represent a revertant fiber. In this study, we used NT-dystrophin-recognizing antibodies, corresponding to exons 18–25, to avoid detecting most revertant fibers. It has been reported that more than 80% of revertant fibers have lost the domain between exons 21 and 30 (Lu et al., 2000). Indeed, there were very few fibers detectable with this NT-dystrophin-recognizing antibodies in saline-injected mdx TA muscles, whereas an increasing number of dystrophin-positive fibers were recognized by CT-dystrophin-detecting antibodies in serial sections.

The expression of the full-length human dystrophin protein is in line with the findings of the nested RT-PCR analysis of RNA harvested from treated muscle using human-specific primers. The presence of an amplicon of the human sequence from exon 23 within vector1-dys1 to exon 26 within vector2-dys2 suggests that there was a successful trans-splicing event between two split DMD cDNAs derived from two AAV vectors. Moreover, the amplicons from exon 54 within vector2-dys2 to exon 54/55 across the vector2-dys2/vector3-dys3 junction were also detectable by seminested RT-PCR with human-specific primers. The second trans-splicing event between vector2 and vector3 should only occur sequentially after trans-splicing between vector1 and vector2 constructs, since vector2 and vector3 do not contain promoters, while vector1 carrying dys1 does. Therefore, the event of the trans-splicing between vector2 and vector3 may be of limited efficiency relative to that occurring between vector1 and vector2. The results presented on the nested RT-PCR analysis using human-specific primers indeed show that this is the case; the human-specific trans-spliced amplicon from vector2 and vector3 is only evident in one out of the three TA muscles that showed trans-splicing event between vector1 and vector2.

The potential for homologous recombination (HR) between vector1-dys1 and endogenous mouse exon 23 harboring the mdx point mutation is negligible. With respect to the possibility of HR events, we would suggest that the exonic regions of homology are much too small and sequence-divergent to result in HR events; this is supported by an extensive HR literature. Even with long, fully homologous sequences, the efficiency of HR is reported as being very low in the absence of a double-stranded breaks induced by endonucleases (Jensen et al., 2011; Popplewell et al., 2013). In addition, the junctional and LongAmp PCR results using human-specific primer sets coupled with sequencing of amplicons confirm that the triple trans-spliced DMD mRNA is completely homologous with the human DMD sequence.

In summary, our study demonstrates that triple trans-splicing vectors have the potential to overcome the size limitation of AAV vector vehicles for the delivery of large genes. This is a true reflection on the efficacy of this novel, proof-of-concept, first-generation AAV triple trans-splicing system for the expression of a full-length human dystrophin-eGFP fusion protein. There are no other prior data published in the field, and all vector modifications and optimizations reflect the use and conceptualization of a triple/multiple rather than the currently existing dual trans-splicing system. The aim now is to optimize the design of triple trans-splicing vectors further so that the splicing between the three vectors is more efficient and expression of the dystrophin protein is enhanced. Strategies to increase the level of an mRNA product in future would include codon optimization, more effective splicing signals, suitable AAV pseudotyping, and/or ITR preselection. This study may provide new triple (or multiple) trans-splicing or similar strategies to overcome the current size limitation of AAV vector vehicles for large therapeutic gene delivery, and thus form a novel potential treatment strategy for a variety of genetic diseases.

Author Contributions

T.K. and L.P. performed experiments. T.A., T.K., and G.D. conceptualized and designed the vector strategy. All authors contributed to the writing/editing of the article.

Footnotes

Acknowledgments

The authors would like to thank the Muscular Dystrophy Campaign, United Kingdom for supporting this work, and also Dr. Helen Foster (Royal Holloway University of London) for assisting in injections.

Author Disclosure Statement

No conflicts of interest exist.

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