16 Design of Trans-Splicing Adeno-Associated Viral Vectors for Duchenne Muscular Dystrophy Gene Therapy Yi Lai, Dejia Li, Yongping Yue, and Dongsheng Duan Summary The development of trans-splicing vectors opens the door for delivering a large therapeutic gene with adeno-associated viral vectors (AAV). One potential application is to deliver the 6 kb mini-dystrophin gene for Duchenne muscular dystrophy (DMD) gene therapy. However, early attempts have been very disappointing because of low transduction efficiency. We have recently identified mRNA accumulation as a critical barrier for the trans-splicing AAV vectors. This barrier can be overcome by rational selection of the gene splitting site. Here we outline a detailed RNase protection assay-based strategy to determine the optimal gene splitting site for the mini-dystrophin gene. We also provide methods to evaluate transduction efficiency of the mini-dystrophin trans-splicing vectors in mdx mouse, a model for DMD. Key Words: AAV; adeno-associated virus; Duchenne muscular dystrophin; mini-dystrophin; trans-splicing; gene therapy; muscle; mdx. dystrophy; 1. Introduction Duchenne muscular dystrophy (DMD) is the most common lethal childhood muscle-wasting disease affecting all body muscles. It is caused by mutations in the dystrophin gene. Current treatment can only alleviate certain symptoms but cannot cure the disease. An ultimate solution to DMD requires replacing the mutated dystrophin gene with a functional gene. To achieve this goal, one needs to have a vehicle that can efficiently deliver a therapeutic gene to all muscles in the body. Adeno-associated viral vector (AAV) seems an ideal tool From: Methods in Molecular Biology, vol. 433: Volume 1: Production and In Vivo Applications of Gene Transfer Vectors Edited by: J. M. Le Doux © Humana Press, Totowa, NJ 259 260 Lai et al. because it readily transduces muscle fibers either by direct muscle injection or through systemic delivery. The challenge with AAV-mediated DMD gene therapy lies in the small viral packaging capacity. Wild type AAV carries a ∼4.7 kb single-stranded DNA genome. It contains two flanking inverted terminal repeats (ITRs) at the ends and two internal open reading frames encoding viral capsid (cap gene) and replication proteins (rep gene), respectively. In a recombinant vector, cap and rep genes can be replaced by a reporter and/or therapeutic expression cassette. This dictates a ≤5 kb vector genome size for an AAV vector. The full-length dystrophin protein is 427 kD and contains the N-terminal, central rod, cysteine-rich, and C-terminal domains. It is encoded in an 11.6 kb coding sequence. Apparently, this exceeds the packaging capacity of a single AAV virion. To solve this problem, investigators have developed the mini- and micro-dystrophin genes (see Fig. 1A) (1,2). The microgenes are less than 4 kb and do not carry the C-terminal domain. They Fig. 1. Dystrophin isoform structure and the effect of the gene splitting site on mRNA accumulation in mini-dystrophin. (A) Schematic outline of the full-length, micro- and mini-dystrophin proteins. Four functional domains in the full-length protein are the N-terminal domain (N), the rod domain (including 24 spectrin-like repeats and four hinges, H1 to H4), the cysteine-rich domain (CR) and the C-terminal domain. Regions that are deleted in micro- and mini-dystrophins are shown as white boxes. Specific force data are derived from Harper et al. (2002) Nat Med (2) (B) Relative mRNA levels according to RPA results from the mini-cassette depicted in Fig. 2B. The data are derived from Lai et al. (2005) Nat Biotech (7). The cartoon on the top of the panel B depicts the exon/intron/exon junctions where the minigene is split in each pair of the trans-splicing vectors. The dotted lines refer to the corresponding locations in the rod domain. Trans-Splice the Dystrophin Gene 261 can fit into a single AAV vector but cannot restore specific force to the normal level (2). The 6 kb minigene is an optimized version of a truncated, but fairly functional human dystrophin gene (3). It contains all the functional domains of the full-length protein. The original gene is capable of supporting daily activity in a 60-year-old patient (3). The optimized gene performed even better than the original gene, and it resulted in a muscle-specific force indistinguishable from that of wildtype mice (2,4). Efficient delivering of the 6 kb minigene with AAV vector may lead to a cure to DMD. The trans-splicing dual vector approach effectively doubles the AAV packaging capacity and raises the hope of delivering the 6 kb minigene with AAV vector (5–7). In this context trans -splicing is defined as the reconstruction of an intact transcript by splicing in “trans” between two covalently linked vector genomes each carrying a part of the transgene (see Fig. 2). This approach stems from a unique feature in AAV transduction biology. After entering cells, viral genomes form head-to-tail concatamers through ITRmediated recombination. By taking advantage of cellular splicing machinery, one can engineer splicing signals at the viral genome junction to remove the noncoding component at the junction and restore a functional expression cassette (see Fig. 2). The trans-splicing system is composed of two vectors. Fig. 2. Trans-splicing AAV-mediated expression of the mini-dystrophin gene. (A) Schematic outline of the transgene reconstitution in mini-dystrophin trans-splicing vectors. (B) The mini-cassette used in RPA to screen for the best gene splitting site. The dotted boxes in panels A and B represent the same region. SD, splicing donor; SA, splicing acceptor; ITR, AAV inverted terminal repeat; dD-ITR, the double D ITR structure formed in recombination. 262 Lai et al. An AV.Donor vector carries the promoter, the 5´ transgene, and the splicing donor signal. An AV.Acceptor vector carries the splicing acceptor signal, the 3´ transgene, and the polyA sequence. Successful transduction of the trans-splicing vectors depends on efficient co-infection of AV.Donor and AV.Acceptor, efficient viral genome recombination, and mRNA production from the reconstituted genome. We have previously demonstrated that co-infection is not a barrier. However, mRNA accumulation represents a predominant rate-limiting step (8). On the basis of this observation, we have developed an RNase-protection assay (RPA)-based screening strategy to rationally design the trans-splicing vectors for the 6 kb minigene. Using the conserved splicing value as a guide, we have analyzed a series of potential gene splitting sites in the mini-dystrophin gene. Our screening has yielded a pair of extremely efficient minigene trans-splicing vectors. This pair of vectors is based on splitting the gene at endogenous intron 60. Their transduction efficiency has reached that of a single AAV vector (see Fig. 3). In this protocol, we outline the procedures used in screening the gene splitting site, generating the mini-dystrophin trans-splicing vectors, and confirming mini-dystrophin expression in the mdx mouse model of DMD. Fig. 3. Transduction efficiency of the intron 60 trans-splicing vectors reaches that of the single intact vector. (A) Representative photomicrographs of the mdx EDL muscles infected by AV.Micro-dys (single intact vector). (B) Representative photomicrographs of the mdx EDL muscles co-infected by AV.Mini-dys.Donor and AV.Minidys.Acceptor (the intron 60 trans-splicing vectors). (C) Quantitative comparison of the transduction efficiency of the single intact vector and the trans-splicing vectors. Data are means ± standard error of mean. N = 8 for AV.Micro-dys infection; N =15 for trans-splicing vector infection; p = 0.43. Trans-Splice the Dystrophin Gene 263 2. Materials 2.1. Generating the Mini-dystrophin Trans-Splicing Vectors 2.1.1. Selection of the Potential Gene Splitting Sites There is no special material needed besides the dystrophin gene sequence itself. 2.1.2. RPA-Mediated Screening of the Potential Gene Splitting Site 1. 293 cells (ATCC). This is an adenovirus E1 gene transformed human kidney cell line. They are used as a packaging cell line for recombinant AAV production. These cells are propagated in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin G/streptomycin (see below). 2. Cell culture medium: DMEM, high glucose with l-glutamine (Gibco-BRL, Grand Island, NY, USA). Store at 4 ºC. 3. FBS (Hyclone). Store at –20 ºC. 4. Penicillin G-Streptomycin: Penicillin 100 U/ml DMEM culture medium and Streptomycin 100 μg/ml DMEM culture medium (Gibco-BRL). Store at –20 ºC. 5. 1× Trypsin–EDTA: 0.25% Trypsin, 1 mM EDTA/4Na (Gibco-BRL). Store at 4 ºC. 6. Lipofectamine and Plus reagent (Invitrogen, Carlsbad, CA, USA). 7. Mini-dystrophin gene plasmid pH2-R19. This plasmid is obtained from Dr. Jeffrey Chamberlain (University of Washington, Seattle, WA, USA). 8. pcDNA3.1(+) (Invitrogen). 9. pGL3-control (Promega, Madison, WI, USA). 10. pGEM3Z (Promega). 11. pTRI--actin-human antisense control template (Ambion, Austin, TX, USA). 12. Guanidine isothiocyanate (Amresco, Solon, OH, USA). 13. Cesium chloride (Research Products International Corp, Mount. Prospect, IL, USA). 14. SP6 RNA polymerase (Invitrogen). 15. RNasin (RNase inhibitor, Roche Applied Science, Indianapolis, IN, USA). 16. 5 mM rNTP (Amersham Pharmacia, Piscataway, NJ, USA). 17. P32 rUTP (Perkin Elmer, Boston, MA, USA). 18. Vanadyl Ribonucleoside complex (VRC) (Gibco-BRL), an RNase inhibitor used during anti-sense RNA probe purification. 19. RQ1 DNase (Promega) to remove the DNA template after RNA probe is generated from in vitro transcription. 20. tRNA (Sigma). 21. RNase A (Roche Applied Science). 22. RNase T1 (Roche Applied Science). 264 Lai et al. 2.1.3. Generating the cis-Plasmids for Recombinant AAV Packaging 1. Vent DNA Polymerase (New England Biolabs): This is a high-fidelity thermophilic DNA polymerase. It has a low polymerase chain reaction (PCR)related mutation rate. 2. Electroporation-competent Escherichia coli SURE cells (Stratagene, LA Jolla, CA, USA): AAV ITRs can be rearranged or deleted by endogenous DNA repair systems when they are propagated in bacterial cells. SURE cells are defective in DNA repair and recombination pathways (such as uvrC, umuC, SbcC, RecJ, recB, and recJ). Store at –80 ºC. 3. E. coli Pulser (Bio-Rad). 4. Gene Pulser cuvets, 0.1 cm gap (Bio-Rad). 5. 14-ml polypropylene round-bottomed tubes (Becton Dickinson Labware, Franklin Lakes, NJ, USA). 6. S.O.C. medium (Gibco-BRL). Store at room temperature. 7. Amp selection LB agar plates (100 μg/ml, Ampicillin). Store at 4 ºC. 8. Standard materials for large-scale plasmid preparation. To achieve the best transfection efficiency for AAV production, we recommend preparing the plasmids by CsCl2 /ethidium bromide equilibrium centrifugation. 2.1.4. Recombinant AAV-6 Production 1. Adenoviral helper plasmid: (pHelper, Stratagene). 2. Helper plasmid for AAV-2 rep gene (pMT-Rep2): This plasmid provides the Rep gene required for recombinant AAV replication and packaging (9). AAV2 rep proteins can fully support AAV-6 production. This plasmid is obtained from Dr. A. Dusty Miller (Fred Hutchinson Cancer Research Center, Seattle, WA, USA). 3. Helper plasmid for AAV-6 capsids (pCMVCap6): This plasmid provides the Cap gene required for rAAV-6 packaging (9). This plasmid is obtained from Dr. A. Dusty Miller (Fred Hutchinson Cancer Research Center). 4. 2.5 M CaCl2 . Sterilize by filtration and store at –20 ºC. 5. 2× HBS buffer: 0.3 M NaCl, 1.5 mM Na2 HPO4 , and 40 mM HEPES, pH 7.05 ± 0.05. Sterilize by filtration and store at –20 ºC. As pH affects transduction efficiency, it is highly suggested to double check pH before each usage. 6. DNase I (Sigma, 11 mg protein/vial, total 33 K [kuniz] units). 7. 0.25% Trypsin (Gibco-BRL). Store at 4 ºC. 8. 10% Sodium deoxycholate. Store at room temperature. 9. Misonic Cell Disruptor S3000 (Misonix, NY). 10. Cell lifter (Corning Incorporated, Corning, NY, USA). 11. HEPES AAV dialysis buffer: 20 mM HEPES, 150 mM NaCl, pH 7.8. Filtersterilize and store at 4 ºC. 12. Dialysis tubing: 12,000 MW cutoffs (Gibco-BRL). Store at 4 ºC. 13. AAV digestion buffer: 0.4 M NaOH, 20 mM EDTA. Freshly made before use. Trans-Splice the Dystrophin Gene 265 14. Slot blot hybridization solution [5× SSC, 5× Denhardts’s solution, 1% sodium dodecyl sulfate (SDS), and 50 % formamide, add 100 μg/ml denatured salmon sperm DNA just before use]. 15. HiTrap heparin column (Amersham). 2.2. In Vivo Evaluation of the Mini-Dystrophin Trans-Splicing Vectors 2.2.1. Local Muscle Injection Two-month-old dystrophin-null mdx mice (C57BL/10ScSn-Dmdmdx/J; The Jackson Laboratory). Dystrophin expression is aborted in this inbreed strain because of a nonsense mutation in exon 23 (10,11). (see Note 1). 1. Anesthetic reagents and instruments for local muscle injection: 4 μl/g body weight anesthetic cocktail (ketamine 25 mg/ml, xylazine 2.5mg/ml, acepromazine 0.5mg/ml in physiological saline). Sterile forceps and scissors (World Precision Instruments, Inc., Sarasota, FL, USA), needle holders (Accurate Surgical & Scientific Instruments Corp., Westbury, NY, USA) and Guthrie double hook retractor (Fine Science Tools, Inc., Foster City, CA, USA). 5-0 sofsilk suture (Auto Suture Company, Norwalk, CT, USA). 33G gas-tight Hamilton syringe and needle (Hamilton Company Reno, NV, USA). 2. Polyethylene mouse Elizabethan collar (E-collar, Harvard Apparatus). 3. Banamine (also called Flunixin, 1.5 mg/kg) (Schering-Plough Animal Health Corp., Union, NJ, USA) 2.2.2. Quantifying Mini-Dystrophin Expression by Western Blot 1. Muscle homogenization solution: 20 mM Na4 P2 O4 , 20 mM NaHPO4 , 1 mM MgCl2 , 0.5 mM EDTA, and 303 mM sucrose. 2. Muscle microsome preparation wash buffer: 20 mM Tris–HCl (pH 7.0), 60 mM KCl, 303 mM sucrose. 3. Muscle microsome storage solution: 20 mM Tris–HCl (pH 7.0) and 303 mM sucrose. 4. Cheese-cloth (Fisher Scientific, Pittsburgh, PA, USA). 5. Tissue-Tek OCT compound (Sakura Finetek Inc., Torrance, CA, USA). 2.2.3. Quantifying Mini-Dystrophin Expression by Immunostaining 1. Mouse monoclonal antibodies for different regions of the dystrophin protein: Dys-3 (clone Dy10/12B2, IgG2a), an antibody that specifically recognizes the hinge 1 region of human dystrophin (Novocastra, Newcastle, UK). Dys-2 (clone Dy8/6C5, IgG1), an antibody that reacts with the dystrophin C-terminal domain (Novocastra). Mandys-8 (clone 8H11, IgG2b), an antibody that specifically recognizes spectrin-like repeat 11 (exon 32) (Sigma) (see Note 2). 2. Polyclonal antibodies for different regions of the dystrophin protein: rabbit anti-dystrophin N-terminal antibody, an affinity-purified antibody obtained from 266 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Lai et al. Dr. Jeffrey Chamberlain’s laboratory (University of Washington, Seattle, WA, USA). Goat anti-dystrophin C-terminal antibody (Santa Cruz). Rabbit antidystrophin spectrin-like repeat 4–6 antibody (Santa Cruz). 2-methylbutane (Sigma). Tissue-Tek OCT (Sakura Finetek Inc., USA). Rabbit anti-mouse IgG (Sigma, final concentration 0.46 mg/ml). Papain (Sigma, final concentration 45 μg/ml). L-cystein (Sigma, final concentration 22 mM). Iodoacetic acid (Sigma, final concentration 10 mM). 20% rabbit serum (Jackson ImmunoResearch Laboratories, Inc.). Alex 488 conjugated rabbit anti-mouse antibodies (Molecular Probe). Alex 594 conjugated rabbit anti-mouse antibodies (Molecular Probe). KPBS: 356 μM KH2 PO4 , 1.64 mM K2 HPO4 , 160 mM NaCl. Gelatin (Sigma). M.O.M kit (Vector Laboratories, Inc.). 3. Methods 3.1. Generating the Mini-dystrophin Trans-Splicing Vectors 3.1.1. Selection of the Potential Gene Splitting Sites There are several considerations when deciding on the potential sites to split the mini-dystrophin gene. These include the size of the individual vector, the gene structure, and the relative efficiency of transcription and splicing. Specifically, both AV.Donor and AV.Acceptor have to fit into a single AAV virion themselves, respectively; the minigene should be split at a natural exon/exon junction rather than within an exon; and the site should yield the highest level of mRNA in the context of a reconstituted/recombined viral genome. 1. Preliminary selection of the gene splitting site according to the size constraint of a single AAV virion. There are a total of 79 exons in the full-length dystrophin gene. However, the minigene only contains the coding sequence from 44 exons. The 5´ half of the minigene is ∼2.2 kb (exon 1 to the beginning of exon 17) spanning the region from the N-terminus to repeat 3. The 3´ half of the minigene is ∼3.8 kb (the end of exon 49 to the beginning of exon 79) covering the region from hinge 3 to the C-terminus. Considering the size of the ITR (two ITRs, total ∼0.3 kb), the CMV promoter (∼0.6 kb), the pA signal (∼0.4 kb), and the splicing signals (∼ 0.3 kb), any exon boundary between exons 51 and 65 would be appropriate to divide the minigene. 2. Preliminary screening of the gene splitting site according to the conserved splicing value in the wildtype dystrophin gene. Sironi et al. (12) has systematically calculated the conserved splicing value (CV) and U1 small nuclear RNA binding energy for all the exon/intron/exon junctions in the dystrophin gene (see Note 3). The mean CVs for the 5´ and 3´ splicing sites are (0.83 ± 0.07) and (0.87 ± 0.09), Trans-Splice the Dystrophin Gene 267 respectively. The mean binding energy is –7.76 ± 2.21 kcal/mole (12). We selected three sites that have the highest CV and lowest binding energy as potential sites for gene splitting. These are 56/56/57 (exon/intron/exon), 60/60/61, and 63/63/64 junctions. Their respective CVs and binding energy are (in the order of 5´ splicing site CV, 3´ splicing site CV, and binding energy) 0.84. 0.97, and –10.4 kcal/mole (for the 56/56/57 junction), 0.94, 0.98, and –11.7 kcal/mole (for the 60/60/61 junction), and 0.95, 0.90, and –10.6 kcal/mole (for the 63/63/64 junction). As a control, we also selected the 53/53/54 junction, which has an average binding energy (–7.7 kcal/mole) and moderate CVs (0.90 and 0.60 for the 5´ and 3´ splicing sites, respectively). 3.1.2. RPA-Mediated Screening of the Potential Gene Splitting Site Formation of the double-D ITR structure is a unique feature in the recombined AAV genome (13). Importantly, the double-D ITR structure in intron down-regulates mRNA accumulation (8). An ideal gene splitting site should yield the highest level of mRNA in the presence of the double-D ITR structure. To identify the best site, we constructed a series of mini-cassettes to mold the reconstituted vector genome. An RPA assay was then used to quantify the relative level of mRNA accumulation (see Fig. 2). 1. Cloning the endogenous dystrophin intron splicing signal from a human cell line. Use 293 cell genomic DNA as the PCR template. Design the primers to amplify the exon/intron junctions. The primer sequences for each junction can be found in the online supplementary material in Lai et al. (http://www.nature.com/nbt/journal/v23/n11/suppinfo/nbt1153_S1.html) (7). To facilitate subsequent cloning, include restriction site overhangs in the primers as needed (see Note 4). 2. Reconstituting the mini-cassette in the backbone of pcDNA3.1(+). First, introduce the respective exon/intron junctions in pcDNA3.1(+). For the 53/53/54 junction, insert the splicing donor between the Kpn I/BamH I sites and insert the splicing acceptor between the Xho I/Xba I sites. For the 56/56/57 junction, insert the splicing donor between the Hind III/Kpn I sites and insert the splicing acceptor between the EcoR V/Xba I site. For the 60/60/63 junction, insert the splicing donor between the Kpn I/BamH I sites and insert the splicing acceptor between the Xho I/Xba I sites. For the 63/63/64 junction, insert the splicing donor between the Hind III/Kpn I sites and insert the splicing acceptor between the Xho I/Xba I sites. After inserting splicing signals, clone the double-D ITR structure in the intron region between the EcoR I/EcoR V sites (see Fig. 2B). 3. Generating the RPA probe. To quantify both spliced and unspliced RNA products, generate an anti-sense RNA probe for each potential site to hybridize with the respective 5´ exon and the intron donor sequence (see Fig. 2B). Then insert the PCR products into the EcoR I/BamH I sites in pGEM3Z accordingly. After linearization at the EcoR I site, synthesize the RPA probes by in vitro transcription with SP6 RNA polymerase and [-32 P]-rUTP as the substrate (7,8). 268 Lai et al. 4. RPA screening. Co-transfect the plasmids carrying the mini-cassette (4 μg) and a transfection control plasmid (0.5 μg; we used a luciferase plasmid pDD12. In this plasmid, the entire expression cassette from pGL3-control (Promega) was cloned into an AAV packaging plasmid) into 70–80% confluent 293 cells using Lipofectamine and Plus reagent (Invitrogen). About 48 h later, extract protein from one-fifth cells for luciferase assay to normalize transfection efficiency. Lyse remaining cells in 4 M guanidine isothiocyanate and extract total RNA in 5.7 M CsCl2 by ultra-centrifugation at the speed of 116,200 × g for at least 12 h at 20 ºC. In each RPA reaction, use10 μg total RNA to hybridize with excessive amount of 32 P-labeled probe and digest unprotected RNA by RNaseT1 (0.923 μg/ml) and RNaseA (at 18.46 μg/ml). Elecctrophorese the reaction products in 8% denaturing polyacrylamide gel and quantify the bands with the Molecular Imager FX and Quantity One (version 4.2.2 image software; Bio-Rad, Heracules, CA). Normalize the RPA reaction with endogenous human -actin using an independent probe generated from pTRI--actin-human antisense. Normalize RNA signal intensity by transfection efficiency, RPA reaction and the number of 32 P-labeled uridine in each protected band. The 60/60/61 junction produces the highest level of mRNA and the 63/63/64 junction is the second best (∼50% of that from the 60/60/61 junction) (see Fig. 1B). 3.1.3. Generating the cis -Plasmids for Recombinant AAV Packaging Generate two pairs of the trans-splicing plasmids representing gene splitting sites at the 60/60/61 and the 63/63/64 junctions, respectively. Each pair includes a cis-donor plasmid and a cis-acceptor plasmid. To facilitate comparison, both pairs are under transcriptional regulation of the CMV promoter and the SV40 polyA. Use standard molecular cloning methods to generate these constructs. The detailed step-by-step cloning procedure can be found in the online supplementary material in Lai et al. (http://www.nature.com/nbt/journal/ v23/n11/suppinfo/nbt1153_S1.html) (7). 3.1.4. Recombinant AAV-6 Production see Notes 5 and 6 . 1. Two days before viral production, split 293 cells at 1:6 to 150 mm culture plates. Seed a total of 15 plates for one production. Change to fresh culture media about 1–2 h before transfection. 2. Preparing DNA-calcium-phosphate precipitate. Co-transfect four plasmids to make AAV-6 vector. These include the cis-plasmid, pMT-Rep2, pCMVCap6, and pHelper. For each vector preparation (15 × 150 mm plates), use 187.5 μg of the cis-plasmid, 187.5 μg pMT-Rep2, 562.5 μg pCMVCap6, and 562.5 μg pHelper (at a ratio of 1:1:3:3). Mix all plasmids thoroughly in 15.2 ml H2 O. Add 1.68 ml of 2.5 M CaCl2 to a final concentration of 250 mM. Generate DNA-calciumphosphate precipitate by slowly dropping the DNA/CaCl2 mixture to 16.8 ml of Trans-Splice the Dystrophin Gene 3. 4. 5. 6. 7. 8. 9. 269 2× HBS. In general, it takes 15–30 min room temperature incubation to form a high quality precipitate (see Note 7). Gently apply the DNA-calcium-phosphate precipitate to 293 cells drop-by-drop while swirling the culture plate. At 72 h after transfection, collect cell lysate with a cell lifter. After a 20 min spin in a bench-top centrifuge (1,800 × g at 4 ºC), resuspend cell pellet in 9 ml of 10 mM Tris–HCl (pH 8.0). Freeze/thaw cell lysate 8–10 times using dry ice/ethanol and a 40 ºC water bath. Sonicate cell lysate at the power output of 5.5 for 10 min (on ice). Digest cell lysate with DNase I at 37 ºC for 45 min (we normally use half vial of Sigma DNase I for each 15 plates viral preparation). Sonicate cell lysate again at the power output of 5.5 for 7 min (on ice). Digest lysate with one-tenth volume of 0.05% trypsin and 10% sodium deoxycholate for 30 min at 37 ºC. Clear cell lysate by spinning at 3,200 × g for 30 min at 4 ºC. Carefully transfer supernatant to a new tube. Adjust the volume to 29 ml with 10 mM Tris–HCl (pH 8.0) and add 18.2 g CsCl2 (this is equal to 0.613 g/ml). The final volume should be about 32.5ml. Incubate for 30 min at 37 ºC to dissolve CsCl2 . Spin at 3200 × g for 30 min at 4 ºC. Carefully load the supernatant into six 5-ml Beckman ultracentrifugation tubes in an SW55Ti rotor. Spin at 200,000 × g for 40 h at 4 ºC. Collect fractions from the bottom of the tube with a 20-G needle. Identify the viral containing fractions by slot blot (see below). Combine fractions with the highest viral titer and centrifuge again at 200,000 × g for 40 h at 4 ºC. Collect fractions as described in step 6 and identify highest viral fraction by slot blot (see below). Dialyze viral stock in HEPES buffer (4 ºC for 2 × 24 h). Slot blot viral titer determination. Use duplicated sets of viral stock aliquots (1, 5, and 10 μl) and plasmid copy number controls (107 , 108 , 109 , 1010 , 1011 molecules/μl) in slot blot. Denature samples in 50 μl AAV digestion buffer at 100 ºC for 10 min. Then immediately chill samples on ice and bring up volume to 400 μl with digestion buffer. Load samples onto Hybond-N plus membrane with a Bio-Dot SF manifold microfiltration apparatus. After blotting, crosslink DNA to the membrane with UV irradiation. Prehybridize the membrane. Then hybridize the membrane with a 32 P-labeled transgene-specific probe in the slot blot hybridization solution. Determine the viral particle titer by comparing the intensity of the viral stock band to that of the plasmid standards. 3.2. In Vivo Evaluation of the Mini-Dystrophin Trans-Splicing Vectors 3.2.1. Local Muscle Injection For all animal experiments, get approval from the institute Animal Care and Use Committee and follow NIH guidelines. Two muscles have been used to evaluate AAV transduction efficiency including the tibialis anterior (TA) muscle and the extensor digitorum longus (EDL) muscle. In trans-splicing 270 Lai et al. vector transduction studies, mix equal amount of AV.Donor and AV.Acceptor particles before muscle injection. Then anesthetize experimental animal. 1. Gene delivery to the TA muscle. Expose the proximal end of the TA muscle with a 2∼3 mm incision. Insert a 33-G Hamilton needle into the middle belly of the TA muscle. Slowly inject 30 μl trans-splicing AAV vectors into the muscle while slowly backing out the injection needle. Suture the wound and monitor the animal until it recovers. 2. Gene delivery to the EDL muscle. Make a 0.5 × 5 mm incision along the longitudinal axis in the lateral surface of the distal hind limb. Separate the EDL tendon from the TA tendon and then gently pull the TA muscle aside with a Guthrie double hook retractor to expose the EDL muscle. Perform two injections from the proximal and the distal ends respectively with a 33G Hamilton syringe. In each injection, directly inject 5 μl virus into the muscle body (a total of 10 μl AAV for each EDL muscle). Suture the wound and monitor the animal until it recovers (see Note 8). 3.2.2. Quantifying Mini-Dystrophin Expression by Western Blot 1. Muscle microsome preparation. Pulverize muscle in liquid nitrogen by hand grinding. Resuspend the muscle powder in 6 ml muscle homogenization solution. Transfer muscle lysate to a clean Oakridge tube. Spin at 250,000 × g for 15 min at 4 ºC in a Sorval RC-5B supercentrifuge. Filter supernatant through six layers of cheese cloth. Load the filtered supernatant (∼5 ml) to a 5.2-ml SW55 centrifuge tube. Spin at 110,000 × g for 30 min at 4 ºC. Discard supernatant. Gently resuspend the pellet in 5 ml wash buffer. Spin again at 110,000 × g for 30 min at 4 ºC. Resuspend the pellet (muscle microsome) in 200 μl storage solution and store at –80 ºC. 2. Western blot. Electrophorese 50 μg of muscle microsome preparation in a 6% SDS-polyacrylamide gel. Detect mini-dystrophin with either Dys-2 (1:100 dilution) or Dys-3 (1:20 dilution) antibodies. 3.2.3. Quantifying Mini-Dystrophin Expression by Immunostaining 1. Snap freeze freshly isolated muscle sample in liquid nitrogen-cooled 2-methylbutane in Tissue-Tek OCT. 2. Immunostaining with monoclonal antibody. To use mouse monoclonal antibody in murine tissue, one must first block the binding of the secondary antibody to endogenous mouse immunoglobulins (Igs) (14). We used papain digested rabbit anti-mouse IgG (including Fab and Fc fragments) for blocking (see Note 9). Briefly, digest rabbit anti-mouse IgG with Papain in the presence of 1 mM EDTA and 22 mM l-cystein at 37 ºC for 16 h. Stop digestion reaction with iodoaceticacid. For immunostaining, first block 8 μm air-dried cryosections in anti-mouse IgG blocking solution at room temperature for 60 min. After washing in PBS, block the cryosections again with 20% rabbit serum at room temperature for 30 Trans-Splice the Dystrophin Gene 271 min. After washing in PBS, apply the primary antibody (diluted in 1% rabbit serum, such as Dys-3, Dys-2, or Mandy-8) at a dilution of 1:100 and incubate at 4 ºC overnight. After washing in PBS, detect signals with Alex 488 or Alex 594 conjugated rabbit anti-mouse antibodies, respectively. 3. Immunostaining with polyclonal antibody. Briefly rinse 8 μm air-dried cryosections with KPBS. Block with 1% goat serum KPBS at room temperature for 15 min. Wash with 0.2% gelatin KPBS. Apply primary antibody at 4 ºC overnight (1:600 for Chamberlain’s affinity purified N-terminal antibody; 1:200 for Santa Cruz’s C-terminal antibody, and 1:400 for Santa Cruz’s repeat 4–6 antibody). Wash with 0.2% gelatin KPBS. Detect signal with Alex 488 or Alex 594 conjugated goat anti-rabbit antibody (for the N-terminal and repeat 4–6 primary antibodies) or rabbit anti-goat antibody (for the C-terminal primary antibody) (see Notes 10 and 11). 4. Notes 1. Despite the nonsense mutation, occasionally dystrophin positive revertant fibers can be detected in mdx muscle (15). These are called revertant fibers. In revertant fibers, the mutated exon 23 is skipped due to alternative splicing. Revertant fibers can be distinguished from AAV-mediated mini-dystrophin expression by the following criteria. First, revertant fibers can often be detected with antibodies against the missing regions in the mini-dystrophin gene such as Mandy-8 (Sigma, recognizes repeat 11) and Repeats 4–6 polyclonal antibody (Santa Cruz). Second, revertant fibers cannot be recognized by human dystrophin-specific antibody Dys-3. 2. In addition to the commercially available monoclonal antibodies mentioned here, more than 60 monoclonal antibodies have been published. These antibodies react with the different regions of the dystrophin protein. A collection of these antibodies can be found in the website http://www.dmd.nl/antibody.html. A large number of epitope-specific antibodies can be obtained from Prof. Glenn E. Morris (Centre for Inherited Neuromuscular Disease, Leopold Muller ARC Building, RJAH Orthopaedic Hospital, OSWESTRY, Shropshire, SY10 7AG, UK). 3. The conserved splicing value for a particular exon/intron/exon junction can be calculated according to the following formula (16). For the 5´ splicing site, CV = (X – 47)/548. X = the sum of all the reference values for the positions X and x (see Table 1). For the 3´ splicing site, CV = (Y – 57)/640 + (Z – 11)/624. Y = the sum of the eight highest values for all the y positions (see Table 1). Z = the sum of all the reference values for the positions z and Z (see Table 1). 4. As mutations in intron may alter splicing profile and mutations in exon may change amino acid sequence, we strongly recommend using high fidelity Taq polymerase for all PCR-mediated cloning. It is also essential to confirm the cloned PCR product by sequencing. A C G T 10 4 78 8 X 0 0 100 0 x 0 0 0 100 x 57 2 39 2 x 71 8 12 9 x 5 6 84 5 x 16 15 22 47 x 9 31 15 45 y 9 33 13 45 y 7 31 11 51 y 7 35 7 51 y 10 35 7 47 y 10 35 11 44 y 7 43 7 42 y b Adapted from Shapiro and Senapathy 1987 Nucleic Acid Res 15(17):7155. Capital letters represent nucleotides in exon. Small letters represent nucleotides in intron. c Values at this nucleotide position is not used in calculation. n/a, not applicable. a 58 13 15 15 Xb Table 1 Reference Value for CV Calculation in a Primate Genea 9 41 8 42 y 6 39 6 48 y 6 40 8 46 y n/a n/a n/a n/a oc 3 74 1 22 z 100 0 0 0 z 0 0 100 0 z 28 13 49 10 Z Trans-Splice the Dystrophin Gene 273 5. AAV-6 has been shown as the best serotype for muscle transduction following direct local injection (17,18). However, for systemic delivery one may also consider other newly developed AAV serotypes such as AAV-8 and AAV-9. 6. In this protocol, we described a CsCl2 ultracentrifugation-based protocol for AAV-6 purification. However, AAV-6 can also be purified with a HiTrap heparin column using HPLC or FPLC (19). 7. A high-quality precipitate is essential to high viral yield. We recommend routinely monitoring calcium phosphate precipitate on a coverslip using a phase contrast microscope. Alternatively, the quality of the transfection reagents (such as CaCl2 and 2× HBS) can be pre-tested with reporter plasmids such as GFP, LacZ, or alkaline phosphatase plasmids. 8. To prevent gnawing on the suture and improve wound healing, we recommend placing a polyethylene mouse Elizabethan collar around the mouse neck. This device blocks the head from access to the rest of the body, but still enables eating, drinking, and comfortable movement. To alleviate pain and discomfort at the operation site, we suggest subcutaneous injection of a non-steroid analgesic drug Banamine. 9. For immunostaining with monoclonal antibody, one can also use M.O.M kit. 10. To confirm trans-splicing AAV vector-mediated mini-dystrophin expression, it is essential to perform immunostaining with at least three antibodies. Minidystrophin should be lighted up with antibodies against the N-terminal and the C-terminal domains, respectively, but it will not be detected with antibody against the regions that are deleted in mini-dystrophin (such as repeat 4–6). 11. The advantage of the polyclonal antibody is its low background staining in mouse muscle. However, polyclonal antibody cannot distinguish between endogenously mouse dystrophin in revertant myofibers and human mini-dystrophin expressed from AAV vectors. Only Dys-3 monoclonal antibody can definitively diagnose human originated mini-dystrophin. Acknowledgments We thank Dr. Jeffrey Chamberlain for providing the pH2-R19 minidystrophin plasmid. We also thank Dr. A. Dusty Miller for the AAV-6 packaging plasmids (pMT-Rep2 and pCMVCap6). This work is supported by grants from the National Institutes of Health (AR-49419, DD) and the Muscular Dystrophy Association (DD). We thank Mr. Arka Ghosh for optimizing AAV-6 preparation protocol. 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