Persistent expression of FLAG-tagged micro dystrophin in nonhuman primates following intramuscular and vascular delivery

Louise R Rodino-Klapac, Chrystal L Montgomery, William G Bremer, Kimberly M Shontz, Vinod Malik, Nancy Davis, Spencer Sprinkle, Katherine J Campbell, Zarife Sahenk, K Reed Clark, Christopher M Walker, Jerry R Mendell, Louis G Chicoine, Louise R Rodino-Klapac, Chrystal L Montgomery, William G Bremer, Kimberly M Shontz, Vinod Malik, Nancy Davis, Spencer Sprinkle, Katherine J Campbell, Zarife Sahenk, K Reed Clark, Christopher M Walker, Jerry R Mendell, Louis G Chicoine

Abstract

Animal models for Duchenne muscular dystrophy (DMD) have species limitations related to assessing function, immune response, and distribution of micro- or mini-dystrophins. Nonhuman primates (NHPs) provide the ideal model to optimize vector delivery across a vascular barrier and provide accurate dose estimates for widespread transduction. To address vascular delivery and dosing in rhesus macaques, we have generated a fusion construct that encodes an eight amino-acid FLAG epitope at the C-terminus of micro-dystrophin to facilitate translational studies targeting DMD. Intramuscular (IM) injection of AAV8.MCK.micro-dys.FLAG in the tibialis anterior (TA) of macaques demonstrated robust gene expression, with muscle transduction (50-79%) persisting for up to 5 months. Success by IM injection was followed by targeted vascular delivery studies using a fluoroscopy-guided catheter threaded through the femoral artery. Three months after gene transfer, >80% of muscle fibers showed gene expression in the targeted muscle. No cellular immune response to AAV8 capsid, micro-dystrophin, or the FLAG tag was detected by interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot (ELISpot) at any time point with either route. In summary, an epitope-tagged micro-dystrophin cassette enhances the ability to evaluate site-specific localization and distribution of gene expression in the NHP in preparation for vascular delivery clinical trials.

Figures

Figure 1
Figure 1
Micro-dystrophin.FLAG improves muscle function. (a) Immunostaining with anti-FLAG antibody reveals robust micro-dystrophin.FLAG expression in mdx mice. Inlay: contralateral limb muscle reveals no FLAG staining. (b) Immunostaining with N-terminal dystrophin antibody Dys3 confirms the presence of micro-dystrophin. Note: Dys3 antibody will only recognize human N-terminal dystrophin and does not crossreact with mouse dystrophin. Inlay: contralateral limb muscle reveals no dystrophin staining. Bar = 100 µm. (c) mdx diaphragms treated with AAV8.MCK.micro-dystrophin.FLAG are significantly protected from muscle fatigue compared to untreated mdx controls (***two-way analysis of variance, P < 0.001), but did not fully restore normal function compared to C57/BL10 strain controls.
Figure 2
Figure 2
Two-month muscle biopsy reveals widespread micro-dys.FLAG expression. (a) A biopsy of all three primates from the site of gene transfer revealed robust staining of micro-dystrophin FLAG localized to the sarcolemmal membrane compared to no staining in contralateral control (a′). Bar = 400 µm for montage and 200 µm for ×20 images. (b) Percentage of micro-dys.FLAG transduced muscle fibers from each biopsy. (c,d) Quantitative comparison of CD4 and CD8 T cells present in the biopsy of treated muscle versus the contralateral limb muscle. Error bars, standard deviation. Student's t-test was used to determine significance between sides (P > 0.05).
Figure 3
Figure 3
Persistent expression micro-dys.FLAG at 5 months. (a) Excision of the entire gene transfer site revealed sustained expression in all three primates. Bar = 200 µm. (a′) Contralateral control shows no FLAG staining. (b) Mean percentage of micro-dys.FLAG transduced muscle fibers from tissue blocks (six blocks each, ~0.5 × 0.5 × 0.5 cm) harvested from injection site for each subject (error bars ± SD). (c,d) Quantitative comparison of CD4 and CD8 T cells present in the biopsy tissue versus the contralateral muscle (error bars ± SD). Student's t-test was used to determine significance between sides (P > 0.05).
Figure 4
Figure 4
Localization of micro-dys.FLAG protein to the sarcolemma. (a) Immunofluorescence labeling for dystrophin C-terminus (Dys2) and micro-dys.FLAG (anti-FLAG) revealed colocalization to the sarcolemma (arrow). Intensity of micro-dys.FLAG expression was variable in some muscle fibers as demonstrated in merged images. Bar: ×20 = 200 µm, ×40 = 100 µm. (b) Western blot analysis using N-terminal (Dys3) and C-terminal (Dys2) antibodies to dystrophin indicate both full-length dystrophin (427 kd) and micro-dystrophin.FLAG (138 kd) in treated (T) samples. A Dys3 positive band at 138 kd is absent in contralateral control (C) tissue, whereas the endogenous full-length 427 kd band is intact. Dys2 positive band at 427 kd present in all samples is detecting full-length dystrophin. Actin is shown as loading control (42 kd). DMD, Duchenne muscular dystrophy.
Figure 5
Figure 5
Cellular immune response. (a) Enzyme-linked immunosorbent spot assay for detection of transgene and/or capsid-specific T cells in PBMCs by interferon-γ secretion. Fifty reactive spots per million cells (dotted line) represent the threshold for a significant response. Black arrow indicates the time of biopsy. The only marginally positive responses to be noted were at day 14 for RQ6661 and day 42 for RQ6698. These were not sustained and had no influence on gene expression. (b) AAV8 binding Ab titers. Antibodies to AAV8 capsid in serum were quantified by enzyme-linked immunosorbent assay and monitored every 2 weeks. AAV8 Ab ratio = ratio OD with AAV8 coat/without AAV8 coat. All three animals demonstrated rapid seroconversion in AAV8 Abs after immunization with rAAV8.MCK.micro-dys.FLAG. Ab, antibody; HBV, hepatitis B virus; OD, optical density; PBMC, peripheral blood mononuclear cell; SFC, spot-forming colonies.
Figure 6
Figure 6
Targeted vascular delivery of micro-dystrophin.FLAG in nonhuman primates. (a) The targeted gastrocnemius muscle from all four primates revealed robust staining of micro-dystrophin.FLAG localized to the sarcolemmal membrane at 3 months after gene transfer. Bar = 200 µm, ×10 images. (a′) Contralateral control shows no FLAG staining. (b) Hematoxylin and eosin staining from isolated muscle tissue at the site of gene transfer revealed no evidence of tissue damage or cellular infiltration. (c) Enzyme-linked immunosorbent spot assay for detection of transgene and/or capsid-specific T cells in PBMCs. Pool 1 is composed of aa14-240 (actin-binding domain), aa253-327 (hinge 1), and aa337-427 (first part of spectrin repeat 1). Pool 2 is composed of aa428-447 (remainder of spectrin repeat 1), aa448-556 (spectrin repeat 2), aa557-667 (spectrin repeat 3), aa668-717 (hinge 2), and aa2932-3040 (spectrin repeat 24). Pool 3 is composed of aa3041-3112 (hinge 4), aa3080-3360 (cysteine repeat region), plus the sequence of the FLAG tag. Two marginally positive responses against micro-dys pool 3 in animal 03E101 were noted at days 15 and 71. These responses were not sustained and had no influence on long-term gene expression. HBV, hepatitis B virus; PBMC, peripheral blood mononuclear cell; SFC, spot-forming colonies.

References

    1. Rodino-Klapac LR, Chicoine LG, Kaspar BK., and , Mendell JR. Gene therapy for duchenne muscular dystrophy: expectations and challenges. Arch Neurol. 2007;64:1236–1241.
    1. Wang Z, Chamberlain JS, Tapscott SJ., and , Storb R. Gene therapy in large animal models of muscular dystrophy. ILAR J. 2009;50:187–198.
    1. Harper SQ, Hauser MA, DelloRusso C, Duan D, Crawford RW, Phelps SF, et al. Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy. Nat Med. 2002;8:253–261.
    1. Liu M, Yue Y, Harper SQ, Grange RW, Chamberlain JS., and , Duan D. Adeno-associated virus-mediated microdystrophin expression protects young mdx muscle from contraction-induced injury. Mol Ther. 2005;11:245–256.
    1. Rodino-Klapac LR, Janssen PM, Montgomery CL, Coley BD, Chicoine LG, Clark KR, et al. A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy. J Transl Med. 2007;5:45.
    1. Bostick B, Yue Y, Lai Y, Long C, Li D., and , Duan D. Adeno-associated virus serotype-9 microdystrophin gene therapy ameliorates electrocardiographic abnormalities in mdx mice. Hum Gene Ther. 2008;19:851–856.
    1. Bostick B, Yue Y, Long C, Marschalk N, Fine DM, Chen J, et al. Cardiac expression of a mini-dystrophin that normalizes skeletal muscle force only partially restores heart function in aged Mdx mice. Mol Ther. 2009;17:253–261.
    1. Townsend D, Blankinship MJ, Allen JM, Gregorevic P, Chamberlain JS., and , Metzger JM. Systemic administration of micro-dystrophin restores cardiac geometry and prevents dobutamine-induced cardiac pump failure. Mol Ther. 2007;15:1086–1092.
    1. Blankinship MJ, Gregorevic P, Allen JM, Harper SQ, Harper H, Halbert CL, et al. Efficient transduction of skeletal muscle using vectors based on adeno-associated virus serotype 6. Mol Ther. 2004;10:671–678.
    1. Mah C, Fraites TJ Jr, Cresawn KO, Zolotukhin I, Lewis MA., and , Byrne BJ. A new method for recombinant adeno-associated virus vector delivery to murine diaphragm. Mol Ther. 2004;9:458–463.
    1. Gregorevic P, Allen JM, Minami E, Blankinship MJ, Haraguchi M, Meuse L, et al. rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice. Nat Med. 2006;12:787–789.
    1. Yue Y, Ghosh A, Long C, Bostick B, Smith BF, Kornegay JN, et al. A single intravenous injection of adeno-associated virus serotype-9 leads to whole body skeletal muscle transduction in dogs. Mol Ther. 2008;16:1944–1952.
    1. Wherry EJ, Barber DL, Kaech SM, Blattman JN., and , Ahmed R. Antigen-independent memory CD8 T cells do not develop during chronic viral infection. Proc Natl Acad Sci USA. 2004;101:16004–16009.
    1. Hopp TP, Prickett KS, Price VL, Libby RT, March CJ, Cerretti DP, et al. A short polypeptide marker sequence useful for recombinant protein identification and purification. Biotechnology (N Y) 1988;6:1204–1210.
    1. Louboutin JP, Reyes BA, Agrawal L, Van Bockstaele E., and , Strayer DS. Strategies for CNS-directed gene delivery: in vivo gene transfer to the brain using SV40-derived vectors. Gene Ther. 2007;14:939–949.
    1. Terpe K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol. 2003;60:523–533.
    1. Ishizaki M, Suga T, Kimura E, Shiota T, Kawano R, Uchida Y, et al. Mdx respiratory impairment following fibrosis of the diaphragm. Neuromuscul Disord. 2008;18:342–348.
    1. Stedman HH, Sweeney HL, Shrager JB, Maguire HC, Panettieri RA, Petrof B, et al. The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature. 1991;352:536–539.
    1. Fisher KJ, Jooss K, Alston J, Yang Y, Haecker SE, High K, et al. Recombinant adeno-associated virus for muscle directed gene therapy. Nat Med. 1997;3:306–312.
    1. Kessler PD, Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, et al. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Proc Natl Acad Sci USA. 1996;93:14082–14087.
    1. Corrado K, Rafael JA, Mills PL, Cole NM, Faulkner JA, Wang K, et al. Transgenic mdx mice expressing dystrophin with a deletion in the actin-binding domain display a “mild Becker” phenotype. J Cell Biol. 1996;134:873–884.
    1. Gao GP, Alvira MR, Wang L, Calcedo R, Johnston J., and , Wilson JM. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci USA. 2002;99:11854–11859.
    1. Li H, Lin SW, Giles-Davis W, Li Y, Zhou D, Xiang ZQ, et al. A preclinical animal model to assess the effect of pre-existing immunity on AAV-mediated gene transfer. Mol Ther. 2009;17:1215–1224.
    1. Li H, Murphy SL, Giles-Davis W, Edmonson S, Xiang Z, Li Y, et al. Pre-existing AAV capsid-specific CD8+ T cells are unable to eliminate AAV-transduced hepatocytes. Mol Ther. 2007;15:792–800.
    1. Mendell JR, Rodino-Klapac LR, Rosales-Quintero X, Kota J, Coley BD, Galloway G, et al. Limb-girdle muscular dystrophy type 2D gene therapy restores alpha-sarcoglycan and associated proteins. Ann Neurol. 2009;66:290–297.
    1. Rabinowitz JE, Rolling F, Li C, Conrath H, Xiao W, Xiao X, et al. Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity. J Virol. 2002;76:791–801.
    1. Clark KR, Liu X, McGrath JP., and , Johnson PR. Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses. Hum Gene Ther. 1999;10:1031–1039.

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