Gene therapy offers a promise for treating inherited muscle disorders. The advantages of recombinant adeno-associated virus (rAAV) gene delivery vector include nonpathogenicity and long-term gene expression after a single administered dose. However, rAAV predominantly transduces the liver after systemic administration, reducing its efficiency for gene transfer to the heart and skeletal muscle. The question of how to deliver the therapeutic genes into most of the diseased myofibers becomes a challenge. The goal of this project is to develop an efficient and muscle-specific AAV vector for systemic delivery. Here, the muscle-targeting peptide ASSLNIA was incorporated into AAV2 capsid after residue 587 without heparin-binding motif (587 TG MTP vector) or with heparin-binding motif (588 HB MTP vector) since heparan sulfate is the primary cellular receptor of AAV2. The efficiencies and selectivities of muscle targeting of modified rAAVs were evaluated in vitro and in vivo. This study demonstrated that the peptide-modified vectors maintained their myotube transduction ability. Peptide-engineered AAVs decreased their transductions in non-muscle cell lines. In addition, the 587 TG MTP vector did not require the heparin-dependent mechanism for muscular targeting. The C2C12 myotube transductions of 587 TG MTP and 588 HB MTP vectors were inhibited 47%~58% in the presence of free ASSLNIA peptide while unmodified rAAV2 transduction was only suppressed by 25%. To explore the muscle-targeting abilities of modified rAAVs in vivo, mice were injected intravenously via a tail vein with a viral dose containing 9x10^11 genomic particles. After four weeks, mouse organs were harvested for the luciferase assay. The 587 TG MTP vector demonstrated enhanced muscle and heart transduction compared to unmodified rAAV2. Importantly, the 587 TG MTP virus significantly reduced its transduction of liver, lungs, and spleen. The vector biodistribution in organs was also determined by real-time PCR and peptide-modified vectors showed similar targeting effects. Moreover, this study found that both 587 TG MTP and 588 HB MTP vectors were resistant to antibody neutralization. These results indicate that this muscle-targeting peptide facilitates the generation of an efficient and muscle-specific AAV vector for systemic gene delivery in the treatment of muscle diseases to provide clinical and public health benefits.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-04112007-143609 |
| Date | 22 June 2007 |
| Creators | Yu, Chi-Yi |
| Contributors | Xiao Xiao, Phalguni Gupta, Paula R. Clemens, Frank J. Jenkins, Simon M. Barratt-Boyes |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-04112007-143609/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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