Delivery of alpha- and beta-sarcoglycan by recombinant adeno-associated virus: efficient rescue of muscle, but differential toxicity.

The sarcoglycanopathies are a group of four autosomal recessive limb girdle muscular dystrophies (LGMD 2D, 2E, 2C, and 2F), caused by mutations of the alpha-, beta-, gamma-, or delta-sarcoglycan genes, respectively. The delta-sarcoglycan-deficient hamster has been the most utilized model for gene delivery to muscle by recombinant adeno-associated virus (AAV) vectors; however, human patients with delta-sarcoglycan deficiency are exceedingly rare, with only two patients described in the United States. Here, we report construction and use of AAV vectors expressing either alpha- or beta-sarcoglycan, the genes responsible for the most common forms of the human sarcoglycanopathies. Both vectors showed successful short-term genetic, biochemical, and histological rescue of both alpha- and beta-sarcoglycan-deficient mouse muscle. However, comparison of persistence of expression in 51 injected mice showed substantial differences between AAV alpha-sarcoglycan (alpha-SG) and beta-sarcoglycan (beta-SG) vectors. AAV-beta-SG showed long-term expression with no decrease in expression for more than 21 months after injection, whereas AAV-alpha-SG showed a dramatic loss of positive fibers between 28 and 41 days post-injection (p = 0.006). Loss of immunopositive myofibers was correlated with significant inflammatory cell infiltrate, primarily macrophages. To determine whether the loss of alpha-sarcoglycan-positive fibers was due to an immune response or cytotoxic effect of alpha-sarcoglycan overexpression, severe combined immunodeficient (SCID) mouse muscle was assayed for cytotoxicity after injection with AAV-alpha-SG, AAV-beta-SG, or phosphate-buffered saline. The results were consistent with overexpression of alpha-sarcoglycan causing significant cytotoxicity. The cytotoxicity of alpha-sarcoglycan, and not beta- or delta-sarcoglycan overexpression, was consistent with biochemical studies of the hierarchical order of assembly of the sarcoglycan complex. Our data suggest that even closely related proteins might require different levels of expression to avoid toxicity and achieve long-term tissue rescue.

Molecular pathophysiology and targeted therapeutics for muscular dystrophy.

Experimental therapeutics of the muscular dystrophies has made impressive advances on several fronts. Adeno-associated virus has emerged as the clear 'vector of choice' for muscle gene delivery, with successful functional rescue of dystrophic muscle in rodent models. Correction of the dystrophin gene mutation in a dog model has been reported, and several reports of progress on myogenic stem cell characterization are resurrecting cell transplantation as a possible therapeutic approach. The downstream consequences of dystrophin deficiency are being defined quickly using microarray experiments, and drugs targeting specific biochemical pathways are being tested rapidly in animal models. Such targeted drug discoveries, which are discussed in this article, have begun to be implemented in human clinical trials.

RNA differential display of scarless wound healing in fetal rabbit indicates downregulation of a CCT chaperonin subunit and upregulation of a glycophorin-like gene transcript.

BACKGROUND/PURPOSE: Scars form as wounds heal in adult organisms. In addition to disrupting cosmetic appearance, scar tissue can cause significant morbidity, and even death if it blocks vital organ function. Previous work has established that fetal wounds, especially in early to midgestation, can heal without scarring. Because such inherent physiological mechanisms ultimately are under genetic control, a study was initiated to elucidate the differences in gene expression that produce scarless wound healing in the mammalian fetus but scarring in postnatal wounds. Reverse transcription polymerase chain reaction (RT-PCR) differential display (DD) was used to detect differentially expressed mRNA transcripts in a rabbit model of wound healing. METHODS: Adult and 21-day fetal full-thickness rabbit skin specimens from wounded and unwounded sites were harvested 12 hours postwounding. RNA extracted from the tissue was used as a template in DD reactions using anchoring and random primers to generate tissue-specific gene expression fingerprints. The over 2,000 resulting amplimers (gene transcripts) were screened for differential expression among the 4 types of specimens: fetal control (unwounded), fetal wound, adult control, and adult wound. Selected bands distinctly upregulated or downregulated in fetal wound lanes on the DD gels were excised, and the cDNA was extracted, reamplified, cloned into vectors, and sequenced. DD results were confirmed by limiting-dilution RT-PCR using sequence-specific primers. RESULTS: Differential display (DD) showed 22 amplimers that were significantly upregulated in all fetal wound samples as compared with little or no expression in fetal control, adult control, or adult wound tissues. Conversely, 5 transcripts were downregulated in the fetal wound specimens but highly expressed in the 3 comparison tissues. Reamplification of selected transcripts by PCR, followed by cloning and DNA sequencing, yielded 7 distinct sequences, each representing a gene expressed differently in fetal wound than in the other 3 tissues. A transcript that was downregulated in fetal wound showed very high sequence homology to part of the human gene for the eta subunit of the hetero-oligomeric particle CCT (the chaperonin containing T-complex polypeptide 1 or TCP-1). An upregulated amplimer showed significant DNA sequence homology to glycophorins A and B. One sequence was identified as 28S rRNA. The remaining 4 candidate sequences showed no significant homology to known genes, but 1 had high homology to expressed sequence tags of unknown function. CONCLUSIONS: With careful experimental design and proper controls and verifications, differential display of RNA expression is a potentially powerful method of finding genes that specifically regulate a particular physiological process such as fetal wound healing. No a priori knowledge of what genes might be involved, or why, is necessary. This study indicates that downregulation of a gene that codes for a chaperonin subunit and upregulation of several other genes may be involved in the striking scarless character of wound healing in the mammalian fetus. Results suggest the hypothesis that downregulation of the CCT chaperonin in fetal wound may inhibit the formation of myofibroblasts, a cell type that correlates highly with scarring in postnatal wound healing, by preventing the folding of sufficient alpha-smooth muscle actin to form the stress fibers characteristic of these cells.

Full functional rescue of a complete muscle (TA) in dystrophic hamsters by adeno-associated virus vector-directed gene therapy.

Limb girdle muscular dystrophy (LGMD) 2F is caused by mutations in the delta-sarcoglycan (SG) gene. Previously, we have shown successful application of a recombinant adeno-associated virus (AAV) vector for genetic and biochemical rescue in the Bio14.6 hamster, a homologous animal model for LGMD 2F (J. Li et al., Gene Ther. 6:74-82, 1999). In this report, we show efficient and long-term delta-SG expression accompanied by nearly complete recovery of physiological function deficits after a single-dose AAV vector injection into the tibialis anterior muscle of the dystrophic hamsters. AAV vector treatment led to more than 97% recovery in muscle strength for both the specific twitch force and the specific tetanic force, when compared to the age-matched control. Vector treatment also prevented pathological muscle hypertrophy and resulted in normal muscle weight and size. Finally, vector-treated muscle showed substantial improvement of the histopathology. This is the first report of successful functional rescue of an entire muscle after AAV-mediated gene delivery. This report also demonstrates the feasibility of in vivo gene therapy for LGMD patients by using AAV vectors.

rAAV vector-mediated sarcogylcan gene transfer in a hamster model for limb girdle muscular dystrophy.

The limb girdle muscular dystrophies (LGMD) are a genetically and phenotypically heterogeneous group of degenerative neuromuscular diseases. A subset of the genetically recessive forms of LGMD are caused by mutations in the four muscle sarcoglycan genes (alpha, beta, gamma and delta). The coding sequences of all known sarcoglycan genes are smaller than 2 kb, and thus can be readily packaged in recombinant adeno-associated virus (rAAV) vectors. Previously, we have demonstrated highly efficient and sustained transduction in mature muscle tissue of immunocompetent animals with rAAV vectors. In this report, we utilize recombinant AAV containing the delta-sarcoglycan gene for genetic complementation of muscle diseases using a delta-sarcoglycan-deficient hamster model (Bio 14.6). We show efficient delivery and widespread expression of delta-sarcoglycan after a single intramuscular injection. Importantly, rAAV vector containing the human delta-sarcoglycan cDNA restored secondary biochemical deficiencies, with correct localization of other sarcoglycan proteins to the muscle fiber membrane. Interestingly, restoration of alpha-, as well as beta-sarcoglycan was homogeneous and properly localized throughout transduced muscle, and appeared unaffected by dramatic overexpression of delta-sarcoglycan in the cytoplasm of some myofibers. These results support the feasibility of rAAV vector's application to treat LGMD by means of direct in vivo gene transfer.