Systemic gene delivery into muscle has been a major challenge for muscular dystrophy gene therapy, with capillary blood vessels posing the principle barrier and limiting vector dissemination. Previous efforts to deliver genes into multiple muscles have relied on isolated vessel perfusion or pharmacological interventions to enforce broad vector distribution. We compared the efficiency of multiple adeno-associated virus (AAV) vectors after a single injection via intraperitoneal or intravenous routes without additional intervention. We show that AAV8 is the most efficient vector for crossing the blood vessel barrier to attain systemic gene transfer in both skeletal and cardiac muscles of mice and hamsters. Serotypes such as AAV1 and AAV6, which demonstrate robust infection in skeletal muscle cells, were less effective in crossing the blood vessel barrier. Gene expression persisted in muscle and heart, but diminished in tissues undergoing rapid cell division, such as neonatal liver. This technology should prove useful for muscle-directed systemic gene therapy.

Low temperature is a major factor limiting rice productivity and geographical distribution. Improved cold tolerance and expanded cultivation to high-altitude or high-latitude regions would help meet growing rice demand. Here we explored a QTL for cold tolerance and cloned the gene, CTB4a (cold tolerance at booting stage), encoding a conserved leucine-rich repeat receptor-like kinase. We show that different CTB4a alleles confer distinct levels of cold tolerance and selection for variation in the CTB4a promoter region has occurred on the basis of environmental temperature. The newly generated cold-tolerant haplotype Tej-Hap-KMXBG was retained by artificial selection during temperate japonica evolution in cold habitats for low-temperature acclimation. Moreover, CTB4a interacts with AtpB, a beta subunit of ATP synthase. Upregulation of CTB4a correlates with increased ATP synthase activity, ATP content, enhanced seed setting and improved yield under cold stress conditions. These findings suggest strategies to improve cold tolerance in crop plants.

Adeno-associated viral (AAV) vectors have been broadly used for gene transfer in vivo for various applications. However, AAV precludes the use of most of the original large-sized tissue-specific promoters for expression of transgenes. Efforts are made to develop highly compact, active and yet tissue-specific promoters for use in AAV vectors. In this study, we further abbreviated the muscle creatine kinase (MCK) promoter by ligating a double or triple tandem of MCK enhancer (206-bp) to its 87-bp basal promoter, generating the dMCK (509-bp) and tMCK (720-bp) promoters. The dMCK promoter is shorter but stronger than some previously developed MCK-based promoters such as the enh358MCK (584-bp) and CK6 (589-bp) in vitro in C2C12 myotubes and in vivo in skeletal muscles. The tMCK promoter is the strongest that we tested here, more active than the promiscuous cytomegalovirus (CMV) promoter. Furthermore, both the dMCK and tMCK promoters are essentially inactive in nonmuscle cell lines as well as in the mouse liver (4200-fold weaker than the CMV promoter).The dMCK promoter was further tested in a few lines of transgenic mice. Expression of LacZ or minidystrophin gene was detected in skeletal muscles throughout the body, but was weak in the diaphragm, and undetectable in the heart and other tissues. Similar to other miniature MCK promoters, the dMCK promoter also shows preference for fast-twitch myofibers. As a result, we further examined a short, synthetic muscle promoter C5-12 (312-bp). It is active in both skeletal and cardiac muscles but lacks apparent preference on myofiber types. Combination of a MCK enhancer to promoter C5-12 has increased its strength in muscle by two-to threefold. The above-mentioned compact muscle-specific promoters are well suited for AAV vectors in muscle-directed gene therapy studies.

Duchenne muscular dystrophy (DMD) is the most common disabling and lethal genetic muscle disorder, afflicting 1 of every 3500 males. Patients with DMD experience progressive muscle degeneration and weakness and succumb to respiratory or cardiac failure by their early twenties. No treatment is currently available for DMD. Mutations in the dystrophin gene result in lack of a functional dystrophin protein in striated muscle, which induces instability in the muscle cell membrane leading to persistent muscle injury after contraction. We have previously created novel minidystrophin genes and demonstrated that adeno-associated virus (AAV)-mediated intramuscular delivery of the minigenes effectively ameliorated mdx dystrophic histopathology and led to normal cell membrane integrity for more than 1 year. In this paper, we investigated whether AAV-minidystrophin could also improve mdx muscle contractile function. Two-month-old adult male mdx mice, with established muscular dystrophy, were given a single-dose injection of an AAV-minidystrophin vector in the tibialis anterior (TA) muscle of one leg, with the untreated contralateral leg used as a control. The treated TA muscle showed both (1) a significant increase in isometric force generation and (2) a significant increase in resistance to lengthening activation-induced muscle force decrements. We conclude that AAV-minidystrophin gene treatment is effective in improving mdx muscle contractile function.

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Background-The success of muscular dystrophy gene therapy requires widespread and stable gene delivery with minimal invasiveness. Here, we investigated the therapeutic effect of systemic delivery of adeno-associated virus (AAV) vectors carrying human ␦-sarcoglycan (␦-SG) gene in TO-2 hamsters, a congestive heart failure and muscular dystrophy model with a ␦-SG gene mutation. Methods and Results-A single injection of double-stranded AAV serotype 8 vector carrying human ␦-SG gene without the need of any physical or pharmaceutical interventions achieved nearly complete gene transfer and tissue-specific expression in the heart and skeletal muscles of the diseased hamsters. Broad and sustained (Ͼ12 months) restoration of the missing ␦-SG gene in the TO-2 hamsters corrected muscle cell membrane leakiness throughout the body and normalized serum creatine kinase levels (a 50-to 100-fold drop). Histological examination revealed minimal or the absence of central nucleation, fibrosis, and calcification in the skeletal muscle and heart. Whole-body functional analysis such as treadmill running showed dramatic improvement, similar to the wild-type F1B hamsters. Furthermore, cardiac functional studies with echocardiography revealed significantly increased percent fractional shortening and decreased left ventricular end-diastolic and end-systolic dimensions in the treated TO-2 hamsters. The survival time of the animals was also dramatically extended. Conclusions-Systemic

Low temperature at the booting stage is a serious abiotic stress in rice, and cold tolerance is a complex trait controlled by many quantitative trait loci (QTL). A QTL for cold tolerance at the booting stage in cold-tolerant near-isogenic rice line ZL1929-4 was analyzed. A total of 647 simple sequence repeat (SSR) markers distributed across 12 chromosomes were used to survey for polymorphisms between ZL1929-4 and the cold-sensitive japonica cultivar Towada, and nine were polymorphic. Single marker analysis revealed that markers on chromosome 7 were associated with cold tolerance. By interval mapping using an F(2) population from ZL1929-4 x Towada, a QTL for cold tolerance was detected on the long arm of chromosome 7. The QTL explained 9 and 21% of the phenotypic variances in the F(2) and F(3) generations, respectively. Recombinant plants were screened for two flanking markers, RM182 and RM1132, in an F(2) population with 2,810 plants. Two-step substitution mapping suggested that the QTL was located in a 92-kb interval between markers RI02905 and RM21862. This interval was present in BAC clone AP003804. We designated the QTL as qCTB7 (quantitative trait locus for cold tolerance at the booting stage on chromosome 7), and identified 12 putative candidate genes.