Yongping Yue

Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus

The restriction of viral receptors and coreceptors to the basolateral surface of airway epithelial cells has been blamed for the inefficient transfer of viral vectors to the apical surface of this tissue. We now report, however, that differentiated human airway epithelia internalize rAAV type-2 virus efficiently from their apical surfaces, despite the absence of known adeno-associated virus-2 (AAV-2) receptors or coreceptors at these sites. The dramatically lower transduction efficiency of rAAV infection from the apical surface of airway cells appears to result instead from differences in endosomal processing and nuclear trafficking of apically or basolaterally internalized virions. AAV capsid proteins are ubiquitinated after endocytosis, and gene transfer can be significantly enhanced by proteasome or ubiquitin ligase inhibitors. Tripeptide proteasome inhibitors increased persistent rAAV gene delivery from the apical surface >200-fold, to a level nearly equivalent to that achieved with basolateral infection. In vivo application of proteasome inhibitor in mouse lung augmented rAAV gene transfer from undetectable levels to a mean of 10.4 +/- 1.6% of the epithelial cells in large bronchioles. Proteasome inhibitors also increased rAAV-2-mediated gene transfer to the liver tenfold, but they did not affect transduction of skeletal or cardiac muscle. These findings suggest that tissue-specific ubiquitination of viral capsid proteins interferes with rAAV-2 transduction and provides new approaches to circumvent this barrier for gene therapy of diseases such as cystic fibrosis.

Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy

Sarcolemma-associated neuronal NOS (nNOS) plays a critical role in normal muscle physiology. In Duchenne muscular dystrophy (DMD), the loss of sarcolemmal nNOS leads to functional ischemia and muscle damage; however, the mechanism of nNOS subcellular localization remains incompletely understood. According to the prevailing model, nNOS is recruited to the sarcolemma by syntrophin, and in DMD this localization is altered. Intriguingly, the presence of syntrophin on the membrane does not always restore sarcolemmal nNOS. Thus, we wished to determine whether dystrophin functions in subcellular localization of nNOS and which regions may be necessary. Using in vivo transfection of dystrophin deletion constructs, we show that sarcolemmal targeting of nNOS was dependent on the spectrin-like repeats 16 and 17 (R16/17) within the rod domain. Treatment of mdx mice (a DMD model) with R16/17-containing synthetic dystrophin genes effectively ameliorated histological muscle pathology and improved muscle strength as well as exercise performance. Furthermore, sarcolemma-targeted nNOS attenuated alpha-adrenergic vasoconstriction in contracting muscle and improved muscle perfusion during exercise as measured by Doppler and microsphere circulation. In summary, we have identified the dystrophin spectrin-like repeats 16 and 17 as a novel scaffold for nNOS sarcolemmal targeting. These data suggest that muscular dystrophy gene therapies based on R16/17-containing dystrophins may yield better clinical outcomes than the current therapies.

A new dual-vector approach to enhance recombinant adeno-associated virus-mediated gene expression through intermolecular cis activation

A new dual-vector approach to enhance recombinant adeno-associated virus-mediated gene expression through intermolecular cis activation

Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors

Although adeno-associated virus (AAV)-mediated gene therapy has been hindered by the small viral packaging capacity of the vector, trans-splicing AAV vectors are able to package twice the size of the vector genome. Unfortunately, the efficiency of current trans-splicing vectors is very low. Here we show that rational design of the gene splitting site has a profound influence on trans-splicing vector-mediated gene expression. Using mRNA accumulation as a guide, we generated a set of efficient trans-splicing vectors and achieved widespread expression of the 6-kb ΔH2-R19 mini-dystrophin gene in skeletal muscle of mdx mice, a model for Duchenne muscular dystrophy. The dystrophic phenotype was ameliorated in both adult and aged mice. This demonstrates the use of trans-splicing vectors to efficiently express a large therapeutic structural protein. This strategy should be applicable to other large therapeutic genes or large transcription regulatory elements.

A Single Intravenous Injection of Adeno-associated Virus Serotype-9 Leads to Whole Body Skeletal Muscle Transduction in Dogs

The success of many gene therapy applications hinges on efficient whole body transduction. In the case of muscular dystrophies, a therapeutic vector has to reach every muscle in the body. Recent studies suggest that vectors based on adeno-associated virus (AAV) are capable of body-wide transduction in rodents. However, translating this finding to large animals remains a challenge. Here we explored systemic gene delivery with AAV serotype-9 (AAV-9) in neonatal dogs. Previous attempts to directly deliver AAV to adult canine muscle have yielded minimal transduction due to a strong cellular immune response. However, in neonatal dogs we observed robust skeletal muscle transduction throughout the body after a single intravenous injection. Importantly, systemic transduction was achieved in the absence of pharmacological intervention or immune suppression and it lasted for at least 6 months (the duration of study). We also observed several unique features not predicted by murine studies. In particular, cardiac muscle was barely transduced in dogs. Many muscular dystrophy patients can be identified by neonatal screening. The technology described here may lead to an effective early intervention in these patients.

Microdystrophin Gene Therapy of Cardiomyopathy Restores Dystrophin-Glycoprotein Complex and Improves Sarcolemma Integrity in the Mdx Mouse Heart

Background—More than 90% of Duchenne muscular dystrophy (DMD) patients develop cardiomyopathy, and many die of cardiac failure. Despite tremendous progress in skeletal muscle gene therapy, few attempts have been made to treat cardiomyopathy. Microdystrophin genes are shown to correct skeletal muscle pathological lesions in the mdx mouse model for DMD. Here, we tested the therapeutic potential of adeno-associated virus (AAV)–mediated microdystrophin gene therapy in the mdx mouse heart. Methods and Results—AAV was delivered to the newborn mdx mouse cardiac cavity. The procedure was rapid and well tolerated. Efficient expression was achieved in the inner and the outer layers of the myocardium. The ubiquitous cytomegalovirus promoter resulted in substantially higher expression than the muscle-specific CK6 promoter. The therapeutic effects of microdystrophin were evaluated at 10 months after infection. Immunostaining demonstrated extensive microdystrophin expression and successful restoration of the dystrophin-glycoprotein complex. Importantly, AAV-mediated microdystrophin expression improved the sarcolemma integrity in the mdx heart. Conclusions—We established a simple gene transfer method for efficient and persistent transduction of the mdx mouse heart. AAV-mediated microdystrophin expression restored the critical dystrophin-glycoprotein complex and improved sarcolemma integrity of the mdx heart. Our results revealed the promise of AAV-microdystrophin gene therapy for cardiomyopathy in DMD.

Enhancement of Muscle Gene Delivery with Pseudotyped Adeno-Associated Virus Type 5 Correlates with Myoblast Differentiation

ABSTRACT Adeno-associated virus (AAV)-based muscle gene therapy has achieved tremendous success in numerous animal models of human diseases. Recent clinical trials with this vector have also demonstrated great promise. However, to achieve therapeutic benefit in patients, large inocula of virus will likely be necessary to establish the required level of transgene expression. For these reasons, efforts aimed at increasing the efficacy of AAV-mediated gene delivery to muscle have the potential for improving the safety and therapeutic benefit in clinical trials. In the present study, we compared the efficiency of gene delivery to mouse muscle cells for recombinant AAV type 2 (rAAV-2) and rAAV-2cap5 (AAV-2 genomes pseudo-packaged into AAV-5 capsids). Despite similar levels of transduction by these two vectors in undifferentiated myoblasts, pseudotyped rAAV-2cap5 demonstrated dramatically enhanced transduction in differentiated myocytes in vitro (>500-fold) and in skeletal muscle in vivo (>200-fold) compared to rAAV-2. Serotype-specific differences in transduction efficiency did not directly correlate with viral binding to muscle cells but rather appeared to involve endocytic or intracellular barriers to infection. Furthermore, application of this pseudotyped virus in a mouse model of Duchennes muscular dystrophy also demonstrated significantly improved transduction efficiency. These findings should have a significant impact on improving rAAV-mediated gene therapy in muscle.

Prevention of Dystrophin-Deficient Cardiomyopathy in Twenty-One-Month-Old Carrier Mice by Mosaic Dystrophin Expression or Complementary Dystrophin/Utrophin Expression

A cure for dystrophin-deficient muscular dystrophy requires treating both skeletal muscle and the heart. Whereas mosaic dystrophin expression has been shown to protect skeletal muscle, controversy exists over whether mosaic expression is protective in the heart. We have shown recently that mosaic dystrophin expression prevents stress-induced heart damage in young carrier mice. Although an interesting finding, the clinical relevance remains to be established because young dystrophin-null mdx mice do not have heart disease. On the other hand, heart failure has been reported in human carriers. To resolve this mouse/human discrepancy, we evaluated the cardiac phenotype in 21-month-old mdx, carrier, and normal mice. We found dilated cardiomyopathy in old mdx mice but not in age-matched carrier mice. All anatomical parameters and physiological assay results (ECG and closed-chest Millar catheter) were within the normal range in old carrier mice. Focal myocardial inflammation was found in a small fraction of old carrier mice, but it had no major impact on heart function. Dobutamine stress revealed a near normal hemodynamic profile except for a marginal reduction in systolic pressure in old carrier mice. Immunostaining and Western blot showed dystrophin expression in 50% cardiomyocytes in old carrier mice. Interestingly, utrophin was upregulated in dystrophin-negative heart cells in carrier mice. In summary, we have provided the first clear-cut evidence that dilated cardiomyopathy in old mdx mice was prevented by mosaic dystrophin expression or complementary dystrophin/utrophin expression. Our results raise the hope for ameliorating dystrophic cardiomyopathy through partial gene and/or cell therapy.

Sarcolemmal nNOS anchoring reveals a qualitative difference between dystrophin and utrophin.

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by dystrophin deficiency. In normal muscle, dystrophin helps maintain sarcolemmal stability. Dystrophin also recruits neuronal nitric oxide synthase (nNOS) to the sarcolemma. Failure to anchor nNOS to the membrane leads to functional ischemia and aggravates muscle disease in DMD. Over the past two decades, a great variety of therapeutic modalities have been explored to treat DMD. A particularly attractive approach is to increase utrophin expression. Utrophin shares considerable sequence, structural and functional similarity with dystrophin. Here, we test the hypothesis that utrophin also brings nNOS to the sarcolemma. Full-length utrophin cDNA was expressed in dystrophin-deficient mdx mice by gutted adenovirus or via transgenic overexpression. Subcellular nNOS localization was determined by immunofluorescence staining, in situ nNOS activity staining and microsomal preparation western blot. Despite supra-physiological utrophin expression, we did not detect nNOS at the sarcolemma. Furthermore, transgenic utrophin overexpression failed to protect mdx muscle from exercise-associated injury. Our results suggest that full-length utrophin cannot anchor nNOS to the sarcolemma. This finding might have important implications for the development of utrophin-based DMD therapies.

Adeno-Associated Virus Serotype-9 Microdystrophin Gene Therapy Ameliorates Electrocardiographic Abnormalities in mdx Mice

Adeno-associated virus (AAV)-mediated microdystrophin gene therapy holds great promise for treating Duchenne muscular dystrophy (DMD). Previous studies have revealed excellent skeletal muscle protection. Cardiac muscle is also compromised in DMD patients. Here we show that a single intravenous injection of AAV serotype-9 (AAV-9) microdystrophin vector efficiently transduced the entire heart in neonatal mdx mice, a dystrophin-deficient mouse DMD model. Furthermore, microdystrophin therapy normalized the heart rate, PR interval, and QT interval. The cardiomyopathy index was also significantly improved in treated mdx mice. Our study demonstrates for the first time that AAV microdystrophin gene therapy can ameliorate the electrocardiographic abnormalities in a mouse model for DMD.