Nancy Larochelle

Muscle-specific overexpression of the adenovirus primary receptor CAR overcomes low efficiency of gene transfer to mature skeletal muscle.

ABSTRACT Significant levels of adenovirus (Ad)-mediated gene transfer occur only in immature muscle or in regenerating muscle, indicating that a developmentally regulated event plays a major role in limiting transgene expression in mature skeletal muscle. We have previously shown that in developing mouse muscle, expression of the primary Ad receptor CAR is severely downregulated during muscle maturation. To evaluate how global expression of CAR throughout muscle affects Ad vector (AdV)-mediated gene transfer into mature skeletal muscle, we produced transgenic mice that express the CAR cDNA under the control of the muscle-specific creatine kinase promoter. Five-month-old transgenic mice were compared to their nontransgenic littermates for their susceptibility to AdV transduction. In CAR transgenics that had been injected in the tibialis anterior muscle with AdVCMVlacZ, increased gene transfer was demonstrated by the increase in the number of transduced muscle fibers (433 ± 121 in transgenic mice versus 8 ± 4 in nontransgenic littermates) as well as the 25-fold increase in overall β-galactosidase activity. Even when the reporter gene was driven by a more efficient promoter (the cytomegalovirus enhancer–chicken β-actin gene promoter), differential transducibility was still evident (893 ± 149 versus 153 ± 30 fibers; P < 0.001). Furthermore, a fivefold decrease in the titer of injected AdV still resulted in significant transduction of muscle (253 ± 130 versus 14 ± 4 fibers). The dramatic enhancement in AdV-mediated gene transfer to mature skeletal muscle that is observed in the CAR transgenics indicates that prior modulation of the level of CAR expression can overcome the poor AdV transducibility of mature skeletal muscle and significant transduction can be obtained at low titers of AdV.

Efficient muscle-specific transgene expression after adenovirus-mediated gene transfer in mice using a 1.35 kb muscle creatine kinase promoter/enhancer

Replication-defective (E1+E3-deleted) human adenovirus vectors are a promising means of therapeutic gene delivery to skeletal muscle cells. Since the tropism of adenovirus is nonselective, muscle-specific expression of systemically administered vectors can only be achieved by the use of a tissue-specific promoter/enhancer that is small enough to fit the insert capacity of the vector. We have generated two replication-defective adenovirus recombinants (AV) in which the reporter gene (either firefly luciferase or E. coli β-galactosidase) was driven by a truncated (1.35 kb) muscle creatine kinase (MCK) promoter/enhancer or by the fast troponin I (TnI) promoter/enhancer. Highly efficient and muscle-specific transgene expression was demonstrated in immunodeficient mice after local injection of AV into muscles at an early age. In nonmuscle tissues (brain, liver, kidney, lung), the transgene expression was extremely low even though in these tissues in situ polymerase chain reaction showed as high an infectivity of the cells by the AV as in muscle. The relatively small size, the good efficiency and the muscle specificity of the MCK promoter would make it ideal to drive the 6.3 kb (truncated) dystrophin cDNA in first generation AV (with a limited (8 kb) insert capacity) designed for gene therapy of Duchenne muscular dystrophy.

Impact of the coxsackie and adenovirus receptor (CAR) on glioma cell growth and invasion: Requirement for the C‐terminal domain

Expression of the coxsackie and adenovirus receptor (CAR) is downregulated in malignant glioma cell lines and is barely detectable in high‐grade primary astrocytoma (glioblastoma multiforme). We determined the effect of forced CAR expression on the invasion and growth of the human glioma cell line U87‐MG, which does not express any CAR. Although retrovirally mediated expression of full‐length CAR in U87‐MG cells did not affect monolayer growth in vitro, it did reduce glioma cell invasion in a 3‐dimensional spheroid model. Furthermore, in xenograft experiments, intracerebral implantation of glioma cells expressing full‐length CAR resulted in tumors with a significantly reduced volume compared to tumors generated by control vector‐transduced U87‐MG cells. In contrast, U87‐MG cells expressing transmembrane CAR with a deletion of the entire cytoplasmic domain (except for the first 2 intracellular juxtamembrane cysteine amino acids) had rates of invasion and tumor growth that were similar to those of the control cells. This difference in behavior between the 2 forms of CAR was not due to improper cell surface localization of the cytoplasmically deleted CAR as determined by comparable immunostaining of unpermeabilized cells, equivalent adenoviral transduction of the cells and similar extent of fractionation into lipid‐rich domains. Taken together, these results suggest that the decrease or loss of CAR expression in malignant glioma may confer a selective advantage in growth and invasion to these tumors.

Expression of Dystrophin Driven by the 1.35-kb MCK Promoter Ameliorates Muscular Dystrophy in Fast, but Not in Slow Muscles of Transgenic MDX Mice

Successful gene therapy of Duchenne muscular dystrophy may require the lifelong expression of a therapeutic gene in all affected muscles. The most promising gene delivery vehicles, viral vectors, suffer from several limitations, including immunogenicity, loss of therapeutic gene expression, and a limited packaging capacity. Therefore, various efforts were previously undertaken to use small therapeutic genes and to place them under the control of a strong and muscle-specific promoter. Here we report the effects of a minidystrophin (6.3 kb) under the control of a short muscle-specific promoter (MCK 1.35 kb) over most of the lifetime (4-20 months) of a transgenic mouse model. Dystrophin expression remained stable and muscle-specific at all ages. The dystrophic phenotype was greatly ameliorated and, most importantly, muscle function in limb muscles was significantly improved not only in young adult but also in aged mice compared to nontransgenic littermates. Dystrophin expression was strong in fast-twitch skeletal muscles such as tibialis anterior and extensor digitorum longus, but weak or absent in heart, diaphragm, and slow-twitch muscles. Additionally, expression was strong in glycolytic but weak in oxidative fibers of fast-twitch muscles. This study may have important implications for the design of future gene therapy trials for muscular dystrophy.

Interferons impair early transgene expression by adenovirus-mediated gene transfer in muscle cells

Abstract Recombinant adenovirus (AVR) promises to be an efficient vector in gene therapy for neuromuscular diseases, but in preclinical experiments the expression of therapeutic genes is shorter lived in immunocompetent animals than in immunocompromised hosts. Interferons (IFN), which are known to have a role both in early antiviral activity and in late cytotoxic immunoreaction against the virus or transduced cells, may influence the efficiency of gene transfer. In this study we investigated the role of IFNs in determining the efficiency of gene transfer by AVR. AVRs expressing β-galactosidase (β-gal) from either a cytomegalovirus (CMV) or a troponin-I promoter were used. Muscle cells were infected by AVR after exposure to various IFNs. The αIFN treatment significantly reduced (up to fivefold) the CMV promoter-driven gene expression in muscle cells in vitro and in immature muscles in vivo, while the least effective inhibitor was βIFN. The decrease in gene expression by IFNs was more pronounced with the CMV-driven transgene than troponin-I promoter-driven one and was due to a decrease in transcript level. Intrinsic IFNs that are triggered by AVR administration can decrease the efficiency of gene transfer in muscle cells. Therefore the use of muscle specific promoters in AVR and/or IFN inhibitory agents will likely improve the prospects of effective gene therapy by AVR.

Strategies for muscle-specific targeting of adenoviral gene transfer vectors

Currently, adenoviral transfer of therapeutic genes such as dystrophin is hampered by low transduction efficiency of adult skeletal muscle. This is largely due to the lack of appropriate virus attachment receptors on the myofiber surface. Recent studies in transgenic mice revealed that upregulation of Coxsackie- and adenovirus receptor improves gene transfer efficiency by approximately ten-fold. Conversely, the vector load that needed to be administered to achieve sufficient gene transfer could be lowered significantly. Reduced viral vector loads may help to control virally mediated toxicity and immunogenicity. To date, there are no drugs or methods known to increase Coxsackie- and adenovirus receptor expression in skeletal muscle that would be easily applicable in humans. However, alternative strategies such as vector retargeting are currently being investigated that may allow for an increase in binding of adenoviral vectors to skeletal muscle. Recent experiments have shown that directed mutagenesis of the adenoviral fiber knob allows for a significant reduction in Coxsackie- and adenovirus receptor binding and for introduction of a new binding domain. Therefore, vector retargeting towards efficient and specific infection of skeletal muscle may be achieved by directed genetic alteration of adenoviral capsid proteins.

Inhibition of the Protein Tyrosine Phosphatase SHP-1 Increases Glucose Uptake in Skeletal Muscle Cells by Augmenting Insulin Receptor Signaling and GLUT4 Expression

The protein tyrosine phosphatase (PTPase) Src-homology 2-domain-containing phosphatase (SHP)-1 was recently reported to be a novel regulator of insulins metabolic action. In order to examine the role of this PTPase in skeletal muscle, we used adenovirus (AdV)-mediated gene transfer to express an interfering mutant of SHP-1 [dominant negative (DN)SHP-1; mutation C453S] in L6 myocytes. Expression of DNSHP-1 increased insulin-induced Akt serine-threonine kinase phosphorylation and augmented glucose uptake and glycogen synthesis. Pharmacological inhibition of glucose transporter type 4 (GLUT4) activity using indinavir and GLUT4 translocation assays revealed an important role for this transporter in the increased insulin-induced glucose uptake in DNSHP-1-expressing myocytes. Both GLUT4 mRNA and protein expression were also found to be increased by DNSHP-1 expression. Furthermore, AdV-mediated delivery of DNSHP-1 in skeletal muscle of transgenic mice overexpressing Coxsackie and AdV receptor also enhanced GLUT4 protein expression. Together, these findings confirm that SHP-1 regulates muscle insulin action in a cell-autonomous manner and further suggest that the PTPase negatively modulates insulin action through down-regulation of both insulin signaling to Akt and GLUT4 translocation, as well as GLUT4 expression.

Efficient and fast functional screening of microdystrophin constructs in vivo and in vitro for therapy of duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal genetic disorder affecting the skeletal muscle compartment, and is caused by mutation(s) in the dystrophin gene. Gene delivery of microdystrophin constructs using adeno-associated virus (AAV) and antisense-mediated exon skipping restoring the genetic reading frame are two of the most promising therapeutic strategies for DMD. Both approaches use microdystrophin proteins either directly as a desired construct for gene delivery, using the capacity-limited AAV vectors, or as the therapeutic outcome of gene splicing. Although functionality of the resulting artificial dystrophin proteins can be predicted in silico, experimental evidence usually obtained in transgenic mice is required before human trials. However, the enormous number of potential constructs makes screening assays for dystrophin protein function in vitro and in vivo highly desirable. Here we present data showing that functionality of microdystrophins can be assessed using relatively simple and fast techniques.

Strength and muscle specificity of a compact promoter derived from the slow troponin I gene in the context of episomal (gutless adenovirus) and integrating (lentiviral) vectors

Gutless adenovirus (helper‐dependent adenoviral vector; HDAd) and lentiviral vectors (LV) are attractive vectors for the gene therapy of muscle diseases. Because the organization of their DNA (episomal versus integrated) differs, we investigated whether the strength and specificity of ΔUSEx3, a novel muscle‐specific promoter previously tested with plasmid, were maintained in the context of these vectors.

Genomic integration of adenoviral gene transfer vectors following transduction of fertilized mouse oocytes

Adenoviral vectors (AdV) are popular tools to deliver foreign genes into a wide range of cells. They have also been used in clinical gene therapy trials. Studies on AdV-mediated gene transfer to mammalian oocytes and transmission through the germ line have been reported controversially. In the present study we investigated whether AdV sequences integrate into the mouse genome by microinjecting AdV into the perivitelline space of fertilized oocytes. We applied a newly developed PCR technique (HiLo-PCR) for identification of chromosomal junctions next to the integrated AdV. We demonstrate that mouse oocytes can be transduced by different recombinant adenoviral vectors (first generation and gutless). In one transgenic mouse line using the first generation adenoviral vector, the genome has integrated into a highly repetitive cluster located on the Y chromosome. While the transgene (GFP) was expressed in early embryos, no expression was detected in adult transgenic mice. The use of gutless AdV resulted in expression of the transgene, albeit the vector was not transmitted to progeny. These results indicate that under optimized conditions fertilized mouse oocytes are transduced by AdV and give rise to transgenic founder animals. Therefore, adequate precautions should be taken in gene therapy protocols of reproductive patients since transduction of oocytes or early embryos and subsequent chromosomal integration cannot be ruled out entirely.