Design of tissue-specific regulatory cassettes for high-level rAAV-mediated expression in skeletal and cardiac muscle.

Systemic delivery of recombinant adeno-associated virus (rAAV) 6 vectors mediates efficient transduction of the entire striated musculature, making this an attractive strategy for muscle gene therapy. However, owing to widespread transduction of non-muscle tissues, optimization of this method would benefit from the use of muscle-specific promoters. Most such promoters either lack high-level expression in certain muscle types or are too large for inclusion in rAAV vectors encoding microdystrophin.

rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice.

Mice carrying mutations in both the dystrophin and utrophin genes die prematurely as a consequence of severe muscular dystrophy. Here, we show that intravascular administration of recombinant adeno-associated viral (rAAV) vectors carrying a microdystrophin gene restores expression of dystrophin in the respiratory, cardiac and limb musculature of these mice, considerably reducing skeletal muscle pathology and extending lifespan. These findings suggest rAAV vector-mediated systemic gene transfer may be useful for treatment of serious neuromuscular disorders such as Duchenne muscular dystrophy.

A highly functional mini-dystrophin/GFP fusion gene for cell and gene therapy studies of Duchenne muscular dystrophy.

A promising approach for treating Duchenne muscular dystrophy (DMD) is by autologous cell transplantation of myogenic stem cells transduced with a therapeutic expression cassette. Development of this method has been hampered by a low frequency of cellular engraftment, the difficulty of tracing transplanted cells, the rapid loss of autologous cells carrying marker genes that are unable to halt muscle necrosis and the difficulty of stable transfer of a large dystrophin gene into myogenic stem cells. We engineered a 5.

Systemic delivery of genes to striated muscles using adeno-associated viral vectors.

A major obstacle limiting gene therapy for diseases of the heart and skeletal muscles is an inability to deliver genes systemically to muscles of an adult organism. Systemic gene transfer to striated muscles is hampered by the vascular endothelium, which represents a barrier to distribution of vectors via the circulation. Here we show the first evidence of widespread transduction of both cardiac and skeletal muscles in an adult mammal, after a single intravenous administration of recombinant adeno-associated virus pseudotype 6 vectors.

Helper-Independent Sleeping Beauty transposon-transposase vectors for efficient nonviral gene delivery and persistent gene expression in vivo.

Transposon-based vectors represent promising new tools for chromosomal transgene insertion and establishment of persistent gene expression in vivo. Here, we report the development of helper-independent transposon-transposase (HITT) vectors, which contain on single plasmids (i) a Sleeping Beauty (SB) transposon containing the transgene and (ii) a SB transposase expression cassette. To obtain an optimal level of transposase expression from HITT vectors, we determined the relative strength of a panel of different promoters in mouse liver and used these promoters to drive transposase expression from injected HITT vectors carrying a human alpha(1)-antitrypsin (hAAT) expression cassette flanked by transposon inverted repeats.

In vivo antiviral efficacy of prenylation inhibitors against hepatitis delta virus.

Hepatitis delta virus (HDV) can dramatically worsen liver disease in patients coinfected with hepatitis B virus (HBV). No effective medical therapy exists for HDV. The HDV envelope requires HBV surface antigen proteins provided by HBV.

A potent and specific morpholino antisense inhibitor of hepatitis C translation in mice.

Hepatitis C virus (HCV) is an RNA virus infecting one in every 40 people worldwide. Current treatments are ineffective and HCV is the leading cause of liver failure leading to transplantation in the United States and Europe. Translational control of HCV is a prime therapeutic target.

Preclinical in vivo evaluation of pseudotyped adeno-associated virus vectors for liver gene therapy.

We report the generation and use of pseudotyped adeno-associated viral (AAV) vectors for the liver-specific expression of human blood coagulation factor IX (hFIX). Therefore, an AAV-2 genome encoding the hfIX gene was cross-packaged into capsids of AAV types 1 to 6 using efficient, large-scale technology for particle production and purification. In immunocompetent mice, the resultant vector particles expressed high hFIX levels ranging from 36% (AAV-4) to more than 2000% of normal (AAV-1, -2, and -6), which would exceed curative levels in patients with hemophilia.

Optimization of cis-acting elements for gene expression from nonviral vectors in vivo.

While naked DNA gene transfer in vivo usually results in transient gene expression, in some cases long-term transgene expression can be achieved. Here we demonstrate that cis-acting DNA elements flanking the transgene expression cassette and components in the plasmid backbone can significantly influence expression levels from nonviral vectors. To demonstrate this, we administered our most robust human coagulation factor IX (hFIX) expression cassette placed in two different plasmid backbones, into the livers of mice, by hydrodynamic transfection.

Free DNA ends are essential for concatemerization of synthetic double-stranded adeno-associated virus vector genomes transfected into mouse hepatocytes in vivo.

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in vivo. In hepatocyte nuclei, the incoming single-stranded (ss) vector genomes are converted into various forms of double-stranded (ds) genomes including extrachromosomal linear and circular monomers and concatemers, and a small portion of the vector genomes integrate into chromosomes. The mechanism of genome conversion is not well understood.

Helper-independent and AAV-ITR-independent chromosomal integration of double-stranded linear DNA vectors in mice.

Nonviral plasmid DNA is a promising vector for achieving ex vivo and in vivo gene transfer. However, transgene expression is usually transient, especially in dividing target cells due to loss of vector genomes. Here we describe the use of naked double-stranded (ds) linear DNA as a way to insert exogenous DNA sequences into chromosomes of mouse hepatocytes in vivo, without helper components such as integrase or transposase.

Epstein-Barr virus vectors provide prolonged robust factor IX expression in mice.

We demonstrate that vectors incorporating components from Epstein-Barr virus (EBV) for retention and from human genomic DNA for replication greatly enhance the level and duration of marker gene expression in dividing cultured cells. The same types of vectors were tested in vivo by high-pressure tail vein injection of naked DNA in mice, resulting in liver delivery and expression. The therapeutic gene was a human factor IX (hFIX) minigene comprising genomically derived 5', 3', and intronic sequences that provided relatively good gene expression in vivo.

Site-specific genomic integration produces therapeutic Factor IX levels in mice.

We used the integrase from phage phiC31 to integrate the human Factor IX (hFIX) gene permanently into specific sites in the mouse genome. A plasmid containing attB and an expression cassette for hFIX was delivered to the livers of mice by using high-pressure tail vein injection. When an integrase expression plasmid was co-injected, hFIX serum levels increased more than tenfold to approximately 4 microg/ml, similar to normal FIX levels, and remained stable throughout the more than eight months of the experiment.

Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo.

A major limitation of adenovirus-mediated gene therapy for inherited diseases is the instability of transgene expression in vivo, which originates at least in part from the loss of the linear, extrachromosomal vector genomes. Herein we describe the production of a gene-deleted adenovirus-transposon vector that stably maintains virus-encoded transgenes in vivo through integration into host cell chromosomes. This system utilizes a donor transposon vector that undergoes Flp-mediated recombination and excision of its therapeutic payload in the presence of the Flp and Sleeping Beauty recombinases.

RNA interference in adult mice.

RNA interference is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes. Here we show that transgene expression can be suppressed in adult mice by synthetic small interfering RNAs and by small-hairpin RNAs transcribed in vivo from DNA templates. We also show the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo.

Determinants of hepatitis C translational initiation in vitro, in cultured cells and mice.

Hepatitis C virus (HCV) is an RNA virus infecting 1 in every 40 people worldwide. Development of new therapeutics for treating HCV has been hampered by the lack of small-animal models. We have adapted existing hydrodynamic transfection methods to optimize the delivery of RNAs to the cytoplasm of mouse liver cells in vivo.