John McC Howell

Dystrophin Expression in Muscle Following Gene Transfer with a Fully Deleted (" Gutted" ) Adenovirus Is Markedly Improved by Trans-Acting Adenoviral Gene Products

Helper-dependent adenoviruses (HDAd) are Ad vectors lacking all or most viral genes. They hold great promise for gene therapy of diseases such as Duchenne muscular dystrophy (DMD), because they are less immunogenic than E1/E3-deleted Ad (first-generation Ad or FGAd) and can carry the full-length (Fl) dystrophin (dys) cDNA (12 kb). We have compared the transgene expression of a HDAd (HDAdCMVDysFl) and a FGAd (FGAdCMV-dys) in cell culture (HeLa, C2C12 myotubes) and in the muscle of mdx mice (the mouse model for DMD). Both vectors encoded dystrophin regulated by the same cytomegalovirus (CMV) promoter. We demonstrate that the amount of dystrophin expressed was significantly higher after gene transfer with FGAdCMV-dys compared to HDAdCMVDysFl both in vitro and in vivo. However, gene transfer with HDAdCMVDysFl in the presence of a FGAd resulted in a significant increase of dystrophin expression indicating that gene products synthesized by the FGAd increase, in trans, the amount of dystrophin produced. This enhancement occurred in cell culture and after gene transfer in the muscle of mdx mice and dystrophic golden retriever (GRMD) dogs, another animal model for DMD. The E4 region of Ad is required for the enhancement, because no increase of dystrophin expression from HDAdCMVDysFl was observed in the presence of an E1/E4-deleted Ad in vitro and in vivo. The characterization of these enhancing gene products followed by their inclusion into an HDAd may be required to produce sufficient dystrophin to mitigate the pathology of DMD by HDAd-mediated gene transfer.

The spread of transgene expression at the site of gene construct injection

Seven 2‐day‐old golden retriever pups were given focal intramuscular injections of a first generation adenovirus–dystrophin minigene construct and adenovirus–β‐galactosidase construct as a 2:1 mixture into the left anterior tibial muscle. The spread of transgene expression within the anterior tibial muscle was compared with the spread of methylene blue dye after identical injection into the contralateral muscle. Transgene expression 5–7 days after intramuscular injection was shown to extend between 5.8 and 11.6 mm along the biopsied muscle length (range of biopsy lengths 11.1–12.2 mm). The level of transgene expression at 2–2.5‐mm intervals from the site of injection was significantly related to the distance from the site of injection (dystrophin, P = 0.009; β‐galactosidase, P = 0.015). The spread of methylene blue dye within the anterior tibial muscle ≤24 h after identical intramuscular injection demonstrated a similar pattern to the transgene expression, with dye staining measured between 5.5 and 8.5 mm along the muscle sample length (range of biopsy lengths 5.6–15.6 mm). The greatest transgene expression and dye staining was measured 2–2.5 mm proximal to the site of injection with a maximum of 23% of muscle fibers expressing the dystrophin transgene, 95.2% expressing the β‐galactosidase transgene, and 98% of the tissue section stained with methylene blue dye. These results suggest transgene expression after focal intramuscular injection is relatively localized around the site of injection. Further research is required to develop techniques that will provide transgene expression throughout the length and breadth of a muscle.

Is there a future for gene therapy

The requirements for successful gene therapy are stated and brief details are given of the gene therapy trials in humans which have been approved by the NIH during the years 1989-1997. An indication is given of the gene therapy trials that have been carried out in animal models of Duchenne muscular dystrophy with emphasis on the Golden Retriever dog model. Problems facing somatic gene therapy for inherited muscle diseases are predominantly the following: the extent of the spread of expression from the injection site, the duration of expression and the need for systemic delivery. Brief details of the problems are given and possible ways of overcoming the difficulties are outlined. These include the use of multiple intramuscular injections, increasing the permeability of the extracellular matrix of muscle, inducing mitosis in myoblasts, the use of ex vivo gene transfer, using modified viruses as vectors or synthesized transporter molecules, the use of mechanisms which combat the action of killer T cells, upregulation of isoforms or of alternative proteins such as utrophin for dystrophin and the use of genetic correction methods such as the use of antisense oligonucleotides. It is concluded that there is a potential future for somatic gene therapy in the inherited muscle diseases.

Direct dystrophin and reporter gene transfer into dog muscle in vivo

Bacterial β‐galactosidase cDNA was injected without lipofectin into 41 sites in dog muscle and expression was seen in 22 of them. The cDNA and lipofectin was injected into 35 similar sites and expression was seen in 21. Expression was seen in a maximum of 2.5% of muscle fibers and 23.21% of nonmuscle cells. A total of 106 muscle sites were injected with the minigene with and without lipofectin. In 4 of the 45 sites injected with the minigene without lipofectin human dystrophin was expressed around the periphery of 0.3% of the fibers. Bacterial β‐galactosidase cDNA was injected into the peritoneal cavity of 4 pups, 2 of which also received lipofectin. In all 4, expression was seen in liver, spleen, and mesenteric lymph node. In the 2 pups that received lipofectin, expression was also seen in the diaphragm, intercostal, and abdominal muscles of 1 and in the diagphragm and intercostal muscles of the other. These experiments show that human dystrophin transgene expression can be obtained in dog muscle. However, other methods will be required to increase the degree of expression before gene therapy trials can be undertaken.

773 BOVINE α-GLUCOSIDASE DEFICIENCY: A MODEL FOR ENZYME REPLACEMENT THERAPY

A bovine species has recently been described, whose pathology and genetics are those seen in Pompes disease (Neuropathol. Appl. Neurobiol. 3:45, 1977.). Further studies have proven a deficiency of lysosomal α-glucosidase and accumulation of glycogen (Aust. J. Exp. Biol. Med. Sci. 55:141, 1977.). Bovine hepatic α-glucosidase was purified to homogeneity with a sp. act.= 7 umoles/min/mg, a single band of M.W.= 107,000 by PAGE and a single subunit of M.W.= 59,000 by SDS-PAGE. The 125I-labeled enzyme was injected intravenously into a homozygous α-glucosidase deficient calf. The t½ of disappearance from the plasma compartment was 2 min. with accumulation of activity in the liver. Disappearance from liver (t½= 20 min.) was paralleled by the reappearance of activity (non-TCA precipitable) in the plasma. Less than 1% of the administered dose accumulated in muscle.The establishment of a breeding herd of heterozygotes with α-glucosidase deficiency has allowed the production of affected animals for enzyme replacement studies. Administration of purified bovine α-glucosidase indicates rapid clearance from the plasma with accumulation in liver but not muscle. This model will allow the further study of enzyme targeting via enzyme lipoprotein conjugates.

Heliotrope Alkaloids and Copper

The ingestion of toxic pyrrolizidine alkaloids by sheep may result in the development of a clinical syndrome in which distinctive damage to the liver plays an important part. The same can be said of the ingestion of copper (Cu). However the detail of the syndromes and the nature of the changes in the liver are different. In Australia the ingestion by sheep of pasture containing hepatotoxic pyrrolizidine alkaloids has resulted in outbreaks of disease known as “The Yellows” or “Haemolytic Jaundice” due to the importance of jaundice as a clinical sign (Bull et al. 1956). In these sheep the concentration of Cu in the liver was excessive and it was presumed that the damage caused by the hepatotoxic alkaloids was responsible for this. The hepatotoxic alkaloids and the Cu in the pasture were thought to be acting synergistically.

253. An AAV Vector-Mediated Gene Transfer into Canine Skeletal Muscle

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by mutations in the dystrophin gene (14 kb cDNA). An adeno-associated virus (AAV) vector-mediated gene transfer is one of attractive approaches to the treatment of DMD, but it has a limitation in insertion size up to 4.9 kb. We recently demonstrated that the AAV vector-mediated micro-dystrophin cDNA transfer could ameliorate dystrophic phenotypes in skeletal muscles of dystrophin-deficient mdx mice (Mol Ther 10: 821-828, 2004). For clinical application, it is important to examine therapeutic effects and the safety issue in larger animal models, such as dystrophic dogs. We established a colony of beagle-based canine X-linked muscular dystrophy in Japan (Exp Anim. 52: 93-97, 2003). To investigate transduction efficiency in canine skeletal muscle using an AAV vector, we injected the AAV vector encoding the LacZ gene driven by a CMV promoter (1.0-2.0 |[times]| 1013 vg/ml, 100-500 |[mu]|l/muscle) into skeletal muscles of normal dogs. |[beta]|-galactosidase (|[beta]|-gal) was expressed only in few fibers at 2 weeks after the injection, and not detected at 4 or 8 weeks after the injection. Instead, large numbers of mononuclear cells appeared around |[beta]|-gal-expressing fibers. To clarify mechanisms of low transduction and cellular infiltration in canine muscle after transfer of the AAV vector, we examined viral infectivity in vitro, cytotoxicity and immune responses of AAV vector transduction in vivo. First, we infected the AAV vector into canine primary myotubes. This in vitro study showed that the AAV vector could allow higher transgene expression in canine myotubes than in murine ones. Second, we tested whether injection of AAV particles elicit cytotoxicity or not. When a promoter-less AAV vector expressing no transgene (5 |[times]| 1012 vg/muscle) was injected into canine muscle, almost no infiltrating cells was observed in injected muscle. Third, we investigated immune responses. A lot of CD4- or CD8-positive cells were detected in clusters of infiltrating cells, together with elevated serum level of anti-|[beta]|-gal IgG. To confirm low transduction depending on immune response, dogs received daily oral administration of cyclosporine (20 mg/kg/day) from |[ndash]|5 day of the introduction of the AAV vector. Immunosuppression largely but not completely improves transduction efficiency of the AAV vector. These results suggest that AAV vector-mediated gene transfer elicits stronger immune responses in canine muscle, but immune responses against transgene products can not thoroughly explain the phenomenon. Cellular toxicity of transgene products might also participate in these infiltrations, while cytotoxicity and immunity of the AAV particles themselves can be negligible based on the result of a promoter-less AAV vectors. It is indispensable to know the molecular background of excess immune responses and cellular toxicity in canine models to establish AAV vector-mediated gene transfer in dystrophic patients.