Recombinant AAV vectors containing the foot and mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra neurons.

Recombinant adeno-associated viruses (rAAVs) are promising vectors for gene therapy since they efficiently and stably transduce a variety of tissues of immunocompetent animals. The major disadvantage of rAAVs is their limited capacity to package foreign DNA (< or =5 kb). Often, co-expression of two or more genes from a single viral vector is desirable to achieve maximal therapeutic efficacy or to track transduced cells in vivo by suitable reporter genes. The internal ribosome entry site (IRES) sequence of encephalomyocarditis virus has been widely used to construct bicistronic viral vectors. However, the IRES is rather long and IRES-mediated translation can be relatively inefficient when compared with cap-dependent translation. As an alternative to the IRES for in vivo gene expression, we studied the 16 amino-acid long 2A peptide of foot and mouth disease virus (FMDV). The 2A peptide mediates the primary cis-'cleavage' of the FMDV polyprotein in a cascade of processing events that ultimately generate the mature FMDV proteins. We have generated several different rAAV genomes in which two coding regions are fused in-frame via the FMDV 2A sequence. We show that FMDV 2A efficiently mediates the generation of the expected cleavage products from the artificial fusion proteins in cells. Furthermore, we find that both EGFP and alpha- synuclein are expressed at substantially higher levels from 2A vectors than from the corresponding IRES-based vectors, while SOD-1 is expressed at comparable or slightly higher levels. Finally, we demonstrate for the first time, that the 2A sequence results in effective bicistronic gene expression in vivo after injection of 2A-dependent rAAVs into the rat substantia nigra. We conclude that 2A-containing rAAVs may represent an attractive alternative to IRES-dependent vectors for ex vivo and in vivo gene expression and gene therapy.

Influence of promoter and WHV post-transcriptional regulatory element on AAV-mediated transgene expression in the rat brain.

Recombinant adeno-associated viruses (rAAVs) can transduce several tissues, including the brain. However, in brain the duration of gene expression in different areas is variable, which has been ascribed to viral (CMV) promoter silencing in some regions over time. We have compared expression of enhanced green fluorescent protein (EGFP) in the nigrostriatal pathway of rats mediated by rAAVs containing the CMV or platelet-derived growth factor-beta chain (PDGF-beta) promoter. In addition, we studied the effects of the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) on transgene expression in vivo. The rAAV vectors containing the neuron-specific PDGF-beta chain promoter transduced significantly more dopaminergic neurons than titer-matched vectors carrying the CMV promoter. Moreover, the WPRE further increased EGFP expression, and a rAAV vector incorporating both the PDGF-beta chain promoter and the WPRE resulted in efficient EGFP expression in dopaminergic neurons and their projections in the striatum for at least 41 weeks after virus injection. Our results emphasize the importance of a strong tissue-specific promoter in achieving optimal transgene expression, not only in long-term but also in short-term studies where viral titers may be limiting. Furthermore, they suggest that incorporation of the WPRE into rAAVs, and possibly other types of vectors, is useful to enhance transgene expression in vivo.

Increased motoneuron survival and improved neuromuscular function in transgenic ALS mice after intraspinal injection of an adeno-associated virus encoding Bcl-2.

Mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) underlie some familial cases of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by loss of cortical, brainstem and spinal motoneurons. Transgenic mice over- expressing a mutated form of human SOD1 containing a Gly-->Ala substitution at position 93 (SOD1(G93A)) develop a severe, progressive motoneuron disease. We investigated the potential of recombinant adeno-associated virus (rAAV) to transfer neuroprotective molecules in this animal ALS model. Initial experiments showed that injection of an rAAV vector encoding green fluorescent protein unilaterally into the lumbar spinal cord of wild-type mice leads to expression of the reporter gene in 34.7 +/- 5.2% of the motoneurons surrounding the injection site. Intraspinal injection of an rAAV encoding the anti-apoptotic protein bcl-2 in SOD1 (G93A) mice resulted in sustained bcl-2 expression in motoneurons and significantly increased the number of surviving motoneurons at the end-stage of disease. Moreover, the compound muscle action potential amplitude elicited by nerve stimulation and recorded by electromyographic measurements was higher in the rAAV-bcl-2-treated group than in controls. Local bcl-2 expression in spinal motoneurons delayed the appearance of signs of motor deficiency but was not sufficient to prolong the survival of SOD1 (G93A) mice. To our know-ledge, this study describes the first successful transduction and protection of spinal motoneurons by direct gene transfer in a model of progressive motoneuron disease. Our results support the use of AAVs for the delivery of protective genes to spinal cord moto-neurons as a possible way to enhance motoneuron survival and repair.

Adenoviral and adeno-associated viral transfer of genes to the peripheral nervous system.

Targeted expression of foreign genes to the peripheral nervous system is interesting for many applications, including gene therapy of neuromuscular diseases, neuroanatomical studies, and elucidation of mechanisms of axonal flow. Here we describe a microneurosurgical technique for injection of replication-defective viral vectors into dorsal root ganglia (DRG). Adenovirus- and adeno-associated virus-based vectors with transcriptional competence for DRG neurons led to expression of the gene of interest throughout the first neuron of the sensory system, from the distal portions of the respective sensory nerve to the ipsilateral nucleus gracilis and cuneatus, which contains the synapses to the spinothalamic tracts. Use of Rag-1 ablated mice, which lack all B and T lymphocytes, allowed for sustained expression for periods exceeding 100 days. In immunocompetent mice, long-term (52 days) expression was achieved with similar efficiency by using adeno-associated viral vectors. DRG injection was vastly superior to intraneural injection into the sciatic nerve, which mainly transduced Schwann cells in the vicinity of the site of inoculation site but only inefficiently transduced nerve fibers, whereas i.m. injection did not lead to any significant expression of the reporter gene in nerve fibers. The versatile and efficient transduction of genes of interest should enable a wide variety of functional studies of peripheral nervous system pathophysiology.

Antioxidant and cytoprotective properties of D-tagatose in cultured murine hepatocytes.

D-Tagatose is a zero-energy producing ketohexose that is a powerful cytoprotective agent against chemically induced cell injury. To further explore the underlying mechanisms of cytoprotection, we investigated the effects of D-tagatose on both the generation of superoxide anion radicals and the consequences of oxidative stress driven by prooxidant compounds in intact cells. Primary cultures of hepatocytes derived from male C57BL/6 mice were exposed to the redox cycling drug nitrofurantoin (NFT). Lethal cell injury induced by 300 microM NFT was completely prevented by high concentrations (20 mM) of D-tagatose, whereas equimolar concentrations of glucose, mannitol, or xylose were ineffective. The extent of NFT-induced intracellular superoxide anion radical formation was not altered by D-tagatose, indicating that the ketohexose did not inhibit the reductive bioactivation of NFT. However, the NFT-induced decline of the intracellular GSH content was largely prevented by D-tagatose. The sugar also afforded complete protection against NFT toxicity in hepatocytes that had been chemically depleted of GSH. Furthermore, the ketohexose fully protected from increases in both membrane lipid peroxidation and protein carbonyl formation. In addition, D-tagatose completely prevented oxidative cell injury inflicted by toxic iron overload with ferric nitrilotriacetate (100 microM). In contrast, D-tagatose did not protect against lethal cell injury induced by tert-butyl hydroperoxide, a prooxidant which acts by hydroxyl radical-independent mechanisms and which is partitioned in the lipid bilayer. These results indicate that D-tagatose, which is a weak iron chelator, can antagonize the iron-dependent toxic consequences of intracellular oxidative stress in hepatocytes. The antioxidant properties of D-tagatose may result from sequestering the redox-active iron, thereby protecting more critical targets from the damaging potential of hydroxyl radical.