Corrective GUSB transfer to the canine mucopolysaccharidosis VII brain.

Severe deficiency in lysosomal ß-glucuronidase (ß-glu) enzymatic activity results in mucopolysaccharidosis (MPS) VII, an orphan disease with symptoms often appearing in early childhood. Symptoms are variable, but many patients have multiple organ disorders including neurological defects. At the cellular level, deficiency in ß-glu activity leads to abnormal accumulation of glycosaminoglycans (GAGs), and secondary accumulation of GM2 and GM3 gangliosides, which have been linked to neuroinflammation. There have been encouraging gene transfer studies in the MPS VII mouse brain, but this is the first study attempting the correction of the >200-fold larger and challenging canine MPS VII brain. Here, the efficacy of a helper-dependent (HD) canine adenovirus (CAV-2) vector harboring a human GUSB expression cassette (HD-RIGIE) in the MPS VII dog brain was tested. Vector genomes, ß-glu activity, GAG content, lysosome morphology and neuropathology were analyzed and quantified. Our data demonstrated that CAV-2 vectors preferentially transduced neurons and axonal retrograde transport from the injection site to efferent regions was efficient. HD-RIGIE injections, associated with mild and transient immunosuppression, corrected neuropathology in injected and noninjected structures throughout the cerebrum. These data support the clinical evaluation of HD CAV-2 vectors to treat the neurological defects associated with MPS VII and possibly other neuropathic lysosomal storage diseases.

Long-term in vivo transduction of neurons throughout the rat CNS using novel helper-dependent CAV-2 vectors.

Numerous genetic and environmental causes, variable pathophysiologies, and the blood-brain barrier create a formidable challenge for the study and treatment of neurodegenerative diseases affecting the central nervous system. Although there are many intracellular strategies to address neurodegeneration, for example, which transgene to use, one fundamental criterion for the long-term survival of neurons may be their genetic modification. Here, we describe the generation and in vivo efficacy of helper-dependent canine adenovirus (CAV-2) vectors that preferentially transduced neurons and efficiently trafficked via axonal retrograde transport. We used a flexible strategy and the synergy between Cre/loxP and nonlethal packaging-defective helper vectors to generate high titer helper-dependent vector stocks. One year after striatal injections in the rat brain, we found stable, high-level expression in striatal neurons, ~50% of the dopaminergic neurons of the substantia nigra, and the cholinergic neurons in the basal nuclei of Meynert. Due to the intrinsic properties of helper-dependent CAV-2 vectors (27-kb cloning capacity; low preexisting, innate, and induced immunogenicity; retrograde transport; and long-term transgene expression), they will aid fundamental and applied studies in neurobiology. Moreover, helper-dependent CAV-2 vectors may be clinically relevant for the treatment of many neurodegenerative diseases.

High in vivo production of a model monoclonal antibody on adenoviral gene transfer.

The therapeutic potential of monoclonal antibodies (MAbs) for treating a variety of severe or life-threatening diseases is high. Although intravenous infusion appears to be the simplest and most obvious mode of administration, it is not applicable in many long-term treatments. It might, however, be advantageously replaced by gene/cell therapies, rendering treatments cost-effective and eliminating the short- and long-term side effects associated with injection of massive doses of antibodies. Grafting of ex vivo genetically modified cells of various types has already been used for in vivo production and systemic delivery of MAbs in mice. However, although sustained for long periods of time, serum levels of ectopic MAbs were low. We show here that in vivo administration to mice of a first-generation adenoviral vector expressing a model MAb also permits achievement of the same goal, but with 100 to 200 times better efficiency that in any other case of gene transfer described thus far. We also investigated for possible anti-idiotypic response against the ectopic MAb. None was detected in the animals expressing the lowest levels of ectopic MAb production; a response was detected among the highest producers. In the latter case, however, the response was low and could not exert any significant neutralizing activity. In conclusion, our work indicates that high levels of circulating ectopic MAb can be obtained on direct in vivo gene transfer without inducing an anti-idiotypic response sufficiently robust to exert a neutralizing effect. This observation is encouraging in the perspective of clinical applications of this technology.