Expansive gene transfer in the rat CNS rapidly produces amyotrophic lateral sclerosis relevant sequelae when TDP-43 is overexpressed.

Improved spread of transduction in the central nervous system (CNS) was achieved from intravenous administration of adeno-associated virus serotype-9 (AAV9) to neonatal rats. Spinal lower motor neuron transduction efficiency was estimated to be 78% using the highest vector dose tested at a 12-week interval. The widespread expression could aid studying diseases that affect both the spinal cord and brain, such as amyotrophic lateral sclerosis (ALS). The protein most relevant to neuropathology in ALS is transactive response DNA-binding protein 43 (TDP-43). When expressed in rats, human wild-type TDP-43 rapidly produced symptoms germane to ALS including paralysis of the hindlimbs and muscle wasting, and mortality over 4 weeks that did not occur in controls. The hindlimb atrophy and weakness was evidenced by assessments of rotarod, rearing, overall locomotion, muscle mass, and histology. The muscle wasting suggested denervation, but there was only 14% loss of motor neurons in the TDP-43 rats. Tissues were negative for ubiquitinated, cytoplasmic TDP-43 pathology, suggesting that altering TDP-43's nuclear function was sufficient to cause the disease state. Other relevant pathology in the rats included microgliosis and degenerating neuronal perikarya positive for phospho-neurofilament. The expression pattern encompassed the distribution of neuropathology of ALS, and could provide a rapid, relevant screening assay for TDP-43 variants and other disease-related proteins.

AAV8, 9, Rh10, Rh43 vector gene transfer in the rat brain: effects of serotype, promoter and purification method.

We compared adeno-associated virus (AAV) serotypes for expression levels of green fluorescent protein (GFP) in the adult rat hippocampus by biophotonic imaging. Preparations of AAV serotypes 8, 9, Rh10, and Rh43 incorporating cytomegalovirus (CMV) promoter-driven GFP were purified by a CsCl method. Neither AAV Rh10 nor AAV Rh43 produced greater levels of GFP than AAV8, which was used as a reference. For AAV9, there was an increase relative to AAV8. The CsCl-purified AAV8 displayed an astroglial transduction pattern in contrast to the expected neuronal expression of other AAVs. After preparing the same CMV-GFP plasmid in AAV8 with an iodixanol purification method, the expected neuronal pattern resulted. The astroglial expression with the CsCl AAV8 was probably due to relatively high levels of protein impurities. We compared the CMV promoter with the CMV/chicken beta-actin (CBA) promoter in the context of AAV8, both prepared by iodixanol, and found the CBA promoter to produce stronger GFP expression. At two doses of vectors optimized for serotype, promoter and purification, we did not observe serotype differences among AAV8, AAV9, or AAV Rh10. The purification method can therefore impact the transduction pattern as well as the results when comparing serotype strengths.