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Recombinant adeno-associated viruses (AAVs) are popular in vivo gene transfer vehicles. However, vector doses needed to achieve therapeutic effect are high and some target tissues in the central nervous system remain difficult to transduce. Gene therapy trials using AAV for the treatment of neurological disorders have seldom led to demonstrated clinical efficacy. Important contributing factors are low transduction rates and inefficient distribution of the vector. To overcome these hurdles, a variety of capsid engineering methods have been utilized to generate capsids with improved transduction properties. Here we describe an alternative approach to capsid engineering, which draws on the natural evolution of the virus and aims to yield capsids that are better suited to infect human tissues. We generated an AAV capsid to include amino acids that are conserved among natural AAV2 isolates and tested its biodistribution properties in mice and rats. Intriguingly, this novel variant, AAV-TT, demonstrates strong neurotropism in rodents and displays significantly improved distribution throughout the central nervous system as compared to AAV2. Additionally, sub-retinal injections in mice revealed markedly enhanced transduction of photoreceptor cells when compared to AAV2. Importantly, AAV-TT exceeds the distribution abilities of benchmark neurotropic serotypes AAV9 and AAVrh10 in the central nervous system of mice, and is the only virus, when administered at low dose, that is able to correct the neurological phenotype in a mouse model of mucopolysaccharidosis IIIC, a transmembrane enzyme lysosomal storage disease, which requires delivery to every cell for biochemical correction. These data represent unprecedented correction of a lysosomal transmembrane enzyme deficiency in mice and suggest that AAV-TT-based gene therapies may be suitable for treatment of human neurological diseases such as mucopolysaccharidosis IIIC, which is characterized by global neuropathology. [ABSTRACT FROM AUTHOR]

Abstract: The main plasma membrane folate transporter FT1 of Leishmania belongs to the novel FBT family which is part of the major facilitator superfamily. We have investigated the role of the 10 most conserved charged amino acids of FBTs by site directed mutagenesis. The functions of the mutated proteins were tested for their capacity to transport FA, to sensitize methotrexate resistant cells to methotrexate, for protein production, and for protein localisation. Of the 10 conserved charged amino acids that were mutated to neutral amino acids, all had effects on FT1 transport activities. Only four of the 10 initial mutants (K116L, K133L, R497L, and D529V) retained between 15% and 50% of FT1 activity. The R497 residue was shown to be involved in substrate binding. When the charged conserved residues at position 124, 134, 179, 514, 537 and 565 were changed to neutral amino acids, this led to inactive proteins but the generation of new mutants D124E, R134K, D514E and D537E regained between 20% and 50% of wild-type FT1 activity suggesting that the charge is important for protein function. The mutated protein D179E had, under our standard experimental conditions, no activity, while E565D was completely inactive. The differential activity of the mutated proteins was due either to changes in the apparent K m or V max. Mutagenesis experiments have revealed that charged amino acids were essential for FT1 stability or activity and led to a plausible model for the transport of folic acid through FT1. [Copyright &y& Elsevier]