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Metabotropic glutamate 2/3 (mGlu2/3) receptors are of considerable interest owing to their role in modulating glutamate transmission via presynaptic, postsynaptic and glial mechanisms. As part of our ongoing efforts to identify novel ligands for these receptors, we have discovered (1S,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid; (LY3020371), a potent and selective orthosteric mGlu2/3 receptor antagonist. In this account, we characterize the effects of LY3020371 in membranes and cells expressing human recombinant mGlu receptor subtypes as well as in native rodent and human brain tissue preparations, providing important translational information for this molecule. In membranes from cells expressing recombinant human mGlu2 and mGlu3 receptor subtypes, LY3020371.HCl competitively displaced binding of the mGlu2/3 agonist ligand [3H]-459477 with high affinity (hmGlu2 Ki = 5.26 nM; hmGlu3 Ki = 2.50 nM). In cells expressing hmGlu2 receptors, LY3020371.HCl potently blocked mGlu2/3 agonist (DCG-IV)-inhibited, forskolin-stimulated cAMP formation (IC50 = 16.2 nM), an effect that was similarly observed in hmGlu3-expressing cells (IC50 = 6.21 nM). Evaluation of LY3020371 in cells expressing the other human mGlu receptor subtypes revealed high mGlu2/3 receptor selectivity. In rat native tissue assays, LY3020371 demonstrated effective displacement of [3H]-459477 from frontal cortical membranes (Ki = 33 nM), and functional antagonist activity in cortical synaptosomes measuring both the reversal of agonist-suppressed second messenger production (IC50 = 29 nM) and agonist-inhibited, K+-evoked glutamate release (IC50 = 86 nM). Antagonism was fully recapitulated in both primary cultured cortical neurons where LY3020371 blocked agonist-suppressed spontaneous Ca2+ oscillations (IC50 = 34 nM) and in an intact hippocampal slice preparation (IC50 = 46 nM). Functional antagonist activity was similarly demonstrated in synaptosomes prepared from epileptic human cortical or hippocampal tissues, suggesting a translation of the mGlu2/3 antagonist pharmacology from rat to human. Intravenous dosing of LY3020371 in rats led to cerebrospinal fluid drug levels that are expected to effectively block mGlu2/3 receptors in vivo. Taken together, these results establish LY3020371 as an important new pharmacological tool for studying mGlu2/3 receptors in vitro and in vivo. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

Adeno-associated virus 2 (AAV), a non-pathogenic human parvovirus, is gaining attention as a vector for its potential use in human gene therapy. However, few studies have examined the safety and the efficacy of this vector system in vivo. We report here that recombinant AAV vectors, when directly injected intravenously in mice, accumulated predominantly in liver cells, suggesting that AAV may possess in vivo organ-tropism for liver. The transduced lacZ reporter gene was expressed in hepatocytes in the liver and, at the level examined, did not appear to induce any detectable cytotoxic T lymphocyte response against βGal. AAV-mediated transduction of murine hematopoietic progenitor cells ex vivo followed by transplantation into lethally irradiated syngeneic mice also revealed high-efficiency gene transfer into progeny cells without any observable cytotoxicity or deleterious effect. The transduced reporter gene sequences were also expressed in mice in vivo. The AAV-based vectors may thus prove useful as a potentially safe alternative to the more commonly used retrovirus- and adenovirus-based vector systems.

The adeno-associated virus 2 (AAV) genome is a single-stranded DNA which contains the inverted terminal repeats (ITRs) of 145 nucleotides. The terminal 125 nucleotides of each ITR form palindromic hairpin structures that serve as primers for AAV DNA replication. These hairpin structures also play a crucial role in the integration, as well as the rescue, of the proviral genome from latently-infected cells, or from the recombinant AAV plasmids. However, the ITRs also contain an additional domain, designated the D-sequence, a 20-nucleotide stretch that is not involved in the formation of hairpins. In order to examine the role of the D-sequence in viral DNA rescue and replication, a number of recombinant AAV plasmids were constructed which contained deletions/substitutions in different regions of the ITRs. The results presented here reveal the existence of additional sequences, other than the hairpin structures, which serve as primers for AAV DNA replication. The results also show that whereas the hairpin structures are sufficient for excision and replication of the viral DNA, the D-sequence is crucial for the high efficiency of rescue and replication of the AAV genome. f2