DNA/RNA heteroduplex oligonucleotide technology for regulating lymphocytes in vivo.

Manipulating lymphocyte functions with gene silencing approaches is promising for treating autoimmunity, inflammation, and cancer. Although oligonucleotide therapy has been proven to be successful in treating several conditions, efficient in vivo delivery of oligonucleotide to lymphocyte populations remains a challenge. Here, we demonstrate that intravenous injection of a heteroduplex oligonucleotide (HDO), comprised of an antisense oligonucleotide (ASO) and its complementary RNA conjugated to α-tocopherol, silences lymphocyte endogenous gene expression with higher potency, efficacy, and longer retention time than ASOs. Importantly, reduction of Itga4 by HDO ameliorates symptoms in both adoptive transfer and active experimental autoimmune encephalomyelitis models. Our findings reveal the advantages of HDO with enhanced gene knockdown effect and different delivery mechanisms compared with ASO. Thus, regulation of lymphocyte functions by HDO is a potential therapeutic option for immune-mediated diseases.

Cloning and cortical expression of rat Emx2 and adenovirus-mediated overexpression to assess its regulation of area-specific targeting of thalamocortical axons.

A goal of this study was to use recombinant adenovirus (AdV) to ectopically express Emx2 in the embryonic neocortex as a gain-of-function approach to study its role in the area-specific targeting of thalamocortical axons (TCAs), using the rat as a model. First, we cloned the cDNA for the full-length coding region of rat Emx2, a homologue of Drosophila empty spiracles. We also used this sequence to define the full-length coding region of mouse Emx2 from genomic DNA. Our analysis of Emx2 expression shows that in rat, as reported in mouse, Emx2 is expressed in high caudal to low rostral, and high medial to low lateral, gradients across the cortex throughout cortical neurogenesis, and expression is primarily restricted to progenitors in the neuroepithelium. We also carried out an analysis of the distribution of cells infected with a replication defective recombinant type 5 adenovirus (AdV) containing a CAG/LacZ expression construct, following an injection into the lateral ventricle of the cerebral hemisphere at different stages of embryonic cortical neurogenesis. AdV-infected cells are broadly distributed tangentially, but their laminar distribution is differentially restricted and reflects the temporal sequence of generation of cortical neurons. This finding indicates that the AdV predominantly infects progenitors in the ventricular zone, which leads to a preferential labeling of their immediate progeny, and infects cells that have recently become postmitotic and have yet to move far from the ventricular surface. We then show that AdV-mediated ectopic Emx2 expression results in aberrant intracortical pathfinding and areal targeting of TCAs from the dorsal lateral geniculate nucleus. These findings indicate that EMX2 imparts positional information normally associated with caudal cortical areas, such as the primary visual area, that influences the area-specific targeting of TCAs. These results are consistent with a role for EMX2 in areal specification of the neocortex as suggested by recent analyses of Emx2 null mutants.