Negative impact of rAAV2 mediated expression of SOCS3 on the regeneration of adult retinal ganglion cell axons.

Intravitreal injections of recombinant ciliary neurotrophic factor (rCNTF) protect adult rat retinal ganglion cells (RGCs) after injury and stimulate regeneration, an effect enhanced by co-injection with a cAMP analogue (CPT-cAMP). This effect is partly mediated by PKA and associated signaling pathways, but CPT-cAMP also moderates upregulation of suppressor of cytokine signaling (SOCS) pathways after rCNTF injection, which will also enhance the responsiveness of RGCs to this and perhaps other cytokines. We now report that intravitreal injections of CPT-cAMP do not potentiate RGC axonal regeneration when CNTF is expressed via an adeno-associated viral vector (rAAV2), and concomitantly we show that increases in retinal SOCS mRNA expression are less when CNTF is delivered using the vector. We also directly tested the impact of elevated SOCS3 expression on the survival and regeneration of injured adult RGCs by injecting a bicistronic rAAV2-SOCS3-GFP vector into the vitreous of eyes in rats with a peripheral nerve graft sutured onto the cut optic nerve. Overexpression of SOCS3 resulted in an overall reduction in axonal regrowth and almost complete regeneration failure of RGCs transduced with the rAAV2-SOCS3-GFP vector. Furthermore, rAAV2-mediated expression of SOCS3 abolished the normally neurotrophic effects elicited by intravitreal rCNTF injections. In summary, CNTF delivery to the retina using viral vectors may be more effective than bolus rCNTF injections because the gene therapy approach has a less pronounced effect on neuron-intrinsic SOCS repressor pathways. Our new gain of function data using rAAV2-SOCS3-GFP demonstrate the negative impact of enhanced SOCS3 expression on the regenerative potential of mature CNS neurons.

Cellular tropism and transduction properties of seven adeno-associated viral vector serotypes in adult retina after intravitreal injection.

Recombinant adeno-associated virus (rAAV) vectors are increasingly being used as tools for gene therapy, and clinical trials have begun in patients with genetically linked retinal disorders. Intravitreal injection is optimal for the transduction of retinal ganglion cells (RGCs), although complete selectivity has not been achieved. There may also be advantages in using intravitreal approaches for the transduction of photoreceptors. Here we compared the cellular tropism and transduction efficiency of rAAV2/1, -2/2, -2/3, -2/4, -2/5, -2/6 and -2/8 in adult rat retina after intravitreal injection. Each vector encoded green fluorescent protein (GFP), and the number, laminar distribution and morphology of transduced GFP(+) cells were determined using fluorescent microscopy. Assessment of transduced cell phenotype was based on cell morphology and immunohistochemistry. rAAV2/2 and rAAV2/6 transduced the greatest number of cells, whereas rAAV2/5 and rAAV2/8 were least efficient. Most vectors primarily transduced RGCs; however, rAAV2/6 had a more diverse tropism profile, with 46% identified as amacrine or bipolar cells, 23% as RGCs and 22% as Müller cells. Müller cells were also frequently transduced by rAAV2/4. The highest photoreceptor transduction was seen after intravitreal rAAV2/3 injection. These data facilitate the design and selection of rAAV vectors to target specific retinal cells, potentially leading to an improved gene therapy for various human retinal pathologies.

Fate of adult neural precursors grafted to adult cortex monitored with a Y-chromosome marker.

Freshly dissected and dissociated subventricular zone cells from adult male mice were transplanted into the right cerebral cortex of adult female mice. Host mice were perfused 3 or 7 weeks later. To detect donor male cells, paraffin sections (10 microm) were hybridized in situ with a Y-chromosome-specific probe. Some hybridized sections were also immunostained with antibodies against glial fibrillary acidic protein (GFAP), NeuN or beta-tubulin. Grafted cells were identified in 80% of host female brains; they were found in both grey and white matter and were located close to the initial injection site. Double-labelling studies showed that, of the surviving cells containing hybridized male nuclei, 5-10% expressed a glial (GFAP) marker and 6-7% expressed neuronal (NeuN or beta-tubulin) markers.

The use of enzyme-linked immunosorbent assay to determine the appearance, specificity and immunoglobulin class of antibodies to the structural proteins of influenza A virus in mice.

The immune response of mice to sublethal infection with influenza A virus was investigated by enzyme-linked immunosorbent assay, haemagglutination-inhibition and neutralization tests. The specificity and immunoglobulin class of antibodies were determined by competition enzyme-linked immunosorbent assay. A rise in geometric mean antibody levels was observed between 4 and 16 days by haemagglutination-inhibition, and between 8 and 32 days by neutralization. A positive correlation was found between the geometric mean antibody responses measured by the three assay systems, but there was no consistent relationship between the titres of individual sera by each assay method. More antibodies to the strain-specific determinants of haemagglutinin were observed 8 days after infection than antibodies to the common determinants, but by day 16 there were equal amounts of antibody to strain-specific and common determinants of haemagglutinin and to neuraminidase. Antibodies to matrix and nucleoprotein antigens were not detected until 64 days after infection. The antibody response 8 days after infection was composed of IgM and IgA immunoglobulin classes, but IgG predominated from 16 days after infection.