Time Course of Transgene Expression After Intrastriatal Pseudotyped rAAV2/1, rAAV2/2, rAAV2/5, and rAAV2/8 Transduction in the Rat

Abstract

In vivo recombinant adeno-associated viral vector (rAAV)-mediated transduction of various tissues including brain has been characterized by slow onset and gradual increase in gene expression before reaching stable long-term protein levels. The early time course of transgene expression has not been quantified using newly available rAAV capsid serotypes. In this experiment, the onset of expression of green fluorescent protein (GFP) after intrastriatal injection of rAAV2-based pseudotyped vectors (rAAV1, rAAV5, and rAAV8 capsids) was quantified. Native GFP fluorescence displayed a delayed onset of expression of at least 7 days for all the pseudotyped rAAV vectors. However, GFP immunohistochemical staining revealed significant transgene expression by 4 days after transduction for all serotypes and stable GFP(+) neuronal populations mediated by all serotypes within 14 days post transduction at the latest. rAAV2/1 and rAAV2/2 displayed no time-dependent increase of GFP(+) striatal neurons; reaching maximal striatal cell GFP(+) counts at 4 days after injection. All serotypes displayed peak transgene expression by 4 weeks post injection where native GFP(+) neurons were equal to immunostained striatal GFP(+) neurons. The inflammatory response to these rAAV vectors was present up to 4 weeks after transduction but was not apparent 9 months post injection. Thus, rAAV-mediated transgene expression begins earlier than previously thought.

Evaluation of the specificity and sensitivity of ferritin as an MRI reporter gene in the mouse brain using lentiviral and adeno-associated viral vectors

“…Our findings are in accordance with the results of several groups that have found that rAAV injections do not induce astrogliosis or infiltration of microglia into the brain [35][36][37] or to a very low extent. 38 The better performance of AAVs in terms of background contrast generation might be explained by the fact that AAV particles are not enveloped, and can therefore withstand more extensive concentration and purification procedures resulting in higher titer and purity. Owing to the higher expression levels combined with the reduced background contrast, AAV-mediated MRI reporter gene delivery to the rodent brain resulted in a better distinction between the contrast generated by the vector encoding for FerrH from a control vector injection (eGFP) and hence a better contrast to background.…”

Efficient transduction of non-human primate motor neurons after intramuscular delivery of recombinant AAV serotype 6

“…These findings are encouraging considering that intraparenchymal injections of therapeutically-relevant doses of rAAV have been shown to induce innate and adaptive immune responses in rodents and non-human primates. [18][19][20] Such immune responses lead to the elimination of transgeneexpressing cells and/or silencing of the transgene, and may account for the poor translation of gene therapy approaches observed in preclinical and clinical studies. 21 Indeed, eradication of rAAV2/2-transduced hepatocytes by cell-mediated immunity has been observed in a Phase I clinical trial for hemophilia.…”

Imaging Intracellular Ca²⁺ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

“…Only C57BL/6 mice of ~8 weeks old were used throughout this study. It has been also shown that viral expression of GFP in striatal neurons and astrocytes in rats could be detected 4 days post injection, reached a plateau by 2 -4 weeks post injection, and then remained stable for 9 months post injection 21 . The time course of cytoGCaMP3 expression has not been precisely determined here, but at least two weeks for virus expression are recommended.…”