Dataset Information

Gene delivery to intestinal epithelial cells in vitro and in vivo with recombinant adeno-associated virus types 1, 2 and 5.

ABSTRACT: Intestinal disorders such as inflammatory bowel disease (IBD) result in chronic illness requiring lifelong therapy. Our aim was to evaluate the efficacy of recombinant adeno-associated virus (AAV) vector-mediated gene delivery to intestinal epithelial cells in vitro and in vivo. Human colon epithelial cell lines and colon biopsies were transduced using AAV pseudotypes 2/1, 2/2, and 2/5 encoding green fluorescence protein (GFP). Mice were administered the same vectors through oral, enema, intraperitoneal (IP) injection and superior mesenteric artery (SMA) injection routes. Tropism and efficiency were determined by microscopy, flow cytometry, immunohistochemistry and PCR. Caco2 cells were more permissive to AAV transduction. Human colon epithelial cells in organ culture were more effectively transduced by AAV2/2. SMA injection provided the most effective means of vector gene transfer to small intestine and colonic epithelial cells in vivo. Transgene detection 80 days post AAV treatment suggests transduction of crypt progenitor cells. This study shows the feasibility of AAV-mediated intestinal gene delivery, applicable for the investigation of IBD pathogenesis and novel therapeutic options, but also revealed the need for further studies to identify more efficient pseudotypes.

SUBMITTER: Polyak S

PROVIDER: S-EPMC3896329 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Gene delivery to intestinal epithelial cells in vitro and in vivo with recombinant adeno-associated virus types 1, 2 and 5.

Polyak Steven S Mah Cathryn C Porvasnik Stacy S Herlihy John-David JD Campbell-Thompson Martha M Byrne Barry J BJ Valentine John F JF

Digestive diseases and sciences 20071013 5

Intestinal disorders such as inflammatory bowel disease (IBD) result in chronic illness requiring lifelong therapy. Our aim was to evaluate the efficacy of recombinant adeno-associated virus (AAV) vector-mediated gene delivery to intestinal epithelial cells in vitro and in vivo. Human colon epithelial cell lines and colon biopsies were transduced using AAV pseudotypes 2/1, 2/2, and 2/5 encoding green fluorescence protein (GFP). Mice were administered the same vectors through oral, enema, intrape ...[more]

PMID: 17934813

Similar Datasets

Project description:Adeno-associated viruses (AAVs) provide advantages in long-term, cardiac-specific gene expression. However, AAV serotype specificity data is lacking in experimental models relevant to cardiac electrophysiology and cardiac optogenetics. We aimed to identify the optimal AAV serotype (1, 6, or 9) in pursuit of scalable rodent and human models using genetic modifications in cardiac electrophysiology and optogenetics, in particular, as well as to elucidate the mechanism of virus uptake. In vitro syncytia of primary neonatal rat ventricular cardiomyocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were infected with AAVs 1, 6, and 9 containing the transgene for eGFP or channelrhodopsin-2 (ChR2) fused to mCherry. In vivo adult rats were intravenously injected with AAV1 and 9 containing ChR2-mCherry. Transgene expression profiles of rat and human cells in vitro revealed that AAV1 and 6 significantly outperformed AAV9. In contrast, systemic delivery of AAV9 in adult rat hearts yielded significantly higher levels of ChR2-mCherry expression and optogenetic responsiveness. We tracked the mechanism of virus uptake to purported receptor-mediators for AAV1/6 (cell surface sialic acid) and AAV9 (37/67 kDa laminin receptor, LamR). In vitro desialylation of NRVMs and hiPSC-CMs with neuraminidase (NM) significantly decreased AAV1,6-mediated gene expression, but interestingly, desialylation of hiPSC-CMs increased AAV9-mediated expression. In fact, only very high viral doses of AAV9-ChR2-mCherry, combined with NM treatment, yielded consistent optogenetic responsiveness in hiPSC-CMs. Differences between the in vitro and in vivo performance of AAV9 could be correlated to robust LamR expression in the intact heart (neonatal rat hearts as well as adult human and rat hearts), but no expression in vitro in cultured cells (primary rat cells and hiPS-CMs). The dynamic nature of LamR expression and its dependence on environmental factors was further corroborated in intact adult human ventricular tissue. The combined transgene expression and cell surface receptor data may explain the preferential efficiency of AAV1/6 in vitro and AAV9 in vivo for cardiac delivery and mechanistic knowledge of their action can help guide cardiac optogenetic efforts. More broadly, these findings are relevant to future efforts in gene therapy for cardiac electrophysiology abnormalities in vivo as well as for genetic modifications of cardiomyocytes by viral means in vitro applications such as disease modeling or high-throughput drug testing.

Project description:Recombinant adeno-associated virus vectors are an increasingly popular tool for gene delivery to the CNS because of their non-pathological nature, low immunogenicity, and ability to stably transduce dividing and non-dividing cells. One of the limitations of rAAVs is their preferential tropism for neuronal cells. Glial cells, specifically astrocytes, appear to be infected at low rates. To overcome this limitation, previous studies utilized rAAVs with astrocyte-specific promoters or assorted rAAV serotypes and pseudotypes with purported selectivity for astrocytes. Yet, the reported glial infection rates are not consistent from study to study. In the present work, we tested seven commercially available recombinant serotypes- rAAV1, 2, and 5 through 9, for their ability to transduce primary rat astrocytes [visualized via viral expression of green fluorescent protein (GFP)]. In cell cultures, rAAV6 consistently demonstrated the highest infection rates, while rAAV2 showed astrocytic transduction in some, but not all, of the tested viral batches. To verify that all rAAV constructs utilized by us were viable and effective, we confirmed high infectivity rates in retinal pigmented epithelial cells (ARPE-19), which are known to be transduced by numerous rAAV serotypes. Based on the in vitro results, we next tested the cell type tropism of rAAV6 and rAAV2 in vivo, which were both injected in the barrel cortex at approximately equal doses. Three weeks later, the brains were sectioned and immunostained for viral GFP and the neuronal marker NeuN or the astrocytic marker GFAP. We found that rAAV6 strongly and preferentially transduced astrocytes (>90% of cells in the virus-infected areas), but not neurons (∼10% infection rate). On the contrary, rAAV2 preferentially infected neurons (∼65%), but not astrocytes (∼20%). Overall, our results suggest that rAAV6 can be used as a tool for manipulating gene expression (either delivery or knockdown) in rat astrocytes in vivo.

Dataset Information

Gene delivery to intestinal epithelial cells in vitro and in vivo with recombinant adeno-associated virus types 1, 2 and 5.

Publications

Gene delivery to intestinal epithelial cells in vitro and in vivo with recombinant adeno-associated virus types 1, 2 and 5.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets