MicroRNA-based recombinant AAV vector assembly improves efficiency of suicide gene transfer in a murine model of lymphoma.

Recent success in clinical trials with recombinant Adeno-associated virus (AAV)-based gene therapy has redirected efforts in optimizing AAV assembly and production, to improve its potency. We reasoned that inclusion of a small RNA during vector assembly, which specifically alters the phosphorylation status of the packaging cells may be beneficial. We thus employed microRNAs (miR-431, miR-636) identified by their ability to bind AAV genome and also dysregulate Mitogen-activated protein kinase (MAPK) signaling during vector production, by a global transcriptome study in producer cells. A modified vector assembly protocol incorporating a plasmid encoding these microRNAs was developed. AAV2 vectors packaged in the presence of microRNA demonstrated an improved gene transfer potency by 3.7-fold, in vitro. Furthermore, AAV6 serotype vectors encoding an inducible caspase 9 suicide gene, packaged in the presence of miR-636, showed a significant tumor regression (~2.2-fold, P < .01) in a syngeneic murine model of T-cell lymphoma. Taken together, we have demonstrated a simple but effective microRNA-based approach to improve the assembly and potency of suicide gene therapy with AAV vectors.

Optimized AAV rh.10 Vectors That Partially Evade Neutralizing Antibodies during Hepatic Gene Transfer.

Of the 12 common serotypes used for gene delivery applications, Adeno-associated virus (AAV)rh.10 serotype has shown sustained hepatic transduction and has the lowest seropositivity in humans. We have evaluated if further modifications to AAVrh.10 at its phosphodegron like regions or predicted immunogenic epitopes could improve its hepatic gene transfer and immune evasion potential. Mutant AAVrh.10 vectors were generated by site directed mutagenesis of the predicted targets. These mutant vectors were first tested for their transduction efficiency in HeLa and HEK293T cells. The optimal vector was further evaluated for their cellular uptake, entry, and intracellular trafficking by quantitative PCR and time-lapse confocal microscopy. To evaluate their potential during hepatic gene therapy, C57BL/6 mice were administered with wild-type or optimal mutant AAVrh.10 and the luciferase transgene expression was documented by serial bioluminescence imaging at 14, 30, 45, and 72 days post-gene transfer. Their hepatic transduction was further verified by a quantitative PCR analysis of AAV copy number in the liver tissue. The optimal AAVrh.10 vector was further evaluated for their immune escape potential, in animals pre-immunized with human intravenous immunoglobulin. Our results demonstrate that a modified AAVrh.10 S671A vector had enhanced cellular entry (3.6 fold), migrate rapidly to the perinuclear region (1 vs. >2 h for wild type vectors) in vitro, which further translates to modest increase in hepatic gene transfer efficiency in vivo. More importantly, the mutant AAVrh.10 vector was able to partially evade neutralizing antibodies (~27-64 fold) in pre-immunized animals. The development of an AAV vector system that can escape the circulating neutralizing antibodies in the host will substantially widen the scope of gene therapy applications in humans.

α2u-globulins mediate manipulation of host attractiveness in Toxoplasma gondii-Rattus novergicus association.

Uninfected female rats (Rattus novergicus) exhibit greater attraction to the males infected with protozoan parasite Toxoplasma gondii. This phenomenon is contrary to the aversion towards infected males observed in multitude of other host-parasite associations. In this report, we describe a proximate mechanism for this anomaly. We demonstrate that T. gondii infection enhances hepatic production and urinary excretion of α2u-globulins in rats. We further demonstrate that α2u-globulins are sufficient to recapitulate male sexual attractiveness akin to effects of the infection. This manipulation possibly results in greater horizontal transmission of this parasite between the infected male and the uninfected female. It supports the notion that in some evolutionary niches parasites can alter host sexual signaling, likely leading to an increased rate of sexual transmission.

Ezh2 mediated H3K27me3 activity facilitates somatic transition during human pluripotent reprogramming.

Factor induced reprogramming of fibroblasts is an orchestrated but inefficient process. At the epigenetic level, it results in drastic chromatin changes to erase the existing somatic "memory" and to establish the pluripotent state. Accordingly, alterations of chromatin regulators including Ezh2 influence iPSC generation. While the role of individual transcription factors in resetting the chromatin landscape during iPSC generation is increasingly evident, their engagement with chromatin modulators remains to be elucidated. In the current study, we demonstrate that histone methyl transferase activity of Ezh2 is required for mesenchymal to epithelial transition (MET) during human iPSC generation. We show that the H3K27me3 activity favors induction of pluripotency by transcriptionally targeting the TGF-ß signaling pathway. We also demonstrate that the Ezh2 negatively regulates the expression of pro-EMT miRNA's such as miR-23a locus during MET. Unique association of Ezh2 with c-Myc was required to silence the aforementioned circuitry. Collectively, our findings provide a mechanistic understanding by which Ezh2 restricts the somatic programme during early phase of cellular reprogramming and establish the importance of Ezh2 dependent H3K27me3 activity in transcriptional and miRNA modulation during human iPSC generation.

Role of calcium signaling in the transcriptional regulation of the apicoplast genome of Plasmodium falciparum.

Calcium is a universal second messenger that plays an important role in regulatory processes in eukaryotic cells. To understand calcium-dependent signaling in malaria parasites, we analyzed transcriptional responses of Plasmodium falciparum to two calcium ionophores (A23187 and ionomycin) that cause redistribution of intracellular calcium within the cytoplasm. While ionomycin induced a specific transcriptional response defined by up- or downregulation of a narrow set of genes, A23187 caused a developmental arrest in the schizont stage. In addition, we observed a dramatic decrease of mRNA levels of the transcripts encoded by the apicoplast genome during the exposure of P. falciparum to both calcium ionophores. Neither of the ionophores caused any disruptions to the DNA replication or the overall apicoplast morphology. This suggests that the mRNA downregulation reflects direct inhibition of the apicoplast gene transcription. Next, we identify a nuclear encoded protein with a calcium binding domain (EF-hand) that is localized to the apicoplast. Overexpression of this protein (termed PfACBP1) in P. falciparum cells mediates an increased resistance to the ionophores which suggests its role in calcium-dependent signaling within the apicoplast. Our data indicate that the P. falciparum apicoplast requires calcium-dependent signaling that involves a novel protein PfACBP1.

Transcriptional profiling of growth perturbations of the human malaria parasite Plasmodium falciparum.

Functions have yet to be defined for the majority of genes of Plasmodium falciparum, the agent responsible for the most serious form of human malaria. Here we report changes in P. falciparum gene expression induced by 20 compounds that inhibit growth of the schizont stage of the intraerythrocytic development cycle. In contrast with previous studies, which reported only minimal changes in response to chemically induced perturbations of P. falciparum growth, we find that approximately 59% of its coding genes display over three-fold changes in expression in response to at least one of the chemicals we tested. We use this compendium for guilt-by-association prediction of protein function using an interaction network constructed from gene co-expression, sequence homology, domain-domain and yeast two-hybrid data. The subcellular localizations of 31 of 42 proteins linked with merozoite invasion is consistent with their role in this process, a key target for malaria control. Our network may facilitate identification of novel antimalarial drugs and vaccines.