IntroductionFanconi anemia (FA) is a recessive syndrome characterized by BM failure, congenital anomalies, and a predisposition to malignancy. 1 In vitro myeloid and erythroid colony growth of BM and peripheral blood cells from FA patients is decreased, suggesting the contribution of an intrinsic cellular defect to the BM failure. 2,3 FA cells have a defect in DNA repair that leads to spontaneous chromosomal breakage and increased sensitivity to DNA bifunctional crosslinking agents such as mitomycin C and diepoxybutane. 4 Whereas the precise biochemical function of most FA proteins and the link between defective DNA repair and BM failure remain incompletely understood, human and murine knockout FA cells display G 2 phase arrest, increased sensitivity to oxidative damage, defective p53 induction, and increased apoptosis. 1,5 FA can be classified into 14 complementation groups. A loss of function in any one of these 14 genes, including FANCA, FANCB, FANCC, FANCD1 (BRCA2), FANCD2, FANCE, FANCF, FANCG, FANCL, FANCI, FANCJ, FANCM, FANCN, and FANCP (SLX4), causes the disease phenotype. 6,7 Expression of these cDNAs in cells from patients with FA in vitro corrects all cell-intrinsic defects, including BM progenitor growth, in vitro. 8,9 Gene-transfer studies have shown that correction of defects in mice with gene-targeted deficiency of FA proteins is feasible and corrects a significant engraftment defect-and in some mouse models provides a selective advantage in vivo. 10,11 Two clinical gene-therapy trials involving a total of 6 FA patients have been reported. 12,13 In both studies, the harvest of CD34 ϩ hematopoietic stem and progenitor cells (HSCs) from the BM or mobilized peripheral blood yielded lower than expected cell numbers and compromised in vitro expansion, resulting in a reduced number of cells available for gene transfer and autologous reinfusion. Therefore, whereas gene transfer per se is no longer a limitation to the therapeutic effectiveness of this approach, there are significant deficiencies in the number of autologous FA HSCs that can be collected and used in somatic gene-therapy trials.The unlimited proliferative capacity of iPSCs is particularly attractive with regard to regenerative therapies and disease models in FA. Successful differentiation of corrected iPSCs into transplantable HSCs would allow the generation of unlimited numbers of these cells and pretransplantation molecular characterization of gene-corrected cells. 14 Several recent studies have highlighted the utility of patient-specific iPSCs for in vitro disease modeling. 15,16 With regard to FA, knockdown of FANCA and FANCD2 in embryonic stem cells (ESCs) leads to reduced hemogenic potential after differentiation, suggesting that FA-deficient human pluripotent stem cells may be amenable to in vitro disease modeling. 17 Raya et al recently reported a failure of 4 FA-A and 2 FA-D2 patient samples to undergo direct reprogramming, concluding that restoration of the FA pathway is a prerequisite for iPSC generation from somatic cells of FA ...

Ischemic stroke and factors modifying ischemic stroke responses, such as social isolation, contribute to long-term disability worldwide. Several studies demonstrated that the aberrant levels of microRNAs contribute to ischemic stroke injury. In prior studies, we established that miR-141-3p increases after ischemic stroke and post-stroke isolation. Herein, we explored two different anti-miR oligonucleotides; peptide nucleic acid (PNAs) and phosphorothioates (PS) for ischemic stroke therapy. We used US FDA approved biocompatible poly (lactic-co-glycolic acid) (PLGA)-based nanoparticle formulations for delivery. The PNA and PS anti-miRs were encapsulated in PLGA nanoparticles by double emulsion solvent evaporation technique. All the formulated nanoparticles showed uniform morphology, size, distribution, and surface charge density. Nanoparticles also exhibited a controlled nucleic acid release profile for 48 h. Further, we performed in vivo studies in the mouse model of ischemic stroke. Ischemic stroke was induced by transient (60 min) occlusion of middle cerebral artery occlusion followed by a reperfusion for 48 or 72 h. We assessed the blood-brain barrier permeability of PLGA NPs containing fluorophore (TAMRA) anti-miR probe after systemic delivery. Confocal imaging shows uptake of fluorophore tagged anti-miR in the brain parenchyma. Next, we evaluated the therapeutic efficacy after systemic delivery of nanoparticles containing PNA and PS anti-miR-141-3p in mice after stroke. Post-treatment differentially reduced both miR-141-3p levels in brain tissue and infarct injury. We noted PNA-based anti-miR showed superior efficacy compared to PS-based anti-miR. Herein, we successfully established that nanoparticles encapsulating PNA or PS-based anti-miRs-141-3p probes could be used as a potential treatment for ischemic stroke.

Function of the mammalian olfactory system depends on specialized olfactory sensory neurons (OSNs) that each express only one allele ("monoallelic") of one odorant receptor (OR) gene ("monogenic"). The lysine-specific demethylase-1 (LSD1) protein removes activating H3K4 or silencing H3K9 methylation marks in a variety of developmental contexts, and is thought to be important for proper OR regulation. Most of the focus in the field has been on a potential "activating" function for LSD1; e.g., in the demethylation of H3K9 associated with the expressed OR allele. Here we show that depletion of LSD1 in an immortalized olfactory-placode-derived cell line (OP6) results in multigenic and multiallelic OR transcription per cell, while not seemingly disrupting the ability of these cells to activate new OR genes during clonal expansion. These results are consistent with LSD1 having a role in silencing additional OR alleles, as opposed to being required for the activation of OR alleles, within the OP6 cellular context.