Hh Abdul-Razak

Activation of EphA receptors mediates the recruitment of the adaptor protein Slap, contributing to the downregulation of N-methyl-D-aspartate receptors.

ABSTRACT Regulation of the activity of N-methyl-d-aspartate receptors (NMDARs) at glutamatergic synapses is essential for certain forms of synaptic plasticity underlying learning and memory and is also associated with neurotoxicity and neurodegenerative diseases. In this report, we investigate the role of Src-like adaptor protein (Slap) in NMDA receptor signaling. We present data showing that in dissociated neuronal cultures, activation of ephrin (Eph) receptors by chimeric preclustered eph-Fc ligands leads to recruitment of Slap and NMDA receptors at the sites of Eph receptor activation. Interestingly, our data suggest that prolonged activation of EphA receptors is as efficient in recruiting Slap and NMDA receptors as prolonged activation of EphB receptors. Using established heterologous systems, we examined whether Slap is an integral part of NMDA receptor signaling. Our results showed that Slap does not alter baseline activity of NMDA receptors and does not affect Src-dependent potentiation of NMDA receptor currents in Xenopus oocytes. We also demonstrate that Slap reduces excitotoxic cell death triggered by activation of NMDARs in HEK293 cells. Finally, we present evidence showing reduced levels of NMDA receptors in the presence of Slap occurring in an activity-dependent manner, suggesting that Slap is part of a mechanism that homeostatically modulates the levels of NMDA receptors.

Advances in gene therapy for muscular dystrophies.

Duchenne muscular dystrophy (DMD) is a recessive lethal inherited muscular dystrophy caused by mutations in the gene encoding dystrophin, a protein required for muscle fibre integrity. So far, many approaches have been tested from the traditional gene addition to newer advanced approaches based on manipulation of the cellular machinery either at the gene transcription, mRNA processing or translation levels. Unfortunately, despite all these efforts, no efficient treatments for DMD are currently available. In this review, we highlight the most advanced therapeutic strategies under investigation as potential DMD treatments.

Molecular Evidence of Genome Editing in a Mouse Model of Immunodeficiency

Genome editing is the introduction of directed modifications in the genome, a process boosted to therapeutic levels by designer nucleases. Building on the experience of ex vivo gene therapy for severe combined immunodeficiencies, it is likely that genome editing of haematopoietic stem/progenitor cells (HSPC) for correction of inherited blood diseases will be an early clinical application. We show molecular evidence of gene correction in a mouse model of primary immunodeficiency. In vitro experiments in DNA-dependent protein kinase catalytic subunit severe combined immunodeficiency (Prkdc scid) fibroblasts using designed zinc finger nucleases (ZFN) and a repair template demonstrated molecular and functional correction of the defect. Following transplantation of ex vivo gene-edited Prkdc scid HSPC, some of the recipient animals carried the expected genomic signature of ZFN-driven gene correction. In some primary and secondary transplant recipients we detected double-positive CD4/CD8 T-cells in thymus and single-positive T-cells in blood, but no other evidence of immune reconstitution. However, the leakiness of this model is a confounding factor for the interpretation of the possible T-cell reconstitution. Our results provide support for the feasibility of rescuing inherited blood disease by ex vivo genome editing followed by transplantation, and highlight some of the challenges.

A southern blot protocol to detect chimeric nuclease-mediated gene repair.

Gene targeting by homologous recombination at chromosomal endogenous loci has traditionally been considered a low-efficiency process. However, the effectiveness of such so-called genome surgery or genome editing has recently been drastically improved through technical developments, chiefly the use of designer nucleases like zinc-finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs) and CRISPR/Cas nucleases. These enzymes are custom designed to recognize long target sites and introduce double-strand breaks (DSBs) at specific target loci in the genome, which in turn mediate significant improvements in the frequency of homologous recombination. Here, we describe a Southern blot-based assay that allows detection of gene repair and estimation of repair frequencies in a cell population, useful in cases where the targeted modification itself cannot be detected by restriction digest. This is achieved through detection of a silent restriction site introduced alongside the desired mutation, in our particular example using integration-deficient lentiviral vectors (IDLVs) coding for ZFNs and a suitable DNA repair template.