Restoring RUNX1 deficiency in RUNX1 familial platelet disorder by inhibiting its degradation.

RUNX1 familial platelet disorder (RUNX1-FPD) is an autosomal dominant disorder caused by a monoallelic mutation of RUNX1, initially resulting in approximately half-normal RUNX1 activity. Clinical features include thrombocytopenia, platelet functional defects, and a predisposition to leukemia. RUNX1 is rapidly degraded through the ubiquitin-proteasome pathway.

High-Throughput Screening Uncovers Novel Botulinum Neurotoxin Inhibitor Chemotypes.

Botulism is caused by potent and specific bacterial neurotoxins that infect host neurons and block neurotransmitter release. Treatment for botulism is limited to administration of an antitoxin within a short time window, before the toxin enters neurons. Alternatively, current botulism drug development targets the toxin light chain, which is a zinc-dependent metalloprotease that is delivered into neurons and mediates long-term pathology.

High Efficiency CRISPR/Cas9-mediated Gene Editing in Primary Human T-cells Using Mutant Adenoviral E4orf6/E1b55k "Helper" Proteins.

Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components-Cas9, guide RNAs and recombination templates-to primary cells rendered proficient for homology-directed repair. Here, we demonstrate an electroporation/transduction codelivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with adeno-associated virus (AAV) vectors expressing guide RNAs and recombination templates. By transiently enhancing target cell permissiveness to AAV transduction and gene editing efficiency, this novel approach promotes efficient gene disruption and/or gene targeting at multiple loci in primary human T-cells, illustrating its broad potential for application in translational gene editing.

pEVL: A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly(A) Sequences.

Increasing demand for large-scale synthesis of in vitro transcribed (IVT) mRNA is being driven by the increasing use of mRNA for transient gene expression in cell engineering and therapeutic applications. An important determinant of IVT mRNA potency is the 3' polyadenosine (poly(A)) tail, the length of which correlates with translational efficiency. However, present methods for generation of IVT mRNA rely on templates derived from circular plasmids or PCR products, in which homopolymeric tracts are unstable, thus limiting encoded poly(A) tail lengths to ~120 base pairs (bp).

A high-throughput-compatible FRET-based platform for identification and characterization of botulinum neurotoxin light chain modulators.

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin.

Identification of clinically viable quinolinol inhibitors of botulinum neurotoxin A light chain.

Botulinum neurotoxins (BoNT) are the most potent toxins known and a significant bioterrorist threat. Few small molecule compounds have been identified that are active in cell-based or animal models, potentially due to toxin enzyme plasticity. Here we screened commercially available quinolinols, as well as synthesized hydroxyquinolines.