The RRE-Rev Module Has No Effect on the Packaging Efficiency of Cas9 and Gag Proteins into NanoMEDIC Virus-like Particles.

Delivery of ribonucleoprotein complexes of Cas9 nuclease and guide RNA into target cells with virus-like particles (VLP) is one of the novel methods of genome editing and is suitable for gene therapy of human diseases in the future. The efficiency of genome editing with VLPs depends on the Cas9 packaging into VLPs, the process mediated by the viral Gag protein. To improve the packaging of Cas9 into NanoMEDIC VLPs, plasmid constructs for Cas9 and Gag expression were modified by adding the HIV Rev response element (RRE), which was expected to increase the nuclear export of RRE-containing transcripts into the cytosol via the Rev accessory protein, as described for a Vpr-Cas9-based VLP system. The Cas9 and Gag protein levels in cell lysates were found to increase upon cotransfection with either the Rev-expressing plasmid or the empty control plasmid. The effect was independent of the presence of RRE in the transcript. Moreover, AP21967-induced dimerization of FRB and FKBP12, but not plasmid modification with RRE and/or cotransfection with the Rev-expressing plasmid, was shown to play the major role in Cas9 packaging into NanoMEDIC VLPs. The data indicated that it is impractical to use the RRE-Rev module to enhance the packaging of Cas9 nuclease into VLPs.

Efficient Editing of the CXCR4 Locus Using Cas9 Ribonucleoprotein Complexes Stabilized with Polyglutamic Acid.

Gene editing using the CRISPR/Cas9 system provides new opportunities to treat human diseases. Approaches aimed at increasing the efficiency of genome editing are therefore important to develop. To increase the level of editing of the CXCR4 locus, which is a target for gene therapy of HIV infection, the Cas9 protein was modified by introducing additional NLS signals and ribonucleoprotein complexes of Cas9 and guide RNA were stabilized with poly-L-glutamic acid. The approach allowed a 1.8-fold increase in the level of CXCR4 knockout in the CEM/R5 T cell line and a 2-fold increase in the level of knock-in of the HIV-1 fusion peptide inhibitor MT-C34 in primary CD4+ T lymphocytes.

[What Are the Realities of Prescription and Control of Antithrombotic Therapy in Atrial Fibrillation in Outpatient Practice?].

We analyzed documentation of 648 outpatients with nonvalvular atrial fibrillation receiving ambulatory care in 3 cities in Russia (Moscow, Krasnodar, and Bryansk). Frequency of use of any anticoagulant in patients with AF and high risk of stroke and systemic embolism was low (30.9% overall, novel oral anticoagulants--5.7%). But portions of patients who according to documents received antiaggregants or no antithrombotic drugs at all were high (53.6 and 13.4%, respectively). Among patients receiving warfarin only 19.6% checked international normalized ratio (INR) every month while 75% did it once in 3 months or rarer or did not control this parameter at all. Among patients in whom INR control was sufficiently regular only in 44% percentage of time in the therapeutic range exceeded 60%. Thus persistent effective anticoagulation was achieved only in 12.6% of warfarin treated outpatients.

[What Are the Realities of Prescription and Control of Antithrombotic Therapy in Atrial Fibrillation in Outpatient Practice?]

We analyzed documentation of 648 outpatients with nonvalvular atrial fibrillation receiving ambulatory care in 3 cities in Russia (Moscow, Krasnodar, and Bryansk). Frequency of use of any anticoagulant in patients with AF and high risk of stroke and systemic embolism was low (30.9% overall, novel oral anticoagulants - 5.7%). But portions of patients who according to documents received antiaggregants or no antithrombotic drugs at all were high (53.6 and 13.4%, respectively). Among patients receiving warfarin only 19.6% checked international normalized ratio (INR) every month while 75% did it once in 3 months or rarer or did not control this parameter at all. Among patients in whom INR control was sufficiently regular only in 44% percentage of time in the therapeutic range exceeded 60%. Thus persistent effective anticoagulation was achieved only in 12.6% of warfarin treated outpatients.

[What physicians think and know about antithrombotic therapy in atrial fibrillation].

We conducted an anonymous survey among 382 physicians (58% internists, 42% cardiologists) in order to obtain information on their opinion on various aspects of antithrombotic therapy in atrial fibrillation. The survey revealed low level of awareness about algorithms of stratification of risks of stroke, systemic embolism, and bleeding. Reported rates of clinical use of recommended antithrombotic agents were: warfarin--30, aspirin monotherapy--19, dabigatran--10, rivaroxaban--8, and combination of aspirin and clopidogrel--8%. Rate of use of drugs without sufficient evidence base in AF was 25%. When asked to designate antithrombotic drug of choice 85% of physicians indicated warfarin and 12%--novel anticoagulants (NOAC). The following factors were considered as limiting wide application of NOAC: high cost (59%), lack of data on these drugs (14%), and impossibility to control safety of their administration (9%).