RNA-guided gene editing of the murine gammaherpesvirus 68 genome reduces infectious virus production.

Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) are cancer-causing viruses that establish lifelong infections in humans. Gene editing using the Cas9-guideRNA (gRNA) CRISPR system has been applied to decrease the latent load of EBV in human Burkitt lymphoma cells. Validating the efficacy of Cas9-gRNA system in eradicating infection in vivo without off-target effects to the host genome will require animal model systems. To this end, we evaluated a series of gRNAs against individual genes and functional genomic elements of murine gammaherpesvirus 68 (MHV68) that are both conserved with KSHV and important for the establishment of latency or reactivation from latency in the host. gRNA sequences against ORF50, ORF72 and ORF73 led to insertion, deletion and substitution mutations in these target regions of the genome in cell culture. Murine NIH3T3 fibroblast cells that stably express Cas9 and gRNAs to ORF50 were most resistant to replication upon de novo infection. Latent murine A20 B cell lines that stably express Cas9 and gRNAs against MHV68 were reduced in their reactivation by approximately 50%, regardless of the viral gene target. Lastly, co-transfection of HEK293T cells with the vector expressing the Cas9-MHV68 gRNA components along with the viral genome provided a rapid read-out of gene editing and biological impact. Combinatorial, multiplex MHV68 gRNA transfections in HEK293T cells led to near complete ablation of infectious particle production. Our findings indicate that Cas9-gRNA editing of the murine gammaherpesvirus genome has a deleterious impact on productive replication in three independent infection systems.

Truncated stathmin-2 is a marker of TDP-43 pathology in frontotemporal dementia.

No treatment for frontotemporal dementia (FTD), the second most common type of early-onset dementia, is available, but therapeutics are being investigated to target the 2 main proteins associated with FTD pathological subtypes: TDP-43 (FTLD-TDP) and tau (FTLD-tau). Testing potential therapies in clinical trials is hampered by our inability to distinguish between patients with FTLD-TDP and FTLD-tau. Therefore, we evaluated truncated stathmin-2 (STMN2) as a proxy of TDP-43 pathology, given the reports that TDP-43 dysfunction causes truncated STMN2 accumulation. Truncated STMN2 accumulated in human induced pluripotent stem cell-derived neurons depleted of TDP-43, but not in those with pathogenic TARDBP mutations in the absence of TDP-43 aggregation or loss of nuclear protein. In RNA-Seq analyses of human brain samples from the NYGC ALS cohort, truncated STMN2 RNA was confined to tissues and disease subtypes marked by TDP-43 inclusions. Last, we validated that truncated STMN2 RNA was elevated in the frontal cortex of a cohort of patients with FTLD-TDP but not in controls or patients with progressive supranuclear palsy, a type of FTLD-tau. Further, in patients with FTLD-TDP, we observed significant associations of truncated STMN2 RNA with phosphorylated TDP-43 levels and an earlier age of disease onset. Overall, our data uncovered truncated STMN2 as a marker for TDP-43 dysfunction in FTD.

The application of poorly crystalline silicotitanate in production of 225Ac.

Actinium-225 (225Ac) can be produced from a Thorium-229/Radium-225 (229Th/225Ra) generator, from high/low energy proton irradiated natural Thorium or Radium-226 target. Titanium based ion exchanger were evaluated for purification of 225Ac. Poorly crystalline silicotitanate (PCST) ion exchanger had high selectivity for Ba, Ag and Th. 225Ac was received with trace amounts of 227Ac, 227Th and 223Ra, and the solution was used to evaluate the retention of the isotopes on PCST ion exchanger. Over 90% of the 225Ac was recovered from PCST, and the radiopurity was >99% (calculated based on 225Ac, 227Th, and 223Ra). The capacity of the PCST inorganic ion exchange for Barium and 232Th was determined to be 24.19 mg/mL for Barium and 5.05 mg/mL for Thorium. PCST ion exchanger could separate 225Ac from isotopes of Ra and Th, and the process represents an interesting one step separation that could be used in an 225Ac generator from 225Ra and/or 229Th. Capacity studies indicated PCST could be used to separate 225Ac produced on small 226Ra targets (0.3-1 g), but PCST did not have a high enough capacity for production scale Th targets (50-100 g).