Biallelic, Selectable, Knock-in Targeting of CCR5 via CRISPR-Cas9 Mediated Homology Directed Repair Inhibits HIV-1 Replication.

Transplanting HIV-1 positive patients with hematopoietic stem cells homozygous for a 32 bp deletion in the chemokine receptor type 5 (CCR5) gene resulted in a loss of detectable HIV-1, suggesting genetically disrupting CCR5 is a promising approach for HIV-1 cure. Targeting the CCR5-locus with CRISPR-Cas9 was shown to decrease the amount of CCR5 expression and HIV-1 susceptibility in vitro as well as in vivo. Still, only the individuals homozygous for the CCR5-Δ32 frameshift mutation confer complete resistance to HIV-1 infection. In this study we introduce a mechanism to target CCR5 and efficiently select for cells with biallelic frameshift insertion, using CRISPR-Cas9 mediated homology directed repair (HDR). We hypothesized that cells harboring two different selectable markers (double positive), each in one allele of the CCR5 locus, would carry a frameshift mutation in both alleles, lack CCR5 expression and resist HIV-1 infection. Inducing double-stranded breaks (DSB) via CRISPR-Cas9 leads to HDR and integration of a donor plasmid. Double-positive cells were selected via fluorescence-activated cell sorting (FACS), and CCR5 was analyzed genetically, phenotypically, and functionally. Targeted and selected populations showed a very high frequency of mutations and a drastic reduction in CCR5 surface expression. Most importantly, double-positive cells displayed potent inhibition to HIV-1 infection. Taken together, we show that targeting cells via CRISPR-Cas9 mediated HDR enables efficient selection of mutant cells that are deficient for CCR5 and highly resistant to HIV-1 infection.

Ultrasound Elastography Predicts Thyroid Nodule Volume Reduction Rate After Percutaneous Ethanol Ablation.

OBJECTIVES: To evaluate the safety and efficacy of percutaneous ethanol ablation (PEA) on indeterminate thyroid nodules (Bethesda III and IV) based on ultrasound (US) elastography by assessing the volume reduction rate (VRR), relative reduction in size, resolution of compressive symptoms, and post-procedural complications. MATERIALS AND METHODS: This is a retrospective cohort study of all thyroid nodules treated with PEA by a single surgeon at a North American tertiary referral center. Study variables included demographics, nodule characteristics, Bethesda classification, US elastography, presence of compressive symptoms, thyroid function, and post-procedural complications. Relative volume reductions and VRR were calculated at 3- and 6-month follow-ups. RESULTS: Thirty-four thyroid nodules were evaluated in 22 patients. All thyroid nodules underwent a fine needle aspiration prior to PEA. After 6 months, 45% of all thyroid nodules exhibited a VRR of ≥50%. A significant VRR was achieved in the soft thyroid nodules at 6 months (42.15% ± 31), compared to the stiff nodules with 30.92% ± 91.53, P < .05. Post-PEA thyroid stimulating hormone levels did not significantly change after the procedure. Compressive symptoms resolved in all 5 patients who reported it. One patient developed transient vocal cord paresis that resolved in 3 months. DISCUSSION: To the best of our knowledge, this is the largest series of PEA for thyroid nodules in North America. Ultrasound elastography is a useful adjunct in predicting the success of PEA for nonmalignant thyroid nodules. Percutaneous ethanol ablation is both a safe and effective alternative to surgery for relief of compressive symptoms in select patients.