A Rapid, Functional sgRNA Screening Method for Generating Murine RET and NTRK1 Fusion Oncogenes.

CRISPR/Cas9 gene editing technology is an indispensable and powerful tool in the field of cancer biology. To conduct successful CRISPR-based experiments, it is crucial that sgRNAs generate their designed alterations. Here, we describe a simple and efficient sgRNA screening method for validating sgRNAs that generate oncogenic gene rearrangements. We used IL3-independence in Ba/F3 cells as an assay to identify sgRNA pairs that generate fusion oncogenes involving the Ret and Ntrk1 tyrosine kinases. We confirmed these rearrangements with PCR or RT-PCR as well as sequencing. Ba/F3 cells harboring Ret or Ntrk1 rearrangements acquired sensitivity to RET and TRK inhibitors, respectively. Adenoviruses encoding Cas9 and sgRNAs that catalyze the Kif5b-Ret and Trim24-Ret rearrangements were intratracheally instilled into mice and yielded lung adenocarcinomas. A cell line (TR.1) was established from a Trim24-Ret positive tumor that exhibited high in vitro sensitivity to RET-specific TKIs. Moreover, orthotopic transplantation of TR.1 cells into the left lung yielded well-defined tumors that shrank in response to LOXO-292 treatment. The method offers an efficient means to validate sgRNAs that successfully target their intended loci for the generation of novel murine oncogene-driven tumor models.

Novel approaches in the treatment of Hansen's disease (Leprosy): a case series of multidrug therapy of monthly rifampin, moxifloxacin, and minocycline (RMM) in the United States.

The World Health Organization (WHO) recommends multidrug therapy (MDT) for the treatment of paucibacillary and multibacillary forms of leprosy, also known as Hansen's disease (HD). MDT combinations of dapsone, rifampin, and clofazimine have reduced the prevalence of the disease but are not without adverse effects impacting regimen adherence. Hence, an urgent need exists to consider alternative MDT regimens with an improved safety profile that promotes treatment adherence. Herein, we described a case series of 10 patients with HD (nine patients with multibacillary leprosy and one with pure neural leprosy) treated with monthly rifampin, moxifloxacin, and minocycline (RMM). The United States National Hansen's Disease Program (NHDP) diagnosed and treated patients across US institutions. All patients received a regimen of 12-24 months of RMM. We reviewed the clinical outcomes, adherence, rate of completion, and adverse events of patients treated with monthly RMM from January 2019 to August 2022. Nine patients had multibacillary leprosy, with some having type-2 reactions. One patient had pure neural leprosy with a reversal reaction. In this case series, we identified that all patients completed the RMM regimen without treatment interruptions. None of the patients experienced any skin hyperpigmentation or any significant side effects. All patients tolerated the monthly RMM regimen with rapid improvement of skin lesions and without logistic hurdles. Based on previous clinical evidence and the results of this case series, the NHDP and other programs should consider the RMM regimen as first-line therapy.