Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto-or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.
ObjectivesNeoadjuvant chemotherapy may be considered for women with epithelial ovarian cancer who have poor performance status or a disease burden not amenable to primary cytoreductive surgery. Overlap exists between indications for neoadjuvant chemotherapy and known risk factors for venous thromboembolism, including impaired mobility, increasing age, and advanced malignancy. The objective of this study was to determine the rate of venous thromboembolism among women receiving neoadjuvant chemotherapy for epithelial ovarian cancer.MethodsA multi-institutional, observational study of patients receiving neoadjuvant chemotherapy for primary epithelial ovarian, fallopian tube, or peritoneal cancer was conducted. Primary outcome was rate of venous thromboembolism during neoadjuvant chemotherapy. Secondary outcomes included rates of venous thromboembolism at other stages of treatment (diagnosis, following interval debulking surgery, during adjuvant chemotherapy, or during treatment for recurrence) and associations between occurrence of venous thromboembolism during neoadjuvant chemotherapy, subject characteristics, and interval debulking outcomes. Venous thromboembolism was defined as deep vein thrombosis in the upper or lower extremities or in association with peripherally inserted central catheters or ports, pulmonary embolism, or concurrent deep vein thrombosis and pulmonary embolism. Both symptomatic and asymptomatic venous thromboembolism were reported.ResultsA total of 230 patients receiving neoadjuvant chemotherapy were included; 63 (27%) patients overall experienced a venous thromboembolism. The primary outcome of venous thromboembolism during neoadjuvant chemotherapy occurred in 16 (7.7%) patients. Of the remaining venous thromboembolism events, 22 were at diagnosis (9.6%), six post-operatively (3%), five during adjuvant chemotherapy (3%), and 14 during treatment for recurrence (12%). Patients experiencing a venous thromboembolism during neoadjuvant chemotherapy had a longer mean time to interval debulking and were less likely to undergo optimal cytoreduction (50% vs 80.2%, p=0.02).ConclusionsPatients with advanced ovarian cancer are at high risk for venous thromboembolism while receiving neoadjuvant chemotherapy. Consideration of thromboprophylaxis may be warranted.
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