dVaccinia virus (VACV) continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer since its use for the eradication of smallpox. However, the current method of editing the VACV genome is not efficient. Here, we demonstrate that the CRISPR-Cas9 system can be used to edit the VACV genome rapidly and efficiently. Additionally, a set of 8,964 computationally designed unique guide RNAs (gRNAs) targeting all VACV genes will be valuable for the study of VACV gene functions. Since the eradication of smallpox, vaccinia virus (VACV) has been developed as a vector for vaccines against infectious diseases and immunotherapies for cancer (1-4), including oncolytic virotherapies (5-8). The renewed interest in VACV has driven a number of vaccine and therapeutic candidates to clinical trials, showing especially encouraging results for cancer treatment (5, 7). To improve VACV as a vector for vaccine or cancer therapy, a flexible system is required to delete viral genes or arm the VACV with therapeutic genes. Such a system would also expedite discoveries in cell biology, such as dissection of the signaling pathways used by VACV for its actin-based motility (9). Several strategies have been developed to construct VACV vectors (10-12); the current method for modification of VACV is based on homologous recombination in mammalian cells, but only 1% to 5% of the recombinant plaques contain the inserted DNA (13).The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system is a natural microbial immune mechanism against invading viruses and other genetic elements (14-16). The CRISPR-Cas9 system, consisting of the RNA-guided Cas9 endonuclease (from Streptococcus pyogenes), a single guide RNA (sgRNA), and the trans-activating CRISPR RNA (tracrRNA), has been adapted for genome editing in eukaryotic cells (17,18). The system has been used successfully for efficient generation of genetically modified cells and animal models (17,(19)(20)(21). Recently, the genomes of adenovirus and type I herpes simplex virus were edited using the gRNA-guided Cas9 system (22). We hypothesized that the CRISPR-Cas9 system could specifically generate doublestrand breaks (DSBs) in the target DNA sites of VACV, increasing the efficiency of editing VACV genomes and constructing new VACV vectors expressing therapeutic genes.Given that VACV replicates in the cytoplasm of infected cells, the Cas9 gene without the nuclear localization signal (NLS) was cloned into the pST1374 vector (Fig. 1A). The expression of Cas9 was confirmed (Fig. 1A). Given the important role of the N1L gene in virulence and in regulating the host immune response to VACV (23-25), we chose this as an example to validate the application of CRISPR-Cas9 for editing VACV. Two individual sgRNAs targeting the VACV N1L (LO24) gene were cloned into this guide RNA (gRNA) cloning vector (Fig. 1B) and designated gRNA N1 and N2. To prove that the CRISPR-Cas9 system could improve the efficiency of constructing a VACV vector expressing a therapeuti...

The COVID-19 pandemic has led to unprecedented demands on healthcare with many requiring intubation. Tracheostomy insertion has often been delayed and the enduring effects of this on voice, swallow, and airway outcomes in COVID-19 tracheostomy patients are unknown. The aim of this study was to prospectively assess these outcomes in this patient cohort following hospital discharge.Methods: All COVID-19 patients who had undergone tracheostomy insertion, and were subsequently decannulated, were identified at our institution and followed up 2 months post-discharge. Patient-reported (PROMS) and clinician-reported outcome measures, endoscopic examination, and spirometry were used to assess voice, swallow, and airway outcomes.Results: Forty-one patients were included in the study with a mean age of 56 years and male:female ratio of 28:13. Average duration of endotracheal intubation was 24 days and 63.4% of tracheostomies were performed at day 21 to 35 of intubation. 53.7% had an abnormal GRBAS score and 30% reported abnormal swallow on EAT-10 questionnaire. 81.1% had normal endoscopic examination of the larynx, however, positive endoscopic findings correlated with the patient self-reported VHI-10 (P = .036) and EAT-10 scores (P = .027). 22.5% had spirometric evidence of fixed upper airway obstruction using the Expiratory-Disproportion Index (EDI) and Spearman correlation analysis showed a positive trend between abnormal endoscopic findings and EDI scores over 50 (P < .0001). Conclusion:The preliminary results of this study reveal a high incidence of laryngeal injury among patients who underwent intubation and tracheostomy insertion during the COVID-19 pandemic. As these patients continue to be followed up, the evolution of these complications will be studied.

In the United Kingdom, patients with a peritonsillar abscess are usually managed as inpatients. However, studies in other countries have shown successful management of these patients as outpatients. We designed an evidence based protocol for the management of patients with a peritonsillar abscess. Diagnosis was by aspiration of pus. Initial treatment was with analgesia, 1.2 g co-amoxiclav and 4 mg dexamethasone intravenously. Data were collected prospectively over 11 months on the management of all patients who had been confirmed by aspiration of pus to have a peritonsillar abscess. Four of the 46 patients (9%) treated as per the protocol required admission, two immediately and two later on. Of the 44 patients initially treated as outpatients, 41 said they were happy to have been treated as outpatients and not admitted to hospital (93%). Patients with a peritonsillar abscess can be managed successfully as outpatients with a high degree of patient satisfaction.

BackgroundLocal recurrence and remote metastasis are major challenges to overcome in order to improve the survival of patients with cancer after surgery. Oncolytic viruses are a particularly attractive option for prevention of postsurgical disease as they offer a non-toxic treatment option that can directly target residual tumor deposits and beneficially modulate the systemic immune environment that is suppressed post surgery and allows residual disease escape from control. Here, we report that a novelVaccinia virus(VV), VVΔTKΔN1L (with deletion of both thymidine kinase (TK) and N1L genes) armed with interleukin 12 (IL-12), can prolong postoperative survival when used as a neoadjuvant treatment in different murine and hamster surgical models of cancer.MethodsA tumor-targeted replicating VV with deletion of TK gene and N1L gene (VVΔTKΔN1L) was created. This virus was armed rationally with IL-12. The effect of VVΔTKΔN1L and VVΔTKΔN1L-IL12 on modulation of the tumor microenvironment and induction of tumor-specific immunity as well the feasibility and safety as a neoadjuvant agent for preventing recurrence and metastasis after surgery were assessed in several clinically relevant models.ResultsVVΔTKΔN1L can significantly prolong postoperative survival when used as a neoadjuvant treatment in three different surgery-induced metastatic models of cancer. Efficacy was critically dependent on elevation of circulating natural killer cells that was achieved by virus-induced cytokine production from cells infected with N1L-deleted, but not N1L-intact VV. This effect was further enhanced by arming VVΔTKΔN1L with IL-12, a potent antitumor cytokine. Five daily treatments with VVΔTKΔN1L-IL12 before surgery dramatically improved postsurgical survival. VVΔTKΔN1L armed with human IL-12 completely prevented tumor recurrence in surgical models of head and neck cancer in Syrian hamsters.ConclusionsThese data provide a proof of concept for translation of the regime into clinical trials. VVΔTKΔN1L-IL12 is a promising agent for use as an adjuvant to surgical treatment of solid tumors.