In vitro functional genetic modification of canine adenovirus type 2 genome by CRISPR/Cas9.

Genetically modified oncolytic adenoviruses have been proposed as a vehicle for cancer therapy. However, several concerns, such as toxicity to normal cells and organs, lack of suitable cell surface receptors to allow viral entry to the desired cell type(s), and activation of both innate and adaptive immune systems in patients, restrict the successful clinical application of adenoviral-mediated cancer gene therapy. Successful virotherapy will require efficient transductional and transcriptional targeting to enhance therapeutic efficacy by ensuring targeted adenoviral infection, replication, and/or therapeutic transgene expression. Targeted modification of viral components, such as viral capsid, fiber knob, and the insertion of transgenes for expression, are prerequisites for the necessary transductional and transcriptional targeting of adenovirus. However, the conventional approach to modify the adenoviral genome is complex, time consuming, and expensive. It is dependent on the presence of unique restriction enzyme sites that may or may not be present in the target location. Clustered regularly interspaced short palindromic repeat (CRISPR) along with the RNA-guided nuclease Cas9 (CRISPR/Cas9) is one of the most powerful tools that has been adopted for precise genome editing in a variety of cells and organisms. However, the ability of the CRISPR/Cas9 system to precisely and efficiently make genetic modification, as well as introduce gene replacements, in adenoviral genomes, remains essentially unknown. Herein the ability of in vitro CRISPR/CAS9-mediated editing of the canine adenovirus type 2 (CAV2) genome to promote targeted modification of the viral genome was assessed. To demonstrate the feasibility of this goal, CRISPR/Cas9 has been used to successfully insert the RFP (red fluorescent protein) reporter construct into the CAV2 genome. Initial results demonstrated high efficiency and accuracy for in vitro CRISPR-mediated editing of the large CAV2 genome. Furthermore, this application was expanded, using multiple guide RNAs, to conduct gene replacement in the CAV2 genome by substituting a portion of the E3 gene with a construct designed to express a single chain antibody to canine PD-1. Thus, this work provides a significantly improved and efficient method for targeted editing of adenoviruses to generate altered and potentially therapeutic viral genomes in the shortest possible time.

The Development and Characterization of a Next-Generation Oncolytic Virus Armed with an Anti-PD-1 sdAb for Osteosarcoma Treatment In Vitro.

Osteosarcoma (OS) is a primary bone malignancy characterized by an aggressive nature, limited treatment options, low survival rate, and poor patient prognosis. Conditionally replicative adenoviruses (CRAds) armed with immune checkpoint inhibitors hold great potential for enhanced therapeutic efficacy. The present study aims to investigate the anti-tumor efficacy of CAV2-AU-M2, a CAV2-based CRAd armed with an anti-PD-1 single-domain antibody (sdAb), against OS cell lines in vitro. The infection, conditional replication, cytopathic effects, and cytotoxicity of CAV2-AU-M2 were tested in four different OS cell lines in two-dimensional (2D) and three-dimensional (3D) cell cultures. CAV2-AU-M2 showed selective replication in the OS cells and induced efficient tumor cell lysis and death. Moreover, CAV2-AU-M2 produced an anti-PD-1 sdAb that demonstrated effective binding to the PD-1 receptors. This study demonstrated the first CRAd armed with an anti-PD-1 sdAb. This combined approach of two distinct immunotherapies is intended to enhance the anti-tumor immune response in the tumor microenvironment.

miRNAs: Potential as Biomarkers and Therapeutic Targets for Cancer.

MicroRNAs (miRNAs) are single-stranded, non-coding RNA molecules that regulate gene expression post-transcriptionally by binding to messenger RNAs. miRNAs are important regulators of gene expression, and their dysregulation is implicated in many human and canine diseases. Most cancers tested to date have been shown to express altered miRNA levels, which indicates their potential importance in the oncogenic process. Based on this evidence, numerous miRNAs have been suggested as potential cancer biomarkers for both diagnosis and prognosis. miRNA-based therapies have also been tested in different cancers and have provided measurable clinical benefits to patients. In addition, understanding miRNA biogenesis and regulatory mechanisms in cancer can provide important knowledge about resistance to chemotherapies, leading to more personalized cancer treatment. In this review, we comprehensively summarized the importance of miRNA in human and canine cancer research. We discussed the current state of development and potential for the miRNA as both a diagnostic marker and a therapeutic target.

Biomechanical characteristics of 9 arthroscopic knots.

PURPOSE: To determine the optimal arthroscopic slipknot through comparison of ease of placement, loop security, knot security, and amount of suture material needed using a new suture material. METHODS: Nine commonly used arthroscopic knots (Dines, Field, Nicky, Hu, San Diego, Snyder, Tennessee slider, Triad, and Tuckahoe) were tested by use of modern suture material, FiberWire (Arthrex, Naples, FL), with the Instron materials testing machine (Instron, Norwood, MA) for ease of knot placement (forward and backward sliding), loop security, and knot security. The amount of suture material needed to create the knot was compared by use of the knot weight. Analysis of variance with Kruskal-Wallis analysis and Bonferroni correction (alpha < .01) was used to compare different knots. RESULTS: The Tennessee slider knot sustained the greatest force at failure (269 N), the greatest knot resistance (32 N), and the smallest mass (8.5 mg). The Dines was the only knot superior in all 3 knot placement categories. The Nicky held the most loop force (66 N), and the Tuckahoe had the greatest loop resistance (20 N) (P < .01 for all except mass [P < .05]). CONCLUSIONS: Our study comprehensively presents ease-of-placement and security characteristics of 9 common and new arthroscopic knots using modern FiberWire suture. The Tennessee slider knot showed superior characteristics in knot security and knot mass. The Dines knot was the most ideal knot to place. However, the surgeon will need to review the individual knot characteristics and select the knot most suited to application. CLINICAL RELEVANCE: This study analyzed 9 arthroscopic knots with modern suture material and identified those with superior characteristics.

Does perioperative systemic infection or fever increase surgical infection risks after internal fixation of femur and tibia fractures in an intensive care polytrauma unit?

BACKGROUND: We hypothesized that internal fixation procedures performed on trauma intensive care unit (ICU) patients with systemic infections, some also febrile, would be at increased risk for deep infection. METHODS: A total of 128 patients (mean age, 37.4 years; mean Injury Severity Score [ISS], 34.7) admitted to the ICU with 179 femur or tibia fractures developed systemic infections. Systemic infections included sepsis, pneumonia, urinary tract infections, abdominal infections, and wound infections remote to the fracture. Of the fractures, 33 open and 146 closed underwent 150 intramedullary and 29 plate fixation procedures. Data were gathered regarding antibiotic use, systemic infection timing in relation to the date of fixation, and whether fever (>38.2°C) was present within 24 hours of fixation. Patients were followed up for a mean of 491 days. RESULTS: Twenty-eight procedures were performed a mean of 4.7 days after the diagnosis of a systemic infection, and 151 were performed a mean of 9.3 days before the diagnosis. Forty-five procedures were performed in patients who were febrile within 24 hours. Of the 179 procedures, 10 (5.6%) developed a deep infection. Four patients' implant infection was potentially hematogenously seeded with the same organism as their systemic infection. Neither the timing of the systemic infection in relation to the fixation procedure nor the presence of fever within 24 hours of fixation, days of preoperative antibiotics, location of the fracture, type of fixation (intramedullary nail vs. plate fixation), or type of systemic infection was significantly associated with the development of an infection. The only significant risk factor for developing an orthopedic infection was an open fracture (p < 0.001). CONCLUSION: Internal fixation performed in ICU patients with fever or in close conjunction to the diagnosis of systemic infection led to a 5.6% infection rate, which compares favorably with historic infection rates for fixation of open or closed tibia and femur fractures. LEVEL OF EVIDENCE: Therapeutic, level IV.