ABSTRACT
Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.
Subject(s)
CRISPR-Cas Systems , DNA Breaks , Dependovirus/genetics , Gene Editing/methods , Genetic Vectors , Virus Integration/genetics , Animals , Bacteriophage lambda/genetics , Brain , Cell Line , Chromosome Mapping , Clustered Regularly Interspaced Short Palindromic Repeats , Cochlea , Endonucleases , Gene Targeting/methods , Genetic Therapy/methods , Genome , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muscles , Neurons/virology , Targeted Gene Repair/methods , Treatment OutcomeABSTRACT
PURPOSE: Less invasive left ventricular assist device implantation became feasible with the development of smaller devices. This study evaluated a sutureless aortic anastomosis device to facilitate the implant procedure. DESCRIPTION: The novel anastomotic device deploys and anchors an acute-angled stent in the aortic wall to create a sutureless outflow graft anastomosis in the ascending aorta. Four aortic anastomoses were performed on the beating hearts of two pigs without cross-clamping or cardiopulmonary bypass. EVALUATION: The procedure was fast and simple. The time of anastomosis averaged 8.1 minutes, with merely oral instructions to the operating surgeon. The design of the stent allowed the outflow graft to be implanted with the intended angulation of 45 degrees. CONCLUSIONS: This proof-of-concept study demonstrates the feasibility and short-term success of the proposed sutureless anastomotic device. Further preclinical studies are necessary to evaluate long-term durability of the anastomosis.
Subject(s)
Aorta/surgery , Blood Vessel Prosthesis Implantation , Blood Vessel Prosthesis , Stents , Suture Techniques , Anastomosis, Surgical , Animals , Prosthesis Design , SwineABSTRACT
The ability to characterize the mechanical properties of erythrocytes is important in clinical and research contexts: to diagnose and monitor hematologic disorders, as well as to optimize the design of cardiovascular implants and blood circulating devices with respect to blood damage. However, investigation of red blood cell (RBC) properties generally involves preparatory and processing steps. Even though these impose mechanical stresses on cells, little is known about their impact on the final measurement results. In this study, we investigated the effect of centrifuging, vortexing, pipetting, and high pressures on several markers of mechanical blood damage and RBC membrane properties. Using human venous blood, we analyzed erythrocyte damage by measuring free hemoglobin, phosphatidylserine exposure by flow cytometry, RBC deformability by ektacytometry and the parameters of a complete blood count. We observed increased levels of free hemoglobin for all tested procedures. The release of hemoglobin into plasma depended significantly on the level of stress. Elevated pressures and centrifuging also altered mean cell volume (MCV) and mean corpuscular hemoglobin (MCH), suggesting changes in erythrocyte population, and membrane properties. Our results show that the effects of blood handling can significantly influence erythrocyte damage metrics. Careful quantification of this influence as well as other unwanted secondary effects should thus be included in experimental protocols and accounted for in clinical laboratories.
