Secreted Particle Information Transfer (SPIT) - A Cellular Platform for Genetic Engineering.

A multitude of tools now exist that allow us to precisely manipulate the human genome in a myriad of different ways. However, successful delivery of these tools to the cells of human patients remains a major barrier to their clinical implementation. Here we introduce a new cellular approach for genetic engineering, ecreted article nformation ransfer (SPIT) that utilizes human cells as delivery vectors for genetic engineering.

Enhancement of erythropoietic output by Cas9-mediated insertion of a natural variant in haematopoietic stem and progenitor cells.

Some gene polymorphisms can lead to monogenic diseases, whereas other polymorphisms may confer beneficial traits. A well-characterized example is congenital erythrocytosis-the non-pathogenic hyper-production of red blood cells-that is caused by a truncated erythropoietin receptor. Here we show that Cas9-mediated genome editing in CD34 human haematopoietic stem and progenitor cells (HSPCs) can recreate the truncated form of the erythropoietin receptor, leading to substantial increases in erythropoietic output.

Secreted Particle Information Transfer (SPIT) - A Cellular Platform for Genetic Engineering.

A multitude of tools now exist that allow us to precisely manipulate the human genome in a myriad of different ways. However, successful delivery of these tools to the cells of human patients remains a major barrier to their clinical implementation. Here we introduce a new cellular approach for genetic engineering, ecreted article nformation ransfer (SPIT) that utilizes human cells as delivery vectors for genetic engineering.

A Case Report of Plasmacytoma in a 28-Year-Old Patient: Bridging the Age Gap in a Rare Presentation.

Plasmacytoma, an uncommon malignancy originating from plasma cells, predominantly manifests in the elderly demographic. However, its incidence among young adults remains infrequent. Herein, we present a case involving a 28-year-old young adult diagnosed with solitary bone plasmacytoma.

Demographic and socioeconomic disparities in receipt of ophthalmology consultation for facial trauma.

Objective: Functional outcomes following facial and ocular trauma are time-sensitive and require prompt evaluation to minimise long-term vision loss, yet few studies have systematically evaluated disparities in the management of these cases. This study investigates whether a patient's race/ethnicity, primary language, insurance status, gender or age affects receipt of ophthalmology consultation for facial trauma.

Methods And Analysis: This study was a retrospective cohort analysis of patients from the Elmhurst City Hospital Trauma Registry in Queens, New York who were seen for facial trauma including open globe injuries and orbital fractures between January 2014 and May 2016.

Population-based sequencing of reveals how current population dynamics are shaped by past epidemics.

Transmission is a driver of tuberculosis (TB) epidemics in high-burden regions, with assumed negligible impact in low-burden areas. However, we still lack a full characterization of transmission dynamics in settings with similar and different burdens. Genomic epidemiology can greatly help to quantify transmission, but the lack of whole genome sequencing population-based studies has hampered its application.

Fungal Necrotizing Scleritis After Intravitreal Injection Therapy.

Purpose: To report a case of infectious necrotizing scleritis secondary to Aspergillus terreus after intravitreal injection therapy.

Methods: This is a case report with literature review.

Results: A 98-year-old woman receiving intravitreal aflibercept injections for neovascular age-related macular degeneration in the left eye presented with severe pain, redness, and purulent discharge at the injection site.

Clinically relevant gene editing in hematopoietic stem cells for the treatment of pyruvate kinase deficiency.

Pyruvate kinase deficiency (PKD), an autosomal-recessive disorder, is the main cause of chronic non-spherocytic hemolytic anemia. PKD is caused by mutations in the pyruvate kinase, liver and red blood cell ( ) gene, which encodes for the erythroid pyruvate kinase protein (RPK). RPK is implicated in the last step of anaerobic glycolysis in red blood cells (RBCs), responsible for the maintenance of normal erythrocyte ATP levels.

An Uncharacteristic Presentation of Evans Syndrome Following Treatment With Dupilumab.

Evans syndrome is a rare autoimmune disorder where patients develop autoimmune hemolytic anemia (AIHA), immune thrombocytopenia (ITP), and less commonly immune neutropenia. Patients typically present with fatigue, pallor, jaundice, petechiae, or epistaxis. A 27-year-old man with a history of atopic dermatitis for which he recently began treatment with dupilumab presented to the emergency department with a headache and blurry vision.

Gene replacement of α-globin with β-globin restores hemoglobin balance in β-thalassemia-derived hematopoietic stem and progenitor cells.

β-Thalassemia pathology is due not only to loss of β-globin (HBB), but also to erythrotoxic accumulation and aggregation of the β-globin-binding partner, α-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in β-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize β-globin:α-globin messenger RNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bilineage hematopoietic reconstitution in mice, establishing proof of concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing β-thalassemia.

Cas9-AAV6 gene correction of beta-globin in autologous HSCs improves sickle cell disease erythropoiesis in mice.

CRISPR/Cas9-mediated beta-globin (HBB) gene correction of sickle cell disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a recent paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated previously. Here, we use a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation.

The TRACE-Seq method tracks recombination alleles and identifies clonal reconstitution dynamics of gene targeted human hematopoietic stem cells.

Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs.

Bleeding Isolated Gastric Varices as a Rare Presentation of Pancreatic Neuroendocrine Tumor: Case Report and Literature Review.

Isolated gastric varices (IGV) are gastric varices in the absence of esophageal varices. IGV is one of the rare causes of gastrointestinal bleeding and an uncommon complication of pancreatic neuroendocrine tumors (PNET). The gold standard diagnostic tool of varices is esophagogastroduodenoscopy (EGD).

Cas9-AAV6-engineered human mesenchymal stromal cells improved cutaneous wound healing in diabetic mice.

Human mesenchymal stromal cells (hMSCs) are a promising source for engineered cell-based therapies in which genetic engineering could enhance therapeutic efficacy and install novel cellular functions. Here, we describe an optimized Cas9-AAV6-based genome editing tool platform for site-specific mutagenesis and integration of up to more than 3 kilobases of exogenous DNA in the genome of hMSCs derived from the bone marrow, adipose tissue, and umbilical cord blood without altering their ex vivo characteristics. We generate safe harbor-integrated lines of engineered hMSCs and show that engineered luciferase-expressing hMSCs are transiently active in vivo in wound beds of db/db mice.

High-resolution mapping of tuberculosis transmission: Whole genome sequencing and phylogenetic modelling of a cohort from Valencia Region, Spain.

Background: Whole genome sequencing provides better delineation of transmission clusters in Mycobacterium tuberculosis than traditional methods. However, its ability to reveal individual transmission links within clusters is limited. Here, we used a 2-step approach based on Bayesian transmission reconstruction to (1) identify likely index and missing cases, (2) determine risk factors associated with transmitters, and (3) estimate when transmission happened.

Highly efficient editing of the β-globin gene in patient-derived hematopoietic stem and progenitor cells to treat sickle cell disease.

Sickle cell disease (SCD) is a monogenic disorder that affects millions worldwide. Allogeneic hematopoietic stem cell transplantation is the only available cure. Here, we demonstrate the use of CRISPR/Cas9 and a short single-stranded oligonucleotide template to correct the sickle mutation in the β-globin gene in hematopoietic stem and progenitor cells (HSPCs) from peripheral blood or bone marrow of patients with SCD, with 24.

CRISPR/Cas9 Genome Engineering in Engraftable Human Brain-Derived Neural Stem Cells.

Human neural stem cells (NSCs) offer therapeutic potential for neurodegenerative diseases, such as inherited monogenic nervous system disorders, and neural injuries. Gene editing in NSCs (GE-NSCs) could enhance their therapeutic potential. We show that NSCs are amenable to gene targeting at multiple loci using Cas9 mRNA with synthetic chemically modified guide RNAs along with DNA donor templates.

Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination.

Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for in vitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery.

Identification of preexisting adaptive immunity to Cas9 proteins in humans.

The CRISPR-Cas9 system is a powerful tool for genome editing, which allows the precise modification of specific DNA sequences. Many efforts are underway to use the CRISPR-Cas9 system to therapeutically correct human genetic diseases. The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes.

Missing Cases of Herpes Simplex Virus (HSV) Infection of the Central Nervous System When the Reller Criteria Are Applied for HSV PCR Testing: a Multicenter Study.

Previous studies suggested that herpes simplex virus (HSV) PCR testing can be safely deferred in patients with normal cerebrospinal fluid (CSF) white blood cell (WBC) counts and protein levels as long as they are older than 2 years of age and are not immunocompromised, the so-called Reller criteria. In this multicenter study, we retrospectively assessed the validity of these screening criteria in our setting. A total of 4,404 CSF specimens submitted for HSV PCR testing to the respective microbiology laboratories at the participating hospitals between 2012 and 2018 were included.