dbGuide: a database of functionally validated guide RNAs for genome editing in human and mouse cells.

With the technology's accessibility and ease of use, CRISPR has been employed widely in many different organisms and experimental settings. As a result, thousands of publications have used CRISPR to make specific genetic perturbations, establishing in itself a resource of validated guide RNA sequences. While numerous computational tools to assist in the design and identification of candidate guide RNAs exist, these are still just at best predictions and generally, researchers inevitably will test multiple sequences for functional activity. Here, we present dbGuide (https://sgrnascorer.cancer.gov/dbguide), a database of functionally validated guide RNA sequences for CRISPR/Cas9-based knockout in human and mouse. Our database not only contains computationally determined candidate guide RNA sequences, but of even greater value, over 4000 sequences which have been functionally validated either through direct amplicon sequencing or manual curation of literature from over 1000 publications. Finally, our established framework will allow for continual addition of newly published and experimentally validated guide RNA sequences for CRISPR/Cas9-based knockout as well as incorporation of sequences from different gene editing systems, additional species and other types of site-specific functionalities such as base editing, gene activation, repression and epigenetic modification.

Targeted Mutation of NGN3 Gene Disrupts Pancreatic Endocrine Cell Development in Pigs.

The domestic pig is an attractive model for biomedical research because of similarities in anatomy and physiology to humans. However, key gaps remain in our understanding of the role of developmental genes in pig, limiting its full potential. In this publication, the role of NEUROGENIN 3 (NGN3), a transcription factor involved in endocrine pancreas development has been investigated by CRISPR/Cas9 gene ablation. Precomplexed Cas9 ribonucleoproteins targeting NGN3 were injected into in vivo derived porcine embryos, and transferred into surrogate females. On day 60 of pregnancy, nine fetuses were collected for genotypic and phenotypic analysis. One of the piglets was identified as an in-frame biallelic knockout (Δ2/Δ2), which showed a loss of putative NGN3-downstream target genes: NEUROD1 and PAX4, as well as insulin, glucagon, somatostatin and pancreatic polypeptide-Y. Fibroblasts from this fetus were used in somatic cell nuclear transfer to generate clonal animals to qualify the effect of mutation on embryonic lethality. Three live piglets were born, received colostrum and suckled normally, but experienced extreme weight loss over a 24 to 36-hour period requiring humane euthanasia. Expression of pancreatic endocrine hormones: insulin, glucagon, and somatostatin were lost. The data support a critical role of NGN3 in porcine endocrine pancreas development.

Neurogenin 3 Expressing Cells in the Human Exocrine Pancreas Have the Capacity for Endocrine Cell Fate.

Neurogenin 3 (NGN3) is necessary and sufficient for endocrine differentiation during pancreatic development and is expressed by a population of progenitor cells that give rise exclusively to hormone-secreting cells within islets. NGN3 protein can be detected in the adult rodent pancreas only following certain types of injury, when it is transiently expressed by exocrine cells undergoing reprogramming to an endocrine cell fate. Here, NGN3 protein can be detected in 2% of acinar and duct cells in living biopsies of histologically normal adult human pancreata and 10% in cadaveric biopsies of organ donor pancreata. The percentage and total number of NGN3+ cells increase during culture without evidence of proliferation or selective cell death. Isolation of highly purified and viable NGN3+ cell populations can be achieved based on coexpression of the cell surface glycoprotein CD133. Transcriptome and targeted expression analyses of isolated CD133+ / NGN3+ cells indicate that they are distinct from surrounding exocrine tissue with respect to expression phenotype and Notch signaling activity, but retain high level mRNA expression of genes indicative of acinar and duct cell function. NGN3+ cells have an mRNA expression profile that resembles that of mouse early endocrine progenitor cells. During in vitro differentiation, NGN3+ cells express genes in a pattern characteristic of endocrine development and result in cells that resemble beta cells on the basis of coexpression of insulin C-peptide, chromogranin A and pancreatic and duodenal homeobox 1. NGN3 expression in the adult human exocrine pancreas marks a dedifferentiating cell population with the capacity to take on an endocrine cell fate. These cells represent a potential source for the treatment of diabetes either through ex vivo manipulation, or in vivo by targeting mechanisms controlling their population size and endocrine cell fate commitment.

Evaluation of Delcath Systems' Generation 2 (GEN 2) melphalan hemofiltration system in a porcine model of percutaneous hepatic perfusion.

PURPOSE: A new melphalan hemoperfusion filter (GEN 2) was evaluated in a simulated-use porcine model of percutaneous hepatic perfusion (PHP). The current study evaluated melphalan filtration efficiency, the transfilter pressure gradient, and the removal of specific blood products. MATERIALS AND METHODS: A porcine PHP procedure using the GEN 2 filter was performed under Good Laboratory Practice conditions to model the 60-min clinical PHP procedure. RESULTS: The mean filter efficiency for removing melphalan in six filters was 99.0 ± 0.4 %. The transfilter pressure gradient across the filter averaged 20.9 mmHg for the 60-min procedure. Many blood components, including albumin and platelets, decreased on average from 3.55 to 2.02 g/dL and from 342 to 177 × 10.e3/µL, respectively, during the procedure. CONCLUSION: The increased melphalan extraction efficiency of the new filter is expected to decrease systemic melphalan exposure. In addition, the low transfilter pressure gradient resulted in low resistance to blood flow in the GEN 2 filter, and the changes to blood components are expected to be clinically manageable.

Evaluation of melphalan, oxaliplatin, and paclitaxel in colon, liver, and gastric cancer cell lines in a short-term exposure model of chemosaturation therapy by percutaneous hepatic perfusion.

BACKGROUND: The goal of this study was to determine whether liver, gastric, or colonic cancer may be suitable targets for chemosaturation therapy with percutaneous hepatic perfusion (CS-PHP) and to assess the feasibility of utilizing other cytotoxic agents besides melphalan in the CS-PHP system. MATERIALS AND METHODS: Forty human cell lines were screened against three cytotoxic chemotherapeutic agents. Specifically, the dose-dependent effect of melphalan, oxaliplatin, and paclitaxel on proliferation and apoptosis in each cell line was evaluated. These agents were also evaluated for their ability to induce apoptosis in normal primary human hepatocytes. A high-dose short-term drug exposure protocol was employed to simulate conditions encountered during CS-PHP. RESULTS: The average concentration of melphalan required for inducing significant apoptosis was 61 µM, or about 3-fold less than the theoretical concentration of 192 µM, achieved in the hepatic artery during CS-PHP dosing with melphalan. Additionally, we found that gastric cancer cell lines were 2-5 fold more sensitive to apoptosis than liver cancer cell lines to all three compounds, suggesting that in addition to colonic and gastric cancer metastases to the liver, primary gastric cancer may also be amenable to management by CS-PHP using an appropriate therapeutic agent. Significantly, at concentrations that are predicted using the CS-PHP system, these agents caused apoptosis of colonic, gastric, and liver cancer cells but were not toxic to primary human hepatocytes. CONCLUSION: The compounds tested are potential candidates for use in the CS-PHP system to treat patients with gastric and colonic metastases, and primary cancer of the liver.

Generation of humanized animal livers using embryoid body-derived stem cell transplant.

OBJECTIVE: Animal organs engineered to be chimeric for human cells could contribute significantly to the field of transplantation, including studies of human-specific diseases such as hepatitis-C, as treatment for in-born errors of metabolism, and for development of a renewable source of transplantable organs via modified xenotransplantation. We sought to use human embryoid body-derived stem cells (EBDs) to populate livers in animals for applications in transplant surgery. METHODS: SCID mice and rats underwent liver injury with carbon tetrachloride exposure or partial hepatectomy. Animals received intrasplenic injection of fluorescently labeled human stem cells. Spleen and liver were assessed at 2, 7, 15, and 30 days after transplant for the presence of EBDs and markers of human hepatocyte differentiation. RESULTS: EBDs migrate to and engraft in animal liver after splenic injection under conditions of hepatic injury. EBDs are detectable at 2 days and are in abundance at 1 week after transplant. EBDs persist in rodent liver long term (>1 month), and once engrafted differentiate into functional human hepatocytes as assessed by production of human alpha-feto-protein (AFP) and human albumin. CONCLUSIONS: We developed a novel animal model in which hepatic injury and stem cell transplantation lead to the generation of humanized animal organs. We are currently using our model to study recurrent hepatitis-C after liver transplantation, and as an alternative to whole organ transplantation for treatment of in-born errors of metabolism.

Glucose responsive insulin production from human embryonic germ (EG) cell derivatives.

Type 1 diabetes mellitus subjects millions to a daily burden of disease management, life threatening hypoglycemia and long-term complications such as retinopathy, nephropathy, heart disease, and stroke. Cell transplantation therapies providing a glucose-regulated supply of insulin have been implemented clinically, but are limited by safety, efficacy and supply considerations. Stem cells promise a plentiful and flexible source of cells for transplantation therapies. Here, we show that cells derived from human embryonic germ (EG) cells express markers of definitive endoderm, pancreatic and beta-cell development, glucose sensing, and production of mature insulin. These cells integrate functions necessary for glucose responsive regulation of preproinsulin mRNA and expression of insulin C-peptide in vitro. Following transplantation into mice, cells become insulin and C-peptide immunoreactive and produce plasma C-peptide in response to glucose. These findings suggest that EG cell derivatives may eventually serve as a source of insulin producing cells for the treatment of diabetes.