Harnessing non-Watson-Crick's base pairing to enhance CRISPR effectors cleavage activities and enable gene editing in mammalian cells.

Genomic DNA of the cyanophage S-2L virus is composed of 2-aminoadenine (Z), thymine (T), guanine (G), and cytosine (C), forming the genetic alphabet ZTGC, which violates Watson-Crick base pairing rules. The Z-base has an extra amino group on the two position that allows the formation of a third hydrogen bond with thymine in DNA strands. Here, we explored and expanded applications of this non-Watson-Crick base pairing in protein expression and gene editing.

Preclinical Evaluation of Foamy Virus Vector-Mediated Gene Addition in Human Hematopoietic Stem/Progenitor Cells for Correction of Leukocyte Adhesion Deficiency Type 1.

gene therapy procedures targeting hematopoietic stem and progenitor cells (HSPCs) predominantly utilize lentivirus-based vectors for gene transfer. We provide the first pre-clinical evidence of the therapeutic utility of a foamy virus vector (FVV) for the genetic correction of human leukocyte adhesion deficiency type 1 (LAD-1), an inherited primary immunodeficiency resulting from mutation of the β2 integrin common chain, CD18. CD34 HSPCs isolated from a severely affected LAD-1 patient were transduced under a current good manufacturing practice-compatible protocol with FVV harboring a therapeutic CD18 transgene.

CRISPR/Cas9 ribonucleoprotein-mediated genome and epigenome editing in mammalian cells.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system has revolutionized the ability to edit the mammalian genome, providing a platform for the correction of pathogenic mutations and further investigation into gene function. CRISPR reagents can be delivered into the cell as DNA, RNA, or pre-formed ribonucleoproteins (RNPs). RNPs offer numerous advantages over other delivery approaches due to their ability to rapidly target genomic sites and quickly degrade thereafter.

Developing Biodegradable Lipid Nanoparticles for Intracellular mRNA Delivery and Genome Editing.

Since the U.S. Food and Drug Administration (FDA) granted emergency use authorization for two mRNA vaccines against SARS-CoV-2, mRNA-based technology has attracted broad attention from the scientific community to investors.

Genome editing in human hematopoietic stem and progenitor cells via CRISPR-Cas9-mediated homology-independent targeted integration.

Ex vivo gene correction of hematopoietic stem and progenitor cells (HSPCs) has emerged as a promising therapeutic approach for treatment of inherited human blood disorders. Use of engineered nucleases to target therapeutic transgenes to their endogenous genetic loci addresses many of the limitations associated with viral vector-based gene replacement strategies, such as insertional mutagenesis, variable gene dosage, and ectopic expression. Common methods of nuclease-mediated site-specific integration utilize the homology-directed repair (HDR) pathway.

Robust generation of erythroid and multilineage hematopoietic progenitors from human iPSCs using a scalable monolayer culture system.

One of the most promising objectives of clinical hematology is to derive engraftable autologous hematopoietic stem cells (HSCs) from human induced pluripotent stem cells (iPSCs). Progress in translating iPSC technologies to the clinic relies on the availability of scalable differentiation methodologies. In this study, human iPSCs were differentiated for 21 days using STEMdiff™, a monolayer-based approach for hematopoietic differentiation of human iPSCs that requires no replating, co-culture or embryoid body formation.

Chromosome Preference During Homologous Recombination Repair of DNA Double-Strand Breaks in .

DNA double-strand breaks (DSBs) are especially toxic DNA lesions that, if left unrepaired, can lead to wide-ranging genomic instability. Of the pathways available to repair DSBs, the most accurate is homologous recombination (HR), where a homologous sequence is used as a donor template to restore genetic information at the break site. While much of the biochemical aspects of HR repair have been characterized, how the repair machinery locates and discriminates between potential homologous donor templates throughout the genome remains elusive.

Renal pathology in patients with rheumatoid arthritis.

We studied 76 patients with rheumatoid arthritis who had autopsies performed at the University of Utah and Salt Lake Veterans Administration Medical Center. The most common pathological finding in the kidney was interstitial fibrosis (46%) and internal proliferation of the arterioles in the absence of hypertension (54% of the cases). Renal amyloidosis was only found in 7% of the patients.

Clinical course of patients with scleroderma renal crisis treated with captopril.

Since it has been suggested that the renin-angiotensin axis may play an important role in the severe hypertension and in the acute renal deterioration in scleroderma, we sought to determine the effectiveness of angiotensin blockade in the treatment of this disorder. Captopril controlled blood pressure successfully and easily in 4 consecutive patients with scleroderma renal crisis. Mean serum creatinine was 3.

Early pathologic features of hereditary nephritis: a clinicopathologic correlation.

The histologic, immunofluoresence and electron microscopic features of renal biopsies from twelve patients having hereditary nephritis were examined and correlated with the clinical data. In ten patients with normal renal function, light microscopy showed similar but nonspecific glomerular abnormalities consisting of mild focal and segmental hypercellularity and thickening of capillary walls. Biopsies from two patients with azotemia had diffuse lesions of the glomeruli and associated interstitial fibrosis.

Serum C-peptide in renal failure patients following stimulation of pancreatic secretion.

We have demonstrated persistently elevated serum C-peptide concentrations in patients with chronic renal failure on chronic hemodialysis. A blunted serum C-peptide response to intravenous glucose, glucagon and tolbutamide was also found. However, the response to oral glucose stimulation was greater and more prolonged than in control subjects, probably related to the magnitude of hyperglycemia found in patients with chronic renal failure.

High-dose corticosteroids: their use in treating idiopathic rapidly progressive glomerulonephritis.

To evaluate the response of patients with idiopathic rapidly progressive glomerulonephritis (RPGN) to high-dose corticosteroids, we have studied ten consecutive patients with this disorder. All were given 1 g of methylprednisolone intravenously each day for one week and then placed on a high-dose orally administered prednisone regimen. Four of the ten patients with idiopathic RPGN responded with a sustained reduction in the serum creatinine level of at least 50%.

Cholesterol embolization: a complication of angiography.

Cholesterol embolization is not widely recognized as a complication of major arteriographic procedures. In a retrospective study of 71 autopsies of patients who underwent diagnostic arteriographic procedures (20 with aortograms, 51 with cardiac catheterization and coronary angiography), we found an incidence of cholesterol embolization of 30% and 25.5%, respectively, in comparison with 4.

Adult hemolytic uremic syndrome with renal arteriolar deposition of IgM andC3.

Two cases of idiopathic adult hemolytic uremic syndrome in which deposits of IgM and C3 were identified in renal arterioles showing fibrinoid necrosis are reported. Fibrin was also identified in the lumina of the involved vessels, but there was no laboratory evidence of disseminated intravascular coagulation. In both cases, serum C3 was decreased and C4 was normal, suggesting involvement of the alternate pathway of complement activation.

Abnormalities in the regulation of growth hormone in chronic renal failure.

Carbohydrate intolerance is a common abnormality in patients with chronic renal failure. In this group of patients we investigated the interrelation among glucose, insulin, and growth hormone and confirmed the presence of carbohydrate intolerance and hyperinsulinemia. In addition we demonstrated alterations in growth hormone regulation, characterized by (1) the lack of suppression of growth hormone by orally induced hyperglycemia and paradoxical increase in serum levels of growth hormone after the administration of intravenous glucose or glucagon; (2) lack of release of growth hormone with induced hypoglycemia and an exaggerated response to levodopa administration.

Hereditary nephritis: a re-examination of its clinical and genetic features.

In order to re-evaluate current concepts of hereditary nephritis we studied the urinary findings, the course of the disease, and its genetic transmission in two large pedigrees. We identified 150 patients with hereditary nephritis. Our data show that microscopic hematuria is the most reliable urinary criterion for diagnosing hereditary nephritis in both male and female patients.

Abnormalities in the regulation of prolactin in patients with chronic renal failure.

We have investigated the hypothalamic-hypophyseal regulation of prolactin secretion in patients with chronic renal failure treated with chronic hemodialysis. When compared to control subjects, baseline serum prolactin levels were elevated in the renal failure patients (range 11 to 16 mmicrogram/ml for renal failure patients, 6 to 9 mmicrogram/ml for controls, P less than 0.05).

Speech and lingual behavior before and after mandibular osteotomy.

A procedure was applied to compare the oral behavior of five patients before and after mandibular osteotomy for correction of Class III malocclusions. It was found that observable changes in oral behavior do occur after mandibular osteotomy. Fewer errors were found in sibilant articulation, while more maladaptive lingual behaviors were found in lingual diadochokinetic tasks.

Thyroid dysfunction in uremia: evidence for thyroid and hypophyseal abnormalities.

Disturbances in thyroid function and a high prevalence of goiter develop in patients on chronic hemodialysis. This study shows that in patients on dialysis, mean serum thyroxine and triiodothyronine levels are lower than normal. Patients with chronic renal failure not on dialysis, have mean serum thyroxine levels similar to normal subjects and low mean serum triiodothyronine levels.

Effects of anesthetic doses of morphine on renal function in the dog.

Renal effects of anesthetic doses of morphine (2 mg./kg.) administered intravenously (I.