Haemophilia B curative FIX production from a low dose UCOE-based lentiviral vector following hepatic pre-natal delivery.

The ubiquitous chromatin opening element from the human HNRPA2B1-CBX3 housekeeping gene locus (A2UCOE) is able to provide stable and cell-to-cell reproducible levels of transgene expression regardless of target cell genome integration site with efficacy demonstrated in adult, embryonic and induced pluripotent stem cells and their differentiated progeny in vitro and in vivo. Here we evaluate the ability of A2UCOE-based lentiviral vectors to confer stable expression following pre-natal delivery in mice. Our results show stable post-natal A2UCOE-eGFP and A2UCOE-luciferase lentiviral vector presence in both the liver and haematopoietic system with concomitant persistence of expression demonstrating efficient transduction of both fetal hepatocytes and haematopoietic stem cells. In addition, we find that an A2UCOE-FIX lentiviral vector produces comparable amounts of plasma FIX protein to that obtained from a SFFV-FIX construct. Furthermore, the A2UCOE-FIX vector shows that at a low (0.19) average vector copy number per liver cell, it can provide stable levels of plasma FIX production, which would convert severe haemophilia B ("pii">CGT-EPUB-lt;1%) to a mild phenotype (≈20%). Our results provide proof-of-principle for low dose pre-natal A2UCOE-based LV delivery to the liver as a therapeutic option for haemophilia B and potentially other metabolic conditions.

Calorimetric and Spectroscopic Analysis of the Thermal Stability of Short Duplex DNA-Containing Sugar and Base-Modified Nucleotides.

Base- and sugar-modified analogs of DNA and RNA are finding ever expanding use in medicine and biotechnology as tools to better tailor structured oligonucleotides by altering their thermal stability, nuclease resistance, base-pairing specificity, antisense activity, or cellular uptake. Proper deployment of these chemical modifications generally requires knowledge of how each affects base-pairing properties and thermal stabilities. Here, we describe in detail how differential scanning calorimetry and UV spectroscopy may be used to quantify the melting thermodynamics of short dsDNA containing chemically modified nucleosides in one or both strands. Insights are provided into why and how the presence of highly stable base pairs containing modified nucleosides can alter the nature of calorimetry or melting spectroscopy data, and how each experiment must therefore be conducted to ensure high-quality melting thermodynamics data are obtained. Strengths and weaknesses of the two methods when applied to chemically modified duplexes are also addressed.

Flexible targeting of ErbB dimers that drive tumorigenesis by using genetically engineered T cells.

Pharmacological targeting of individual ErbB receptors elicits antitumor activity, but is frequently compromised by resistance leading to therapeutic failure. Here, we describe an immunotherapeutic approach that exploits prevalent and fundamental mechanisms by which aberrant upregulation of the ErbB network drives tumorigenesis. A chimeric antigen receptor named T1E28z was engineered, in which the promiscuous ErbB ligand, T1E, is fused to a CD28 + CD3ζ endodomain. Using a panel of ErbB-engineered 32D hematopoietic cells, we found that human T1E28z⁺ T cells are selectively activated by all ErbB1-based homodimers and heterodimers and by the potently mitogenic ErbB2/3 heterodimer. Owing to this flexible targeting capability, recognition and destruction of several tumor cell lines was achieved by T1E28⁺ T cells in vitro, comprising a wide diversity of ErbB receptor profiles and tumor origins. Furthermore, compelling antitumor activity was observed in mice bearing established xenografts, characterized either by ErbB1/2 or ErbB2/3 overexpression and representative of insidious or rapidly progressive tumor types. Together, these findings support the clinical development of a broadly applicable immunotherapeutic approach in which the propensity of solid tumors to dysregulate the extended ErbB network is targeted for therapeutic gain.

Death From Liver Disease and Development of Hepatocellular Carcinoma in Patients With Chronic Hepatitis B Virus Infection: A Prospective Study.

Background Patients with chronic hepatitis B virus (HBV) infection are at risk for death from complications of liver disease and development of hepatocellular carcinoma (HCC). To identify the time course and risk factors associated with these events, we conducted a prospective study in chronic hepatitis B patients referred to our clinic. Methods From January 1989 to March 1998, 400 hepatitis B surface antigen (HBsAg)-positive patients were classified into three categories: inactive carriers (N=110), chronic hepatitis (N=151), and cirrhosis (N=139). These patients were observed at 3- to 6-month intervals with liver tests, alpha-fetoprotein (AFP) levels, and ultrasound examinations. The study endpoints were death from liver disease complications and development of HCC. Results The patients were followed for a mean time (± SD) of 83.6 ± 39.6 months. During this period, no liver-related deaths or HCC were noted in inactive carriers. However, 38 of 139 (27.3%) patients with cirrhosis died from non-HCC-related liver complications. Multivariate analysis demonstrated that male sex (odds ratio [OR] 5.9; 95% confidence interval [CI], 2.0-22.6; P=.003), decreased initial serum albumin (OR 69.1; 95% CI, 11.5-486.4; P=.0009), low platelet count (OR 8.8; 95% CI, 0.96-92.9; P=.05), and presence of cirrhosis (OR 14.2; 95% CI, 3.4-111.8; P=.0009) were independently associated with increased mortality from chronic hepatitis B. During the same time period, nine of 151 (6.0%) chronic hepatitis patients and 22 of 139 (15.8%) patients with cirrhosis developed HCC. By multivariate analysis, progression to HCC was associated with advanced age (OR 19.7; 95% CI, 1.9-231.9; P=.01) and presence of cirrhosis (OR 3.6; 95% CI, 1.6-8.9; P=.003). Patients positive for hepatitis B early antigen (HBeAg) and HBeAg antibodies experienced liver-related deaths and developed HCC at similar rates. Conclusions This prospective study from the United States confirms previous observations of the high risk of mortality and development of HCC in patients infected with HBV. To decrease the risk of these complications, antiviral therapy should be initiated early in the course of the disease. In addition, surveillance for HCC must be performed at least every 6 months in patients with chronic hepatitis and cirrhosis.