Long-Term Suppression of Hepatitis B Virus Replication by Short Hairpin RNA Expression Using the Scaffold/Matrix Attachment Region-Based Replicating Vector System pEPI-1

Abstract

Since the emergence of viral resistance of hepatitis B virus (HBV) during treatment is becoming an important issue even with newer drugs, there is a need for alternative treatment options such as, for example, RNA interference (RNAi) technology. While short-term suppression of HBV replication is easily achieved with small interfering RNA oligonucleotides, this is not the case for long-term suppression due to the lack of an optimal vector system. Based on the nonviral scaffold/matrix attachment region (S/MAR)-based vector system pEPI-1, which is free of common side effects and is stably retained as an episome even in the absence of selection, we designed a short hairpin RNA (shRNA) expression vector called pEPI-RNAi for HBV suppression. HBV-replicating HepG2.2.15 cells were transfected with pEPI-RNAi, and the intracellular status of the plasmid was followed by PCR and Southern analysis. HBV replication was measured on the DNA, RNA, and protein level. HBV RNA expression was reduced by almost 85% 3 months posttransfection with pEPI-RNAi. At 8 months posttransfection in the absence of antibiotic selection pressure, the suppression level was still 70% and the vector was retained as an episome. The reduction of total intracellular HBV DNA at this point was 77%, showing a marked suppression of HBV DNA replication. At a comparable level, secretion of viral antigens, as well as progeny HBV virions, was inhibited. The S/MAR-based vector system pEPI-1 allows long-term suppression of HBV replication by the expression of suitable shRNAs. Due to its unique properties compared to commonly used vectors, it provides an interesting option for the treatment of chronically HBV-infected individuals.

Different matrix attachment regions flanking a transgene effectively enhance gene expression in stably transfected Chinese hamster ovary cells

“…The expression level of a reporter gene when two β-globin MARs are flanked by a chloramphenicol acetyltransferase (CAT) expression cassette is significantly higher than that when one β-globin MAR is flanked by a CAT expression cassette in stably transfected Chinese hamster ovary (CHO) cells (Wang et al, 2010). Numerous MARs that improve foreign gene expression are currently used in gene therapy (Argyros et al, 2008;Jenke et al, 2008;Wong et al, 2010). One of the major problems associated with the majority of gene therapy protocols is the requirement of sustaining expression while a transgene is being integrated into a chromosome.…”

Long‐term RNA interference and its application to hepatitis B virus

“…Jenke et al. 48 reported a novel non‐viral scaffold or matrix attachment region‐based vector system pEPI‐1. HBV RNA expression was reduced by 85 percent and 70 percent at 3 and 8 months after transfection with pEPI‐RNAi, respectively.…”

“…Secreted HBsAg was reduced by 94.2 percent in the cell culture and 84.5 percent in mouse serum, whereas the immunohistochemical detection of the HBV core antigen (HBcAg) revealed a more than 99 percent reduction in stained hepatocytes upon RNAi treatment. Jenke et al 48 reported a novel non-viral scaffold or matrix attachment region-based vector system pEPI-1. HBV RNA expression was reduced by 85 percent and 70 percent at 3 and 8 months after transfection with pEPI-RNAi, respectively.…”

Involved field radiation for Hodgkin's lymphoma: The actual dose to breasts in close proximity

“…[1][2][3][4] Despite this, the use of involved field radiation is still often questioned with respect to out-of-field radiation dose, especially to the critical organs in close proximity, namely breasts in female patients. Although multiple publications addressed the out-of-field dose, [5][6][7][8][9] there is a paucity of data in the literature addressing specifically the dose to the breasts in young female patients with HL treated with modern field radiation therapy. We undertook this study to determine the dose of radiation to the breasts located in close proximity of a mediastinal radiation field, using an anthropomorphic phantom.…”