[The construction and evaluation of antisense RNA expression vector targeting at HCV 5'NCR].

Antisense RNA is an important field of antisense technique. In order to explore a novel approach for the treatment of hepatitis C virus (HCV) infection and prove the availability of transgenic cellular model HepG2. 9706, an antisense RNA was designed targeting at the highly conserved 5'NCR and translation initiation site of HCV RNA at nt positions 13-397. It was inserted into the downstream of SV40 promotor sequence of pGL3 control vector in which luciferase gene is deleted. The antisense RNA expression vector (pHCV-asR) was constructed and identified by PCR, endonucleases reaction and DNA sequencing. Its expression in transfected HepG2 cells was tested by RT-PCR. To evaluate the inhibitory activities of pHCV-asR, HepG2. 9706 cells were transfected using this construct via Lipofectin method. Luciferase activity in cell lysates was measured for quantitative determining antiviral effects within the cells. The results showed that the inserted sequence of the pHCV-asR is the same as the designed sequence and can express in HepG2 cells. It was also found that pHCV-asR in HepG2. 9706 has a dose-dependent inhibitory effects on luciferase expression controlled by HCV 5'NCR with a inhibitory rate of 57%.

[The inhibitory effects of hepatocyte targeting pH-sensitive liposome mediated phosphorothioate antisense oligonucleotide on gene expression controlled by HCV 5'NCR].

Antisense oligodeoxynucleotide is a negative charged macromolecule and hence is difficult to penetrate cell membrane and liable to degradation. To increase the effective concentration of antisense drugs in the target cells, a hepatocyte-targeting liposome directed to asialoglycoprotein receptors exclusively expressing on the hepatocyte membrane was designed and prepared based on the receptor-mediated gene transfer. In order to accelerate endosomal exit of nucleic acid drugs, the liposomal formulation with pH-sensitive property was adopted. The hepatocyte-targeting and pH-sensitivity of liposome were analyzed by galactose-receptor competitive inhibition and hemolysis of chicken red blood cell. Antisense oligodeoxynucleotide, HCV363 against HCV 5'NCR, was delivered via prepared liposome to transgenic cell HepG2.9706 and evaluated for its inhibitory effect on luciferase expression controlled by HCV 5'NCR in HepG2.9706 using Luciferase Assay System The results showed that different concentrations (10, 20, 30 mmol/L) of galactose solutions reduce the delivery effects of liposome to some extend that were up to saturation when the concentrations of galactose solution exceed 20 mmol/L. Prepared liposomes mixed with chicken RBC are put into PBS buffers with different pH values(4.0-8.0), it was observed that the amount of heme is greatly released in acidic PBS (pH < 6) due to the fusion of liposome and RBC membranes. Liposome-mediated HCV363 has dose-dependent inhibitory activities on luciferase expression controlled by HCV 5'NCR in HepG2.9706 and the inhibitory rate is 86% at a concentration of 1.0 mumol/L. In conclusion, the liposome is proven to be a hepatocyte-targeting pH-sensitive delivery system that can increase the pharmaceutical effects of antisense drugs.

Monoclonal antibody 55 (CD10) and complement used for purging autologous bone marrow in common acute lymphoblastic leukemia.

A CD10 monoclonal antibody 55 (McAb55) was intended for purging residual common acute lymphoblastic leukemia antigen (CALLA) positive leukemic cells from autotransplants of common acute lymphoblastic leukemia (C-ALL) patients. It was found that after two rounds of McAb55 and complement treatment, 4-5 logs of CALLA+ cells were removed from bone marrow detected by clonogenic assay. The standardization of separation, purgation and preservation of bone marrow for C-ALL patients' autotransplants was then set as follows: Following the carboxymethyl starch sedimentation and Ficoll-Hypaque gradient separation, the isolated mononuclear cells (MNCs) were treated with McAb55 and complement twice and kept in room temperature for 48-72 hours prior to infusion. This procedure resulted in the removal of more than 99% of CALLA+ cells, recovery of 10-30% MNCs, and leaving the hematopoiesis stem cells intact. After the intensive cytoreductive therapy, 4 patients with C-ALL received the purged autotransplants giving timely recovery of the hematopoietic function. The patients were all remaining in remission status for more than 40-250 days so far.