Antiviral Effects Of Antisense Locked Nucleic Acid Targeted At HBV S Gene By Cationic Liposomes In The Hepatitis B Virus Transgenic Mice

China not only has a large population, but also has a lot people who have hepatitis virus. The number of people who have contracted the hepatitis B virus(HBV) is one point three hundred million. There is no specific medicine at present. The effect of traditional Chinese herbal medicine is not good enough, because the pharmacological mechanism isn't clear, and the clinical medicine, such as interfereon-αand lamivudine, has no satisfactory therapeutic effect on HBV with low specificity and inducing the mutations of virus, so it has important significance to find out a new specific medicine of curing chronic hepatitis B of the genetic level. Gene therapy of HBV infection has become the focus of research gradually because of the usage of Antisense oligodeoxynucleotide(ASODN).However, only limited long-term efficacy can be achieved with this therapy because the ASODN can be dissolved easily. Locked nucleic acid(LNA) is a new class of novel nucleic acid, it has more special characteristic such as stronger anti-dissolve capability, higher linkability, light toxicity, and it can be absorbed by the body easily. LNA has been used for the treatment of HIV infection in other countries, but up till now no reports for using it to treat HBV infection.In this study, the cationic liposomes was used as LNA carrier, and was injected into HBV transgenic mice through the tail vein by fluid dynamics, to investigate the curative effects of antisense LNA targeting the S regions in the genome of the hepatitis B virus(HBV).we planed:1. To investigate the existence of ASODN in liposomes and choose the optimum proportion between ASODN and cationic liposomes.2. To investigate the toxicity of cationic liposomes as medicine carriers for gene therapy in mice.3. To investigate the effectual dosage of antisense oligodeoxynucleotide in HBV transgenic mice4. To investigate the curative effects of antisense LNA targeting the S regions in the genome of hepatitis B virus. Objective To investigate the existence of ASODN in liposomes and choose the optimum proportion between ASODN and cationic liposomes.Methods Antisense oligodeoxynucleotide(ASODN) complementary to the S regions in the genome of HBV was synthesized, and diluted into different standard concentrations with ddH2O.The standard curve was designed by UV-2450 Ultraviolet Spectrophotometer in a wavelength of 260nm.The comparative dosage of ASODN/liposome is shown as ug/ul. The drug loaded efficiency was calculated through the formula:(3-mf)/3×100% . Agarose Gel Electrophoresis was used to analyze the existence of ASODN in the liposomes mixture.Results The linear regression equation: A=0.063C+0.073, coefficient of regression was 0.999.The drug loaded efficiencyies were 71.71%, 78.38%, 81.45% and 81.68% when the proportion of ASODN/liposome were 1:0.5, 1:1,1:2,1:3,1:4 respectively. Agarose gel electrophoresis showed three different existence of ASODN in the liposomes formulation: free, absorbed and encapsulated types. Compared with free ASODN, the density of Ethidium Bromide(EB) was significantly different. The level of decreasing was largely depended on ug/ul charge ratio. The density of EB in 1:3~1:4 charge ratio was thinner than 1:0.5~1:2.Conclusion The optimum proportion between ASODN and cationic liposomes was 1:3. The existence of ASODN in liposomes formulation was encapsulated types. Part Tow: Toxicity of cationic liposomes as medicines carriers for gene therapy in miceObjective To investigate the toxicity of cationic liposomes as medicine carriers for gene therapy in mice.Methods The cationic liposomes of difference volume were injected into mice through the tail vein by fluid dynamics,while in the control group,each mouse received the same volume 5% glucose solution in the same way.Venous blood samples were collected separately on the previous day ,the 1st day and the 3rd day after the injection via orbital vein.WBC,LY% and GR% in the anticoagulated blood were detected by automatic globulimeter,while serum Alb,ALT,BUN and CR were measured by the spectrophotometer.3 days later the mice were elthenised and HE staining was used to examine the liver and kidney.Pathological examination of the tissues was performed.Results In comparison with the control group,WBC,BUN and CR were significantly higher in the 24ul/g body weight group(P<0.05),and Alb,ALT didn't show any significant difference(P>0.05).No significant abnormality was found in any of the other groups(P>0.05).In comparison with that before injection, WBC,BUN and CR were significantly higher on the 1 day after the injection(P<0.05),and all targets returned to normal levels except for CR 3 days after the injection(P>0.05). Alb, ALT didn't show any significant difference(P>0.05).No significant abnormality was found in the tissues in all groups.Conclusion The toxicity of cationic liposomes in low dose(<12ul/g body weight)is slightly,so the cationic liposomes can be used to carry nucleotide medicines for gene therapy. Part Three: Effectual dosage of antisense oligodeoxynucleotide in HBV transgenic miceObjective : To investigate the effectual dosage of antisense oligo deoxynucleotide in HBV transgenic mice.Methods: Cationic liposomes was used as drug carrier which targeted antisense oligodeoxynucleotide to mice liver. ASODN was incubated with cationic liposomes for 60 minutes to produce the targeted Lipo-ASODN mixture.8 HBV transgenic mice were randomly divided into 4 equal groups: 3ug/g body weight group, 6ug/g body weight group, 12ug/g body weight group,and 5%GLU control group, to be injected into the caudal vein with corresponding Lipo-ASODN mixture once every other day. while in the control group, each mouse received the same volume 5%GLU solution in the same way. Venous blood samples were collected before,on the 1st day,3rd day, 7th day after the injection. Serum HBsAg and HBeAg were detected by Enzyme Linked Immunosorbent Assay(ELISA).HBV S segment was identified by Polymerase Chain Reaction(PCR) and Agarose Gel Electrophoresis Assay.Results:The production of PCR has 770bp. The inhibition rates of HBsAg 1st day,3rd day,7th day after injection were 22.1%,36.1% , 27.9% in 3ug/g body weight group,27.5%,40.6%,33.9% in 6ug/g body weight group and 31.2%, 44.1%, 28.9% in 12ug/g body weight group.While the inhibition rates of HBeAg were 54.4%,77.9%,83.8% in 3ug/g body weight group,56.2%,77.5%,86.2% in 6ug/g body weight group and 60.0%, 69.3%, 84.0% in 12ug/g body weight group.Conclusion : Effectual dosage of antisense oligodeoxynucleotide in HBV transgenic mice in 6ug/g body weight group. Part Four: Antiviral effects of antisense locked nucleic acid targeted at HBV S gene by Cationic liposomes in the hepatitis B virus transgenic miceObjective: To investigate the curative effects of antisense LNA targeting the S regions in the genome of hepatitis B virus(HBV).Methods: Antisense LNA complementary to the S regions in the genome of HBV was synthesized. Cationic liposomes was used as drug carriers which targeted antisense LNA in mice liver.LNA was incubated with cationic liposomes for 60 minutes to produce the targeted Lipo-LNA mixture.30 HBV transgenic mice were randomly divided into 5 equal groups: blank LNA group, S-ASODN-liposomes group, LNA-liposomes group, blank liposomes control group, and 5%GLU control group, to be injected into the caudal vein with corresponding Lipo-LNA(or S-ASODN) mixture once every other day. while in the control group, each mouse received the same volume 5%GLU solution(or blank liposomes) in the same way. Venous blood samples were collected before,the 1st day and on the 3rd, 7th,14th and 28th day after the injections. Serum HBsAg was detected by Enzyme Linked Immunosorbent Assay(ELISA).HBV DNA was detected by Real Time PCR. while serum Alb, ALT, BUN, CR, CK, CK-MB, ApoA1, ApoB were measured by an automatic biochemistry analyzer.WBC,HGB,RBC,MCV were measured by an Automatic Hematology Analyzer. After 28 days the mice were elthenised and immunohistochemistry was used to examine the HBsAg and HBcAg in the liver tissues.Pathological examination of the tissues was performed.Results:The serum HBsAg concentrations on the 1st,3rd,7th, 14th and 28th day after injection were significantly lower than that before injection in the S-ASODN-liposomes group and LNA-liposomes group(all P <0.05).The inhibition rates in S-ASODN-liposomes group was 20.3%, 31.5%, 37.1%, 24.1% and 15.5% respectively. While the LNA-liposomes group was 41.7%, 52.8%, 57.8%, 30.5% and 27.6% respectively. In comparison with that before the injections, the HBV DNA expression level on the 1st,3rd,7th, 14th and 28th day after injection was significantly lower in the S-ASODN-liposomes group and LNA-liposomes group (all P <0.05). The inhibition rates in S-ASODN-liposomes group was 2.41%,14.06%,27.44%,18.69% and 17.49% respectively. While the LNA-liposomes group was 6.64%, 38.53%, 40.19%, 39.12% and 23.58% respectively.HBsAg and HBcAg expression in liver were significantly lower in LNA-liposomes group in 28 days later. No significant abnormality was found in the tissues in all groups.Conclusion: Antisense LNA targeting the S regions in the genome of HBV inhibits the replication and expression of HBV.