Phsa Mediated Recombinant Retrovirus Targeting Of Liver Cells

Targeting retroviral entry is a central theme in the development of vectors for gene therapy. The attractiveness of the approach is the selective delivery of a therapeutic gene, which would immensely reduce unfavorable side effects and ease the clinical application of gene therapy. Recently, the study on the relation between HDV ribozyme structure and activity have been done at home and abroad, and our laboratory have proved that HDV ribozyme is a new potent of the antisense regent for HBV gene therapy. But how to carry HDV ribozyme gene into defined target cells or tissues has been a tackling problem.The ability of viruses to introduce foreign DNA into target cells is being exploited in many gene therapy strategies aimed at treating genetic diseases, including cancer. Of the various viral vectors developed for this purpose, those based on retroviruses are best understood and the most widely used. These vectors integrate their genomes stably into host cell DNA allowing long term expression of inserted therapeutic genes. The processes of viral entry and genome integration do not require viral protein synthesis. In order to produce vector particles,the viral proteins such as the core protein, the reverse transcriptase and the envelope protein are provided in trans in the packaging cell lines. These cell lines release vector genomes packaged into infectious particles that are free from contaminating helper virus and replication-competent recombinant virus.The host range of the retroviral vector is dependent upon its envelope glycoprotein (Env), which recognizes and binds to a specific cell surface receptor protein. After binding, the envelope protein undergoes conformational changes allowing induction of membrane fusion. The murine leukemia virus (MLV) Env protein, like all retroviral Envs, consists of two subunits: SU, which contains the receptor binding domain, and TM which contains the fusion peptide. Binding of SU to the receptor is thought to induce a conformational change in TM, resulting in the exposure of the fusion peptide and activation of the process of fusion of the viral and cellular membranes, and eventual delivery of the viral core into the cell. So the host range of a virus that does not infect human cells may be extended to a predetermined human cell type. The ecotropic MLV envelope protein has most frequently been used for this approach. Since ecotropic MLV recognizes receptors only on rodent cells and not on human cells, its host range is restricted to rodent cells. This targeting approach requires the inclusion of a novel attachment site and the induction of fusion via a novel receptor interaction.The mechanism for the entry of Hepatitis B Virus (HBV)-particles into target cells, in particular into hepatocytes is yet not understood. Several receptors on liver cells have been previously suggested which could bind either to wild-type HBV particles or to genetically engineered virus. The envelope of the HBV particle is composed of three related surface (S) proteins. The major or small S protein is 226 amino acids in length. The two other S proteins include the small S sequence at their carboxyl termini. In addition, the middle S protein contains an amino-terminal extension to the small S protein of 55 amino acids (PreS2). The large S protein contains a further amino-terminal extension to the middle S protein of 108 to 119 (depending on subtype ay or ad, respectively) amino acids (PreS1). It has been suggested that the large S protein performs the attachment function since a synthetic peptide containing a PreSl amino acid sequence and an antiserum raised against this peptide inhibited the attachment of cultured hepatoma (HepG2) cells to immobilized HBsAg particles. It also has been suggested that the middle S protein, which binds to polymerized human serum albumin (pHSA), may use pHSA to attach to hepatocytes, which express albumin receptors. In this study, we have investigated whether Moloney murine leukemin virus (MoMLV)-based retroviral vector that contains the HBV PreS2 peptide fused to aa +1 at the N-terminus of Env could be applied to target hepatocytes.Amphotropic murine leukemin virus (A-MLV) and MoMLV use sodium-dependent phosphate symporters, Pit1 and Pit2 as receptors for infection. Studies of naturally occurring MLVs have identified the amino-terminal domain of the SU glycoprotein as responsible for receptor recognition and binding. Specially, residues Tyr-60 and Val-61 of MoMLV VRA are critical for receptor recognition. We use site-specific mutagenesis to investigate the new infectivity of recombinant retroviruses, mediated by HBV PreS1 or PreS2 peptide.PurposeTo explore the possibility of targeting hepatocytes mediated by pHSA by recombinant retrovirus vector which carries pHSA-R. Methods1. The construction of recombinant retrovirus-associated expression vectors: The env gene of vector pcDNA3.1(-)-env-preS2 was mutated by PCR and additional preS1-associated expression vectors were constructed.2. To obtain the supernatant of the packaging cell lines. The plasmids pL–EGFP and pcDNA4/HisMaxA-gag-pol were cotransfected into the packaging 293T cell lines in combined with env-associated expression vectors, respectively, by calcium phosphate precipitation method.3. The detection of recombinant retroviral vectors infectivity was studied in HepG2215, NIH3T3 and HEK cells by real-time quantitative PCR.Results1. The design of expression vectors. Four eukaryote expression vectors pcDNA3.1(-)-envm-preS2, pcDNA3.1(-)-envm, pcDNA3.1(-)-envm-preS1 and pcDNA3.1(-)-envm-preS1+S2, were successfully constructed and identified by DNA sequencing and digestion with restriction enzymes.2. Transient transfection was performed on 293T cells by calcium phosphate treatment. The high-titer retrovirus carrying eGFP gene were obtained from supernatant of the packaging cell line.3. The packaged recombinant retrovirus based on pcDNA3.1(-)-envm-preS2 had a better hepatocellular tropism.4. In the presence of polymerized human serum albumin, both preS1 and preS2- associated recombinant retrovirus were capable of infect hepatocytes. preS2-associated recombinant retrovirus had a higher infectivity.5. pHSA could enhance the preS1-associated recombinant retrovirus infectivity to NIH 3T3 and HEK cells.6. preS2-associated recombinant retrovirus could infect NIH 3T3 and HEK cells with or without pHSA with a similar titer, though the infectivity was lower. Our results indicated that the transduction of NIH 3T3 and HEK cells was caused by the interactions between HBV PreS2 peptide and some receptors rather than pHSA-R on cells surface. ConclusionsThe packaged recombinant retrovirus based on pcDNA3.1(-)-envm-preS2 had a better hepatocellular tropism mediated by pHSA. The efficiency of this system in gene transfer might provide an optimal system for hepatocyte gene therpy.