Efficient Gene Delivery and Targeted Expression to HepatocytesIn Vivoby Improved Lentiviral Vectors

Abstract

Safe and efficient genetic modification of liver cells could enable new therapies for a variety of hepatic and systemic diseases. Lentiviral vectors are promising tools for in vivo gene delivery. Previous data suggested that recruitment into the cell cycle was required for transduction of hepatocytes in vivo. We developed an improved vector design that enhanced nuclear translocation in target cells and significantly improved gene transfer performance. Using the new vector and a panel of internal promoters, we showed that rat hepatocytes were transduced ex vivo to high frequency without requirement for proliferation. On intravenous administration of vector into adult severe combined immunodeficient (SCID) mice, we found high levels (up to 30%) of transduction of parenchymal and nonparenchymal cells of the liver, integration of the vector genome in liver DNA and stable expression of the marker green fluorescent protein (GFP)-encoding gene without signs of toxicity. Coadministration of vectors and 5'-bromo-2'-deoxyuridine in vivo proved that cell cycling was not required for efficient transduction of hepatocytes. In addition to the liver, the spleen and the bone marrow were transduced effectively by systemic delivery of vector. GFP expression was observed in all these organs when driven by the cytomegalovirus promoter and by the phosphoglycerate kinase gene promoter. Using the promoter of the albumin gene, we could restrict expression to hepatocytes. By a single vector injection into the bloodstream of SCID mice, we achieved therapeutic-range levels of the human clotting factor IX, stable in the plasma for up to 1 year (the longest time tested), indicating the potential efficacy of improved lentiviral vectors for the gene therapy of hemophilias and other diseases.

Complement regulatory proteins are incorporated into lentiviral vectors and protect particles against complement inactivation

“…Third-generation HIV-1 vectors lacking complement inhibitor proteins were prepared by transient transfection according to published procedures 34 using a mixture of four plasmids in the following amounts per 10 cm dish: pMDLg/pRRE (Gag/Pol expression plasmid) at 6.5 mg, pRSV-Rev 34 at 2.5 mg, Transfer Vector (pRRL.sin.CMV.eGFP.ppt.pre or pRRL.sin.CM-V.eGFP.pre) 35 at 10 mg, and 3.5 mg of envelope plasmid (pMD2.VSVG-Env or pMD2.AcNPV.Env 3 or pMD2.Ampho.Env 36 ). For vectors with complement inhibitor proteins, a fifth plasmid (pMD2-DAF, pMD2-MCP or pMD2-CD59) was added to the transfection reaction at 10 mg per plate.…”

In vivo evaluation of an EIAV vector for the systemic genetic delivery of therapeutic antibodies

“…These observations are in agreement with an earlier study where VSV-G-pseudotyped HIV vectors were found to mainly target the spleen, liver and bone marrow of mice injected i.v. 33,34 Discussion Previous evaluation of HIV-1 lentivectors following systemic delivery have demonstrated long-term expression of the transgene for up to 1 year, depending on the transgene and host. [33][34][35] An alternative to HIV-1 is the EIAV-based vector, which has been developed, in part, because HIV-1 is a human pathogen and as a result may face extra hurdles in clinical use.…”

“…33,34 Discussion Previous evaluation of HIV-1 lentivectors following systemic delivery have demonstrated long-term expression of the transgene for up to 1 year, depending on the transgene and host. [33][34][35] An alternative to HIV-1 is the EIAV-based vector, which has been developed, in part, because HIV-1 is a human pathogen and as a result may face extra hurdles in clinical use. In addition to this, the potential advantages of EIAV are its relative simplicity compared to HIV-1, the inability of the parent virus to replicate in human cells and the nonlethal nature of the infection in its natural host, the genus Equidiae.…”

Liver gene therapy: advances and hurdles

“…40,41 Stimulation of hepatocytes to drag them in the cell cycle greatly improved liver transduction as well as modification of lentivectors such as inclusion of the central purine tract to achieve better transduction. [42][43][44] However, it turned out as well that immune response against the transgene product was elicited when large batches of lentivectors were administered to animals, thus precluding long-term expression of the transgene. 45 It was therefore required to use liver-specific promoters to circumvent induction of immune response after in vivo delivery of lentiviral vectors.…”