Lentiviral Vectors and Gene Therapy
Advances in the knowledge of gene regulation and virus life cycle in the past decade have allowed scientists to make significant progress in using virus-derived vectors in clinical trials of human gene therapy. To take advantage of these advances, we focus on designing vectors derived from lentiviruses, such as human immunodeficiency virus (HIV)-1 and feline immunodeficiency virus (FIV), to deliver genes into mammalian cells for possible treatment of human diseases. Lentiviral vectors can efficiently transduce proliferating, as well as quiescent cells, both in culture and in vivo. The viral genome integrates into host chromosomes, and the inserted gene can be maintained in the cells permanently. Since almost all viral genes are removed, up to 8-kb foreign DNA can be inserted into a lentiviral vector.
Currently, we focus on four specific goals:
For mass vector production, a stable packaging cell line is required. Various studies suggest that vector titers are frequently in direct proportion to the amounts of viral structural proteins, including gag, pol and env proteins expressed in such packaging cell lines. We are testing several approaches to generate human cell lines expressing high-level viral structure proteins.
Since HIV induces acquired immunodeficiency syndrome (AIDS) in humans, we must take steps in vector design to enhance the safety of using lentiviral vectors in humans. We will introduce multiple mutations into lentiviral vectors to minimize the possibility of generating replication-competent helper virus in the vector preparations.
We will target the human FVIII gene for liver specific transduction and gene expression with lentiviral vectors. A deficiency in the blood clotting protein FVIII results in a bleeding disorder in hemophilia A patients. Therapy with human plasma-derived FVIII has resulted in transmission of several human viruses. Even the recombinant FVIII is problematic, due to the high cost of this protein. Somatic gene therapy, therefore, represents an alternative approach to treat this disease. Since the major organ producing FVIII is liver, we will generate lentiviral vectors that specifically target hepatocytes. In addition, liver-specific cis-regulatory sequences will be tested for their ability to drive persistent FVIII gene expression in hepatocytes.
We will then test their efficacy in stem cell-derived myeloid and lymphoid cells, both in culture and in animal models.