4Bioengineering.com

Recombinant retroviruses used for human gene therapy are derived from the wild-type Moloney murine leukemia retrovirus. The recombinant viruses are structurally identical to the wild-type virus but carry a genetically engineered genome that encodes the therapeutic gene and sequences which regulate its expression. Recombinant retroviruses cannot self-replicate, but can infect and integrate their genomes into the chromosomal DNA of the target cell.

Recombinant retroviruses consist of a two-part system composed of a retroviral vector and a packaging cell line. The retroviral vector is essentially the wild-type genome with all the viral genes removed. It encodes the therapeutic gene, regulatory sequences necessary for the expression of the gene, and a packaging sequence required for its efficient incorporation into virus particles.

The second part of the system is the packaging cell line, which expresses all the viral genes required to form an infectious virus particle. Gag encodes the proteins that form a capsid around the viral RNA genome. Pol encodes the enzymatic activities of the virus, including reverse transcriptase. Env encodes the virus attachment proteins (VAPs) that cover the surface of the virus particle.

VAPs are the primary determinant of the host range of the virus because they mediate the binding of the virus to its receptors on the cell surface. Amphotropic retroviruses, from which protrude VAPs that bind to the amphotropic receptor expressed in most human tissues, are used for human gene transfer because they can infect human cells.

Cells that produce recombinant retroviruses are made by transfecting the packaging cell line with the retroviral vector. The proteins encoded by gag and pol recognize the psi packaging sequence in the viral genomic RNA (transcribed from the transfected retroviral vector) and form a capsid around two identical copies of the viral genomic RNA. The capsid buds from the packaging cell line, acquires a lipid-bilayer with an array of protruding VAPs, and is shed into the surrounding culture medium, which is harvested and used as a viral stock.

The viral stock is then used to transduce (i.e., permanently integrate the therapeutic gene into the chromosomal DNA of) the target cells. Successful transduction requires the completion of a complex series of steps. The virus particles must first be transported to the surface of the cells where the VAPs of the viruses bind to their receptors. After the virus particles bind to their cell surface receptors, they enter the cell and release their RNA genomes into the cytoplasm.

The RNA genomes are reverse transcribed from RNA to DNA, transported into the nucleus, and integrated into the chromosomal DNA. Expression of the therapeutic genes is controlled by regulatory sequences genetically engineered into the retroviral vectors, or by viral regulatory sequences encoded in the long terminal repeats that bracket the ends of the integrated viral genomes.