Gene therapy trials to treat severe combined immunodeficiency (SCID) were halted or restricted in the USA when leukemia was reported in three of eleven patients treated in the French X-linked SCID ( X-SCID) gene therapy trial. This problem has recently begun to be addressed by utilizing zinc finger nucleases or by including certain sequences such as the beta-globin locus control region to direct the site of integration to specific chromosomal sites. If the gene happens to be one regulating cell division, uncontrolled cell division (i.e., cancer) can occur.
If genetic material happens to be inserted in the middle of one of the original genes of the host cell, this gene will be disrupted (insertional mutagenesis). One of the problems of gene therapy using retroviruses is that the integrase enzyme can insert the genetic material of the virus in any arbitrary position in the genome of the host. Producing recombinant retroviral vectors. Following insertion of the desired gene into in the retroviral DNA vector, and maintainance of the proper packaging cell line, it is now a simple matter to prepare retroviral vectors (see figure 3).įigure 3. Early packaging cell lines contained replication competent retroviral genomes and a single recombination event between this genome and the retroviral DNA vector could result in the production of a wild type virus. These packaging cell lines have been made so that they contain the gag, pol and env genes. Packaging cell lines provide all the viral proteins required for capsid production and the virion maturation of the vector. In order to produce vector particles a packaging cell is essential. These are replaced by the therapeutic gene. Retroviral vectors are created by removal op the retroviral gag, pol, and env genes. Sometimes the genes of the retrovirus do not express their information immediately. If this host cell divides later, its descendants will all contain the new genes. Now that the genetic material of the virus is incorporated and has become part of the genetic material of the host cell, we can say that the host cell is now modified to contain a new gene. This process is done by another enzyme carried in the virus called integrase (see figure 2).
That is, it must be inserted into the large DNA molecules in the cell (the chromosomes).
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After this DNA copy is produced and is free in the nucleus of the host cell, it must be incorporated into the genome of the host cell. It is carried out by one of the enzymes carried in the virus, called reverse transcriptase. The process of producing a DNA copy from an RNA molecule is termed reverse transcription.
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