Gene Delivery Using Viral Vectors - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 242

recombination of helper sequences with vector or endogenous viral sequences; this is
more likely with longer periods of cell culture. If vector-producing packaging cells are
cocultivated with a packaging cell line that expresses an env gene that does not inter-
fere with the env gene in the first helper cell line, the vector can “ping-pong” between
cells, resulting in multiple reinfection events. Vector titer can be increased by using this
ping-pong amplification procedure, and there is also a greater chance that the vector
will rearrange and, because of that helper virus, will be produced during the amplifica-
tion process. A report of a very high-titer murine retroviral vector (10 10 cfu/mL) pro-
duced by using this technique was not reproducible [170,171] . An increase in titer of
up to 10-fold is typical.
5.4.3 Application of Retroviral Vectors
5.4.3.1 Cell Marking
It is generally useful to follow cells after they have been introduced into a human
patient because the transplanted cells cannot be distinguished from preexisting cells
in the subject; although cells can be marked with radioisotopes or dyes, a genetic tag
is a better marker for long-term assessment of the persistence and distribution of the
transplanted cells.
The first virus-mediated gene transfer trial in humans involved marking lympho-
cytes with a retroviral vector to study their persistence and distribution in patients
[172] . The gene-marking procedure had no toxic effects; however, all patients were ter-
minal cancer patients and thus long-term toxicities could not be adequately evaluated.
In one more example, HIV antigen-specific cytotoxic T lymphocytes, used in an
attempt to kill HIV-infected cells in patients, were marked with a retroviral vector to
study the persistence and distribution of cells, following their infusion into patients.
This technique also allowed the detection of the infused cells for the short term, and
five of the six patients developed cytotoxic-T-lymphocyte responses against cells carr-
ying the marker gene [173] . Retrovirus marking has also been used to detect malig-
nant cells that may still be present in bone marrow used for autologous transplantation.
Marking human marrow used for autologous transplantation with retroviral vectors
has been done to study the role of the infused marrow in long-term hematopoiesis in
humans and to examine gene transfer rates in human hematopoietic cells [174] .
5.4.3.2 Genetic Diseases
Gene therapy relies on the addition of genes to correct inherited genetic defects
because the rates of gene repair by homologous recombination are currently too low.
In the past, the U.S. Recombinant DNA Advisory Committee approved gene therapy
trials to correct a genetic disease, severe combined immunodeficiency due to defects
in the enzyme ADA. ADA-deficient persons have severely reduced levels of T and
B lymphocytes, and they die in childhood from infection. For treatment of this ADA
deficiency disease, lymphocytes were harvested from blood, by leukapheresis (a lab-
oratory procedure in which white blood cells are separated from a sample of blood),
transduced with a retroviral vector expressing ADA and NeoR (Npt), grown to large