Gene Delivery Using Viral Vectors - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 240

A number of methods for concentration of retroviral vectors have been developed,
including centrifugation, hollow fiber filtration, and tangential flow filtration. About a
20-fold increase in viral titer can be achieved but the relative fragility of retroviral env
protein limits the ability to concentrate retroviral vectors, and concentrating the virus
usually results in a poor recovery of infectious virions. This problem can be overcome
by the substitution of the retroviral env protein with the more stable VSV-G protein
that allows more effective vector concentration with better yields. Additionally, high-
titer vectors methods to facilitate vector infection of cells are also vital for maximiz-
ing the efficiency of gene transfer. Polybrene and protamine sulfate are usually used
to facilitate binding and entry of retroviruses and various cationic lipids that can result
in further improvements [162] . Cell transduction is limited by viral diffusion in solu-
tion. Passage of the virus through a porous membrane on which target cells have been
placed can improve transduction rates [163] . Centrifugation of vectors onto cells can
increase transduction rates that bring the virus to the cells or prevent the virus from dif-
fusing away from the cells [164] .
5.4.2.2 Limitations
1. Sequences inserted into retroviral vectors must be compatible with the retroviral life cycle
and, in particular, must allow the efficient transcription of the complete retroviral genome.
2. Even though there appears to be no lower limit on retroviral vector size, there do appear
to be upper limits. The genome of a typical replication-competent murine retrovirus is
about 8.3 kb, whereas that of RSV, which contains src sequences in addition to the normal
complement of viral genes, is about 9.3 kb. The maximum size for a replication-competent
spleen necrosis virus vector is similar, about 10 kb. Although this precludes vector trans-
mission of large genes, most cDNAs can be accommodated.
3. Retroviral vectors based on murine viruses appear to transduce only dividing cells, so these
vectors have limited utility.
4. Retroviral vector expression is subject to suppression in embryonic cells. The retroviral
LTRs and the tRNA-binding site are clearly involved in this effect, but other elements may
also be implicated. Modifications have been made in retroviral vectors to overcome these
effects, but the possibilities for vector alteration are more limited than in simple plasmid-
based vectors.
The most basic example of a retroviral packaging cell line is the SE21Q1b cell
line that expressed a naturally occurring avian sarcoma virus mutant that failed to
package viral genomic RNA [165] . This cell line provided the stimulus for construct-
ing synthetic packaging cell lines. It is always difficult to obtain a reliable rate for
helper virus production from a particular packaging cell line because helper virus
generation is dependent on both the cell line and the vector. Helper virus assays dif-
fer considerably in their sensitivities, and helper virus production can be the result of
contamination of packaging cells with exogenous helper virus. But the use of several
packaging cell lines (e.g., PA317,  CRIP, PG13, and GP  envAm12) for human
gene therapy has also shown that helper virus generation occurs at a very low rate
in the newer packaging cell lines, and it is possible to make hundreds of liters of
helper-free stocks. The older packaging cell lines such as  -2 can also be kept free
of helper by freezing vector-containing cells soon after their construction, passaging