Biomedical Engineering Reference
In-Depth Information
in trans
using engineered 'helper' or 'packaging' cells to express the viral proteins needed,
limiting remobilization and ensuring stability [18]. Usually the packaging cell lines are
stably transfected with plasmids encoding viral proteins. Typically, packaging cells express
the viral proteins required for packaging of vectors but lack a packaging signal. In contrast,
viral vector plasmids contain a packaging signal together with other essential virally
encoded regulatory sequences, together with a strong constitutive (or inducible) promoter
(which may be a viral promoter) and polyadenylation signals. Genes may be encoded on
more than one separate plasmid to minimize replication-competent virus production. Helper
viruses are not used owing to the likelihood that a replication-competent virus could be
generated through high-frequency recombination [19].
Several different virus families have been exploited for gene therapy applications to take
advantage of their efficiency of infection and cell tropism. A brief overview of some of the
more commonly used viral vector systems is outlined below and in Lawson [17].
11.2.3.1 Retroviruses
Retroviruses are RNA viruses that replicate through an integrated DNA intermediate (see
Coffin
et al
. [20] for review). Retroviral particles encapsidate two copies of the full-length
viral RNA with each copy containing the full genetic information needed for virus repli-
cation, including the
gag
(group-specific antigen),
pro
(protease),
pol
(polymerase) and
env
(envelope) genes. Retroviruses can be classified into simple and complex retroviruses.
Complex viruses encode the essential viral genes above as well as several accessory genes.
A further classification of retroviruses is into oncoretroviruses (mostly simple retroviruses,
e.g. murine leukemia virus; MLV), lentivirus (complex retroviruses, e.g. human immunod-
eficiency virus-1; HIV-1) and spumavirus (complex retroviruses, e.g. human foamy virus
(FV)). Currently, all three types are being exploited as gene therapy tools.
11.2.3.2 Oncoretroviruses
The earliest gene therapy trials using retroviral transfer used vectors based on MLV, an
amphotropic (able to infect human cells) oncoretrovirus. The oncoretrovirus genome is
relatively simple and can be easily rearranged to generate replication-defective recombinant
viral vectors. In general, the retroviral LTR sequences are retained, together with a minimal
packaging signal and the gene of interest. Transfection of oncoretroviral vectors (e.g. pMFG;
[21], pBAbe series [6]) into a suitable eukaryotic packaging cell line (e.g. Omega E; GP
E;
GP EnvAm12 [6, 22]) results in production of recombinant replication-defective particles.
Although straightforward to propagate, the main drawback of using these vectors is that only
dividing cells can take up the particles and integrate the DNA into the host cell genome
for long-term expression of the transgene. Therefore, their usefulness is limited clinically,
since many targets for gene therapy are non-dividing cells [23].
+
11.2.3.3 Lentiviruses
Recently, there has been some progress in the development of vectors based on lentivirus,
in particular HIV-1. The lentiviruses have a more complex replication cycle than oncoretro-
viruses and, therefore, a more complex genome. The major advantage of use of vectors based
on lentiviruses for gene therapy applications is that they are able to infect non-dividing and
terminally differentiated cell types, which is a significant improvement over oncoretrovirus
