Gene Delivery Using Viral Vectors - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 239

An alternative approach is to use the design fusion proteins that can then be
expressed from a single ORF [152] . Vectors containing internal promoters have
been widely used to express multiple genes that make it simple to exploit pro-
moter/enhancer combinations other than the viral LTR for driving gene expression.
Multiple internal promoters can be incorporated in a retroviral vector; it is possible
to express at least three different cDNAs with each from its own promoter [153] . In
vectors with internal promoters, that selection for the expression of one gene resulted
in reduced expression from the other gene also present in the vector [154] . This effect
was called promoter suppression. In some cases, it is insignificant, but this effect is
highly dependent on vector construction [155] .
A modification on the general approch of multiple promoters in retroviral vectors is
the insertion of a promoter-cDNA combination (minigene) in the U3 region of the retro-
viral LTR that produces two copies of the minigene in the resulting provirus. It has been
reported that the minigene expression in these “double-copy” vectors is much higher
than that obtained when the minigene is inserted internally in the vector [156] , because
one of the minigenes is located outside of the retroviral transcription unit, which could
reduce any negative effect of viral transcription [157] . A disadvantage of double-copy
vector is that the RNA form of the vector genome contains two copies of the cDNA, and
because the size of the vector RNA is limited to about 10 kb, the size of the cDNA that
can be accommodated is about half of that possible in other retroviral vectors.
Insertion of IRES elements into retroviral vectors is compatible with the retro-
viral replication cycle. IRES elements were first found in the nontranslated 5 ends
of picornaviruses, where they promote cap-independent translation of viral proteins
and allow expression of multiple coding regions from a single promoter [158] . When
IRES elements are located between ORFs in RNA, it allows efficient translation of
the downstream ORF by promoting entry of the ribosome at the IRES element, fol-
lowed by downstream initiation of translation.
5.4.2.1 High-Titer Vector Production
High-titer virus is desirable, especially when a large number of cells must be infected.
Additionally, high titers are a requirement for transduction of a large percentage of
certain cell types; for example, the frequency of human hematopoietic progenitor cell
infection is strongly dependent on vector titer, and useful frequencies of infection occur
only with very high-titer stocks [159] . Helper virus-free vector titers of 10 7 cfu/mL are
accessible with currently available vectors. Comparison of different vector designs
allowed the definition of crucial elements for high-titer viral production. The internal
protein-encoding regions of MLVs could be deleted without abolishing the infectiv-
ity of the vector [160] . These early vectors retained only a small portion of the 3 end
of the env-coding region. Subsequent work has shown that all of the env -gene-coding
sequences can be removed without further reduction in vector titer. Additional experi-
ments showed that preservation of sequences at the 5 end of the gag gene consider-
ably raised viral titers due to an increase in the packaging efficiency of viral RNA into
virions [161] . To obtain high titers (10 6 to 10 7 ), it is important that this larger signal,
called   , be included in retroviral vectors.