Nucleic-acid- and cell-based therapeutics - Pharmaceutical Biotechnology: Concepts and Aapplications - page 428

LTR gag pol env LTR

(a)

gene of interest

LTR LTR

(b)

Figure 14.4 Schematic representation of (a) the proviral genome of a basic retrovirus and (b) the genome of
a basic engineered retroviral vector carrying the gene of interest. Refer to text for further details

order to generate mature virion particles harbouring the vector nucleic acid (Figure 14.4b), this
genetic material must be introduced into a 'packing cell'. These are recombinant cells that have
previously been engineered to contain the gag , pol and env structural genes (Figure 14.5). In this
way, packing cells are capable of producing mature, but replication-defi cient, viral particles, har-
bouring the gene to be transferred (see Section 14.3.3). These viral particles function as so-called
one-time, single-hit gene transfer systems.
More recently, various modifi cations have been introduced to this basic retroviral system. The
inclusion of the 5´ end of the gag gene is shown to enhance levels of vector production by up to
200-fold. Additionally, specifi c promoters have been introduced in order to attempt to control ex-
pression of the inserted gene. Most work has focused upon the use of tissue-specifi c promoters in
an effort to limit expression of the desired gene to a specifi c tissue type.
The most commonly employed (recombinant defi cient) retrovirus used in this regard has been
derived from the Maloney murine leukaemia virus (MoMuLV).
Retroviruses display a number of properties/characteristics that infl uence their potential as vec-
tors in gene therapy protocols. These may be summarized as follows:

• retroviruses as a group have been studied in detail and their biochemistry and molecular biology

are well understood;

• most retroviruses can integrate their proviral DNA only into actively replicating cells;

• the effi ciency of gene transfer to most sensitive cell types is very high, often approaching 100

per cent;

• integrated DNA can be subject to long-term, relatively high-level expression;

• proviral DNA integrates randomly into the host chromosomes;

• retroviruses are promiscuous, in that they infect a variety of dividing cell types;

• complete copies of the proviral DNA are passed on to daughter cells if the original recipient

cell divides;

• good, high-level, titre stocks of replication-incompetent retroviral particles can be produced;

• safety studies using retroviral vectors have already been carried out on various animal species.