The retroviral life cycle

The retroviral life cycle (Figure 14.B1) begins with the entry of the enveloped virus into the cell. The viral reverse transcriptase enzyme then copies the viral RNA genome into a single (minus) DNA strand and, using this as a template, generates double-stranded DNA. The double-stranded DNA is then randomly integrated into the host cell genome (the proviral DNA). Transcription of the proviral genes host cell's transcription machinery yields mRNA that directs synthesis of mature virion particles. The viral particles bud out from the cell's plasma membrane, picking up a membrane-derived outer coat as they do so.

Nucleoprotein

Nucleoprotein

gag (codes for core viral protein),pol (codes for reverse transcriptase) and env (codes for the viral envelope proteins). At either end of the viral genome are the long terminal repeats (LTRs), which harbour powerful promoter and enhancer regions and sequences required to promote integration into the host DNA. Also present, immediately adjacent to the 5' LTR, is the packing sequence (y). This is required to promote viral RNA packaging.

The ability of such retroviruses to (a) effectively enter various cell types and (b) integrate their genome into the host cell genome in a stable, long-term fashion, made them obvious potential vectors for gene therapy.

The construction of retroviruses to function as gene vectors entails replacing the endogenous viral genes, required for normal viral replication, with the exogenous gene of interest (Figure 14.4a). Removal of the viral structural genes means that the resulting vector cannot itself replicate. In

gag pol

gene of interest LTR

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-deficient, viral particles, harbouring 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 modifications 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, specific promoters have been introduced in order to attempt to control expression of the inserted gene. Most work has focused upon the use of tissue-specific promoters in an effort to limit expression of the desired gene to a specific tissue type.

The most commonly employed (recombinant deficient) retrovirus used in this regard has been derived from the Maloney murine leukaemia virus (MoMuLV).

Retroviruses display a number of properties/characteristics that influence their potential as vectors 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 efficiency 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.

Packing cell

Assembled retroviral vectors harboring the target gene which exit the cell

Figure 14.5 The use of packing cells to generate replication-deficient retroviral vectors. The packaging cell is an engineered animal cell into which the retroviral gag (g), pol (p) and env (e) genes have been introduced. The cell line chosen must be one which the (replication-deficient) virus can infect. The engineered retroviral vector genome (which is carrying the target gene; TG) is then incubated with the packing cell. This results in the generation and assembly of mature replication-deficient retroviral vector particles. These exit the cell and will replicate by entering other packaging cells. By completing a number of such replication cycles, large quantities of the desired retroviral vectors are produced

Assembled retroviral vectors harboring the target gene which exit the cell

Figure 14.5 The use of packing cells to generate replication-deficient retroviral vectors. The packaging cell is an engineered animal cell into which the retroviral gag (g), pol (p) and env (e) genes have been introduced. The cell line chosen must be one which the (replication-deficient) virus can infect. The engineered retroviral vector genome (which is carrying the target gene; TG) is then incubated with the packing cell. This results in the generation and assembly of mature replication-deficient retroviral vector particles. These exit the cell and will replicate by entering other packaging cells. By completing a number of such replication cycles, large quantities of the desired retroviral vectors are produced

The fact that they have been well studied, display almost 100 per cent transduction efficacy in sensitive cells and that the transferred genes are usually subject to long-term, fairly high-level expression renders retroviruses powerful potential vectors. These advantages form the basis of their widespread use in this regard.

However, many of their other characteristics serve to curtail the application of retroviruses as gene therapy vectors. From a practical standpoint, retroviral vectors are relatively labile. Thus, although retroviruses are relatively easy to propagate, they are often damaged by subsequent purification and concentration, which are steps essential for their clinical use. In most instances, their ability to infect only dividing cells clearly restricts their use. Their lack of selectivity in terms of the dividing cell types they infect is also a disadvantage. They will not infect all dividing cell types: the entry of any specific retrovirus is dependent upon the existence of an appropriate viral receptor on the surface of a target cell. As the identity of most retroviral receptors remains unknown, it remains difficult to predict the entire range of cell types any retrovirus is likely to infect during a gene therapy protocol. Integration and expression of the exogenous gene in cells other than target cells could result in physiological complications.

An additional drawback with regard to retroviral-based vectors is the propensity of the transferred gene to integrate randomly into the chromosomes of the recipient cells. Integration of the transferred DNA in the middle of a gene whose product plays a critical role in the cell could irrevocably damage cellular function. For example, disruption of a central metabolic enzyme could cause cell death, and disruption of a tumour suppresser gene could give rise to cellular transformation. In addition, integration of the proviral nucleic acid to sites adjacent to quiescent cellular proto-oncogenes could result in their activation.

The theoretical complications posed by random chromosomal integration became a medical reality in 2002, when two children who had received retroviral-based gene therapy 2 years previously developed a leukaemic-like condition. The initial clinical trial aimed to treat X-linked severe combined immunodeficiency (SCID-X1), a hereditary disorder in which T-lymphocytes and NK cells in particular do not develop, due to a mutation in the gene coding for the yc cytokine receptor subunit. The clinical consequence is near abolition of a functional immune system.

The trial entailed retroviral-mediated ex vivo transduction of haematopoietic stem cells from 10 young SCID-X1 sufferers, with subsequent re-infusion of the treated cells. A marked and prolonged clinical response in which the condition was essentially reversed was observed in 9 out of the 10 patients. The prolonged response was likely due to the transduction of pluripotent progenitor cells with self-renewal capacity (Chapter 10). However, the two youngest patients (1 and 3 months old at the time of treatment) developed uncontrolled proliferation of mature T-lymphocytes 30 months and 34 months after gene therapy respectively.

It has subsequently been shown that this leukaemia-like condition was triggered by proviral integration at a site near the LM02 proto-oncogene promoter, leading to gene activation. This development resulted in an initial ban on further retroviral-based gene therapy trials in some world regions, and the proportion of trials undertaken subsequently using retroviral-based systems has dropped significantly.

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