Gene therapy includes any one or more of: the addition, the replacement, the deletion, the supplementation, the manipulation etc. of one or more nucleotide sequences in, for example, one or more targeted sites—such as targeted cells. If the targeted sites are targeted cells, then the cells may be part of a tissue or an organ. General teachings on gene therapy may be found in Molecular Biology.
By way of further example, gene therapy also provides a means by which any one or more of: a nucleotide sequence, such as a gene, can be applied to replace or supplement a defective gene; a pathogenic gene or gene product can be eliminated; a new gene can be added in order, for example, to create a more favourable phenotype; cells can be manipulated at the molecular level to treat cancer or other conditions—such as immune, cardiovascular, neurological, inflammatory or infectious disorders; antigens can be manipulated and/or introduced to elicit an immune response—such as genetic vaccination.
In recent years, retroviruses have been proposed for use in gene therapy. Essentially, retroviruses are RNA viruses with a life cycle different to that of lytic viruses. In this regard, when a retrovirus infects a cell, its genome is converted to a DNA form. In other words, a retrovirus is an infectious entity that replicates through a DNA intermediate. More details on retroviral infection etc., are presented later on.
As mentioned above, retroviruses have been proposed as a delivery system (otherwise known as a delivery vehicle or delivery vector) for inter alia the transfer of a NOI, or a plurality of NOIs, to one or more sites of interest. The transfer can occur in vitro, ex vivo, in vivo, or combinations thereof. When used in this fashion, the retroviruses are typically called retroviral vectors or recombinant retroviral vectors. Retroviral vectors have even been exploited to study various aspects of the retrovirus life cycle, including receptor usage, reverse transcription and RNA packaging.
In a typical recombinant retroviral vector for use in gene therapy, at least part of one or more of the gag, pol and env protein coding regions may be removed from the virus. This makes the retroviral vector replication-defective. The removed portions may even be replaced by a NOI in order to generate a virus capable of integrating its genome into a host genome, but wherein the modified viral genome is unable to propagate itself due to a lack of structural proteins. When integrated in the host genome, expression of the NOI occurs—resulting in, for example, a therapeutic effect. Thus, the transfer of a NOI into a site of interest is typically achieved by: integrating the NOI into the recombinant viral vector; packaging the modified viral vector into a virion coat; and allowing transduction of a site of interest—such as a targeted cell or a targeted cell population.
It is possible to propagate and isolate retroviral vectors (e.g., to prepare suitable titres of the retroviral vector) for subsequent transduction of, for example, a site of interest by using a combination of a packaging or helper cell line and a recombinant vector.
In some instances, propagation and isolation may entail isolation of the retroviral gag, pol and env genes and their separate introduction into a host cell to produce a “packaging cell line”. The packaging cell line produces the proteins required for packaging retroviral RNA. However, when a recombinant vector carrying a NOI and a psi region is introduced into the packaging cell line, the helper proteins can package the psi-positive recombinant vector to produce the recombinant virus stock. This is commonly known as a “producer cell”. The vector can be used to infect cells to introduce the NOI into the genome of the cells. The recombinant virus whose genome lacks all genes required to make viral proteins can infect only once and cannot propagate. Hence, the NOI is introduced into the host cell genome without the generation of potentially harmful retrovirus. A summary of the available packaging lines is presented in “Retroviruses”.
The design of retroviral packaging cell lines has evolved to address the problem of inter alia the spontaneous production of helper virus that was frequently encountered with early designs. As recombination is greatly facilitated by homology, reducing or eliminating homology between the vector and gag/pol has reduced the problem of helper virus production.
More recently, packaging cells have been developed in which the gag/pol and env viral coding regions and the viral vector are carried on separate expression plasmids that are independently transfected into a packaging cell line so that three recombinant events are required for wild type viral production.
Transient transfection can be used to make vectors. In this regard, transient transfection has been used if the vector or retroviral packaging components are toxic to cells. Components typically used to generate retroviral vectors include a plasmid encoding the Gag/Pol proteins, a plasmid encoding the env protein and a plasmid containing a NOI. Vector production involves transient transfection of one or more of these components into cells containing the other required components. If the vector encodes toxic genes or genes that interfere with the replication of the host cell, such as inhibitors of the cell cycle or genes that induce apoptosis, it has proved difficult to generate stable vector-producing cell lines, thus transient transfection can be used to produce the vector before the cells die. However, the aforementioned technique can be problematic in the sense that the titre levels are not always satisfactory, it is difficult to make large batches of virus, and safety tests must be performed on each small batch.
In view of the toxicity of some HIV proteins, e.g., the HIV protease—which can make it difficult to generate stable HIV-based packaging cells —HIV vectors are usually made by transient transfection of vector and helper virus. Some workers have even replaced the HIV env protein with that of vesicular stomatis virus (VSV). A drawback, however, with this approach is that the VSV-G protein is quite toxic to cells.
Thus, and as indicated, retroviral vectors are used extensively in biomedical research and for gene therapy. Current methods for the production of retroviral vectors make use of the fact that the two roles of the wild-type retrovirus genome, that is protein encoding and as a template for new genome copies, can be de-coupled. Protein that is required for the assembly of new virus particles and for enzyme and regulatory functions can be produced by non-genome sequences in, for example, a mammalian packaging cell line. A genome sequence lacking the protein encoding functions is provided, so that the resulting retroviral vector particles are capable of infecting but not of replicating in a target cell. The genome sequence can also be designed for delivery and integration of a therapeutic gene. Standard methods for producing murine leukaemia virus (MLV)-based vectors, for example, include use of stably engineered cell lines expressing the gag-pol and env genes (the packaging components) of MLV. These will package a compatible retroviral vector genome introduced by transfection with an appropriate plasmid. An alternative method for producing HIV based vectors, for example, involves simultaneous transient transfection of gag-pol, env, and vector genome plasmids into suitable cells.
Although the principles of these systems are well understood, in practice the re-constructed virus assembly system often fails to generate the quantity of vector particles required in practice for use in gene therapy. Retroviral vector particles are generally harvested by removing supernatant from a culture of particle-producing cells. The resulting suspension may be concentrated with respect to the vector particles, using physical methods, but only to a limited degree as problems such as aggregation and damage tend to arise. Thus, it may only be possible to concentrate a suspension of vector particles by up to 100-fold.
The same issues arise when trying to produce a recombinant protein which is potentially harmful to the host cell in which it is being expressed. One approach that has been considered is to use an inducible system; however, this does not overcome the basic problem of toxicity, inducible production is only transient and problems have arisen from “leaky” promoters.
Vectors based on human immunodeficiency virus type 1 (HIV-1) offer a means for the delivery of therapeutic transgenes into a wide variety of cell types, both dividing and non-dividing. HIV-1 based vectors have commonly been produced either transiently or using packaging cell lines in which vector production is induced. Until recently no stable packaging cells to continuously produce high titre HIV-1 vectors were available, because it had been difficult to stably express large amounts of HIV-1 gag-pol. Furthermore, it has also proven to be difficult to continuously produce the rhabdovirus vesicular stomatitis virus G protein (VSV-G), which is most commonly used to replace HIV-1's own envelope proteins and ‘pseudotype’ HIV-1 vector particles.