Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Utility Patent
1998-10-21
2001-01-02
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S007100, C435S455000, C435S456000, C435S235100, C435S236000, C435S237000, C435S325000, C435S320100
Utility Patent
active
06168916
ABSTRACT:
This invention relates to improvements in retroviral vectors for gene therapy and other uses. In particular the invention relates to methods for producing improved retroviral vectors, and for producing improved retroviruses which may be used to make retroviral vectors. The invention further relates to the retroviruses and retroviral vectors produced by such methods and to retroviral production systems and packaging cell lines derived using the retroviruses and retroviral vectors.
Within the field of gene therapy retroviral vectors are the most widely used gene delivery system. The most commonly used retroviral vectors are based on murine leukaemia virus (MLV). Furthermore, MLV-based vectors are the only retroviral vectors that have been used in human clinical trials (Morgan & Anderson 1993). MLV is a simple C-type retrovirus, the molecular biology of which has been reviewed extensively. The commonly used vectors are usually produced using packaging cells lines containing integrated copies of a gag-pol expression cassette and an env expression cassette (McClaughlin et al 1990). A plasmid encoding the vector genome (RNA) is transfected into the packaging cell to produce the RNA species that is packaged into the viral particles encoded by gag-pol and env. The retroviral particles so produced generally have one of two distinct tropisms. If the env gene in the packaging cell encodes envelope proteins from an ecotropic virus then the retroviral vector will transduce only murine and rat cells. If the env gene is from an amphotropic virus then the vector will transduce a broader range of cell types including human cells. The determinants of tropism reside substantially, therefore, in the envelope protein(s) (Hunter & Swanstrom, 1990). In particular the variable regions known as VRA and VRB determine whether the surface protein (SU, gp70) binds the ecotropic or amphotropic receptors (Baftini et al, 1992). The ecotropic receptor is the murine basic amino acid transporter (Albritton et al 1989) and the amphotropic receptor is a widely distributed phosphate transporter (Miller et al 1994; VanZeijl et al, 1994). In order to transduce human cells for therapeutic purposes amphotropic vectors are used.
Amphotropic vectors are derived from natural amphotropic MLV variants, isolated in 1976, that are capable of infecting a wide range of cell types (Rasheed et al., 1976; Hartley and Rowe, 1976). These vectors were first produced in the mid-1980s (e.g. Cone and Mulligan, 1984) using components from the original viruses. Although amphotropic vectors transduce human cells these vectors are generally an order of magnitude less efficient at transducing human cell lines compared with their efficiency on murine cells. Also, even though they are being used widely for gene therapy trials in man the efficiency of transduction is very variable from primary cell type to cell type (Rosenberg et al., 1990; Grossman et al., 1994) and this seriously limits the usefulness of these systems. The origin of this variability is not known.
There is a need for retroviral vectors which are more efficient at transducing human cells. There is also a need for retroviral vectors which are more suited than existing vectors for gene therapy in humans or other uses of retroviral vectors such as induction of disease mimics in animals. The invention addresses these needs.
The invention provides in one aspect a method of making a retroviral vector having one or more selected characteristics, which method comprises:
(i) providing a starting retrovirus or retroviral vector, and a host cell for the retrovirus or the retroviral vector;
(ii) subjecting the starting retrovirus or retroviral vector to a selection process in vitro which selection process involves a plurality of rounds of infection of the host cell during which the retrovirus or retroviral vector evolves to attain the selected characteristic or characteristics; and
(iii) where a starting retrovirus is provided in (i), using the retrovirus resulting from (ii) in at least one component of a retroviral vector production system for producing retroviral vectors having the selected characteristic or characteristics.
Thus, by a process which may generally be described as evolution in vitro, a retroviral vector can be produced which has characteristics more suited to a chosen purpose. A selected characteristic according to the invention will be associated with the genome of the retrovirus or retroviral vector resulting from the selection process (ii). The starting retrovirus or retrovirus vector will generally be a population of retrovirus or retrovirus vectors, which population evolves in vitro in the method according to the invention.
In another aspect, the invention provides retroviral vectors made by a method as described herein.
In a further aspect the invention provides a retroviral vector production system, said system having at least one component associated with a selected characteristic or characteristics attained by a method as described herein and transferred from the retrovirus or retroviral vector into the retroviral vector production system.
The retroviral vector production system preferably comprises a packaging cell line transfected with a DNA construct encoding a packagable retroviral vector genome. The selected characteristic or characteristics may be associated with the vector genome or with one or more components of the packaging cell line, or with both.
In still further aspects, the invention provides expression vectors comprising components of retroviral vector production systems, which components are derived from retroviruses or retroviral vectors resulting from a selection process as described herein. The invention also provides methods of making retroviral vector production systems which systems comprise such components.
The starting retrovirus or retroviral vector may be derived from any suitable retrovirus or retroviruses. MLV is presently the most extensively studied retrovirus in this field and MLV-based vectors have been used in gene therapy. The invention is not however limited to MLV-based vectors. Suitable retroviruses include other oncoretroviruses (the sub-group of retroviruses of which MLV is a member), or lentiviruses (which include HIV, SIV, FIV, BLV, EIAV, CEV and visna virus), or retroviruses from other sub-groups.
The term retroviral vector is used here to describe a defective retrovirus which is not by itself replication competent. However, it is capable of infection of a host cell, and thus has a genome which is capable of integrating into the host cell genome and has a packaging signal. Thus, a retroviral vector usually lacks at least one of the packaging components gag-pol, and env. When a starting retroviral vector is used in the method according to the invention, the host cell will need to contain the missing packaging component or components in order that infection of the host cell by the vector may result in the production of further retroviral vector particles.
A starting retrovirus contains functional env and gag-pol in its genome and is replication competent. Even so, a starting retrovirus is not necessarily a wild type retrovirus; generally it will have been modified in some way while still remaining replication competent. The use of modified retroviruses is preferred because these can be well-characterised and designed to meet certain needs. For example, for practical reasons it may be necessary to include a selectable marker in the genome of the starting virus, so that infected host cells can be selected for. Also, it may be preferable for the virus genome to be under the transcriptional control of a high efficiency promoter such as the cytomegalovirus (CMV) promoter, in place of the viral LTR U3 promoter. The purpose of this would be to ensure that viral genome production in infected cells is not a rate-limiting step during the selection process, so that a characteristic such as more efficient infection can be effectively selected for. These features of the genome may be employed in the genome of a starting retroviral vect
Cannon Paula Marie
Kingsman Alan John
Kingsman Susan Mary
Nowak Martin Andreas
Guzo David
Merchant & Gould P.C.
Oxford Biomedica (UK) Limited
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