Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1998-12-28
2001-11-06
Nguyen, Dave T. (Department: 1633)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C435S091400, C435S320100, C435S325000, C435S455000, C514S04400A
Reexamination Certificate
active
06312682
ABSTRACT:
This invention relates to retroviral vector production systems and to retroviral vector particles produced by the systems. In particular, it relates to systems and vector particles from which certain retroviral auxiliary factors are absent. The invention also relates to uses of retroviral vectors, in particular for gene therapy.
Retroviral vectors have been the vehicle of choice for clinical gene transfer because of their efficacy, safety, and stable long-term gene expression. According to the United States National Institutes of Health RAC report issued in September 1996 (Ross et al., 1996), 76 out of 107 trials reviewed by the NIH were based on vector systems derived from the murine leukaemia virus (MLV).
One major drawback of these vectors is their inability to infect non-proliferating cells such as neurons, macrophages and haematopoeitic stem cells. These cells are important targets for gene therapy.
Human immunodeficiency virus type 1 (HIV-1) belongs to a sub-family within the retroviruses, the lentiviruses and in common with other members of this family HIV can infect quiescent cells. This makes lentiviruses attractive vectors for gene therapy.
The viral determinants for HIV-1 infection of non-dividing cells are thought to reside in the p17 matrix protein (MA) and vpr (Gallay et al., 1996). MA has karyophilic properties conferred by a conserved stretch of basic residues, which constitute a nuclear localization signal (NLS) (Bukrinsky et al., 1993). Vpr also contains a distinct NLS (Mahalingam et al., 1995). MA-NLS mutant viruses fail to replicate efficiently in macrophages in the absence of a functional vpr gene (Heinzinger et al., 1994). These data have been interpreted to mean that vpr as well as MA function as karyophilic determinants of HIV-1. In the absence of vpr the transduction efficiency of monocyte-derived macrophages decreases by over 50%, in the presence of functional MA. (Naldini et al., 1996).
Following work reported in Lever et al., 1989 which showed the sequences required for packaging of HIV-1, there has been much interest in the development of an HIV-1 based gene therapy vector. Transfer of foreign genes into a human T-cell line by a replication defective HIV-1 based vector was demonstrated by Poznanski et al (Poznansky et al., 1991). Other groups have designed HIV-1 based vectors that are tat-inducible (Buchschacher, Jr. and Panganiban, 1992) or that use heterologous promoters (Shimada et al., 1991). However, the viral titers obtained with these vectors was low (at most 10
3
infectious particles per ml), and it was not clear whether the vector system could guarantee the production of helper virus-free vectors. More recently, new efforts to produce helper virus-free vectors have been based on three-plasmid cotransfections (Richardson et al., 1995). HIV vectors can be pseudotyped with Vesicular Stomatitis Virus glycoprotein (VSV-G) and these particles retain infectivity after concentraton by ultracentrifugation (Akkina et al., 1996). Pseudotyping with VSV-G confers a broader host range and eliminates the chances of recombination to produce wild type HIV envelope. In vivo transduction of non-dividing neuronal cells has been demonstrated with VSV-G pseudotyping of HIV-1 in a three-plasmid cotransfection system (Naldini et al., 1996 and Naldini et al., 1996a).
HIV-1 contains nine genes, three of which: gag, pol and env are found in all retroviruses. These are the structural genes. The other six: vif, vpu, vpr, nef, tat and rev are referred to as auxiliary genes. Other retroviruses have different sets of auxiliary genes in their wild type genomes. Some of the auxiliary genes of other retroviruses are analogous to those of HIV-1, although they may not always have been given the same names in the literature. Analogous auxiliary genes have homology in their nucleotide sequences and perform the same or similar functions. HIV-2 and SIV strains generally contain env, vpr, vif, tat, and nef genes analogous to those of HIV1. HIV-2 and some strains of SIV also contain vpx which, in some SIV strains lacking vpr can be considered analogous to vpr. Lentiviruses other than HIV-1 also contain auxiliary genes which are not analogous to the HIV-1 auxiliary genes. Retrovirus auxiliary genes are reviewed for example by Tomonaga and Mikami (1996) and by Joag et al. in Fields Virology, Vol 2.
To date all vector systems based on HIV contain some or all of the HIV auxiliary genes. Rev acts as an RNA export protein and tat is a major transactivator of the proviral long terminal repeat (LTR). The auxiliary genes play a crucial role in viral replication and pathogenesis. The auxiliary genes have not been fully characterized nor their function defined.
However some of the auxiliary genes are thought to be involved in the pathogenesis of HIV-1. Tat has been implicated in the development of Kaposi's sarcoma (Barillari et al., 1993; Ensoli et al., 1990). HIV vpr has been shown to cause cell arrest and apoptosis and this has been proposed to be the cause of T-Cell dysfunction seen in AIDS patients (Jowett et al., 1995). Also extracellular Vpr present in peripheral blood has been suggested to contribute to tissue-specific pathologies associated with HIV infection since Vpr induces cell proliferation and differentiation (Levy et al, 1993 and Levy et al, 1995).
Since the roles of the auxiliary genes are not clear and they probably play a major role in pathogenesis their removal from HIV-1 vector production systems is desirable, provided that sufficiently high retrovirus vector titer and ability to transduce non-proliferating cells can be retained.
Naldini et al's data shows that the presence or absence of vpu has no effect on the vector particle titer. That is, a packaging system they used produced a titer of 4×10
5
when pseudotyped with VSV-G and this system was env and vpu negative. In another system which was only env negative they obtained the same titer (Naldini et al. 1996 and Naldini et al. 1996a). However, as already discussed another system of Naldini et al which was vpr negative as well as vpu negative gave a transduction efficiency which was decreased by 50% compared to a vpr positive system.
We have now discovered that leaving some or all of the auxiliary genes out of retrovirus vector production systems does not significantly compromise vector particle titers or the ability of the vector verticles to transduce non-dividing cells.
The invention therefore provides in one aspect a retroviral vector production system for producing lentivirus-based, replication defective vector particles for gene therapy, said vector particles capable of infecting and transducing non-dividing mammalian target cells, which system comprises a set of nucleic acid sequences encoding the components of the vector, wherein one or more functional genes chosen from the HIV-1 auxiliary genes vpr, vif, tat and nef or from the analogous auxiliary genes of other lentiviruses, which auxiliary genes are normally present in the lentivirus on which the vector particles are based, is or are absent from the system. The functional vpu gene may also be absent, with the proviso that when the production system is for an HIV-1 based vector and vpr and vpu are both absent, so also is one of the other auxiliary genes.
In another aspect, the invention provides retroviral vector particles produced by a retroviral vector particle production system described herein.
In yet another aspect, the invention provides a DNA construct for use in a retroviral vector production system described herein, said DNA construct encoding a packagable RNA vector genome for a retroviral vector particle and operably linked to a promoter, wherein all of the functional retroviral auxiliary genes are absent from the construct, other than rev which is optionally present. The DNA construct may be provided as part of a set of DNA constructs also encoding some or all of the structural components of the vector particles.
In further aspects, the invention provides the use of retroviral vector particles as described herein, for gene therapy
Kim Narry
Kingsman Alan John
Kingsman Susan Mary
Mitrophanous Kyriacos
Frommer Lawrence & Haug
Kowalski Thomas J.
Nguyen Dave T.
Oxford BioMedica plc
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