Vector system

This application is a continuation-in-part of U.S. application Ser. No. 10/408,456, filed on Apr. 7, 2003, which is a continuation-in-part of International Application No. PCT/GB01/04433, filed on Oct. 5, 2001, which claims priority to British Application No. GB 0024550.6, filed on Oct. 6, 2000.

This application makes reference to U.S. application Ser. No. 10/008,610, filed on Nov. 8, 2001, which claims priority to U.S. Provisional Application Ser. No. 60/247,604, filed on Nov. 9, 2000.

This application also makes reference to U.S. application Ser. No. 10/841,603, filed on May 7, 2004, which is a continuation-in-part of International application No. PCT/GB03/064665, filed on Feb. 3, 2003,which claims priority to British application Nos. GB 0202403.2, filed on Feb. 1, 2002, and GB 0212768.6, filed on May 31, 2002.

This application also makes reference to U.S. Pat. Nos. 6,312,682 and 6,312,683, the contents of which are expressly incorporated herein by reference.

All of the foregoing applications, as well as all documents cited in the foregoing applications (“application documents”) and all documents cited or referenced in the application documents are incorporated herein by reference. Also, all documents cited in this application (“herein-cited documents”) and all documents cited or referenced in herein-cited documents are incorporated herein by reference. In addition, any manufacturer\'s instructions or catalogues for any products cited or mentioned in each of the application documents or herein-cited documents are incorporated by reference. Documents incorporated by reference into this text or any teachings therein can be used in the practice of this invention. Documents incorporated by reference into this text are not admitted to be prior art.

The present invention relates to a vector system comprising a mutated post-transcriptional regulatory element. In particular, the present invention relates to a mutated WPRE sequence that can efficiently express nucleotides of interest in a retroviral vector system. The present invention also relates to methods of delivering and expressing nucleotides of interest to a target cells.

Retroviral vector systems, such as lentiviral vector systems, have been proposed as a delivery system for, inter alia, the transfer of a nucleotide of interest to one or more sites of interest. Indeed, the concept of using viral vectors for gene therapy is well known (Verma and Somia (1997) Nature 389:239-242). Retrovirus genomes contain accessory genes, such as a rev gene, a tat gene, a vif gene, a nef gene, a vpr gene or an S2 gene. The deletion of such accessory genes, particularly when using retroviral vector systems in gene therapy, is highly advantageous. Firstly, it permits vectors to be produced without genes normally associated with disease in retroviral (e.g. HIV) infections. Secondly, the deletion of accessory genes permits the vector to package more heterologous DNA. Thirdly, genes whose function is unknown such as dUTPase and S2, may be omitted, thus reducing the risk of causing undesirable effects.

We have previously taught, e.g. in WO98/17815, how to remove many of the accessory genes. Further, in WO99/45126, we describe codon optimisation of the gag-pol sequence as a means of seeking to overcome the Rev/RRE requirement for export and to enhance RNA stability. However, the need remains to provide strategies for the provision of useful and safe viral vectors, and efficient means for their production.

WO 98/18934 involves gene transfer systems, such as retroviral vectors; and there are other documents that may involve retroviral vectors (See, e.g., Naldini et al., 1996 Science 272, 263; PCT/GB96/01230; Bowtell et al., 1988 J. Virol. 62, 2464; Correll et al., 1994 Blood 84, 1812; Emerman and Temin 1984 Cell 39, 459; Ghattas et al., 1991 Mol. Cell. Biol. 11, 5848; Hantzopoulos et al., 1989 PNAS 86, 3519; Hatzoglou et al., 1991 J. Biol. Chem 266, 8416; Hatzoglou et al., 1988 J. Biol. Chem 263, 17798; Li et al., 1992 Hum. Gen. Ther. 3, 381; McLachlin et al., 1993 Virol. 195, 1; Overell et al., 1988 Mol. Cell Biol. 8, 1803; Scharfman et al., 1991 PNAS 88, 4626; Vile et al., 1994 Gene Ther 1, 307; Xu et al., 1989 Virol. 171, 331; Yee et al., 1987 PNAS 84, 5197; WO99/15683; Verma and Somia (1997) Nature 389:239-242; page 446, Chapter 9 of Coffin et al “Retroviruses” 1997 Cold Spring Harbour Laboratory Press).

One shortcoming of retroviral vectors, whether based on retroviruses or lentiviruses, is their frequent inability to generate high levels of gene expression, particularly in vivo. Many steps, both transcriptional and post-transcriptional, are involved in regulating gene expression. Therefore, it is possible to enhance expression of transgenes delivered by retroviral vectors through the addition of elements known to post-transcriptionally increase gene expression. The best-known example is the inclusion of introns within the expression cassette (Choi, T. et al, (1991) Mol. Cell. Biol. 9: 3070-3074). Many gene transfer experiments, performed both in vitro and in vivo, have demonstrated that the presence of an intron can facilitate gene expression.

Other types of elements can also be used to stimulate heterologous gene expression post-transcriptionally. These elements, unlike introns, are advantageous in that they do not require splicing events. For instance, previous studies have suggested that the hepatitis B virus (HBV) post-transcriptional regulatory element (PRE) and an intron are functionally equivalent (Huang, Z. M. and Yen, T. S. (1995) Mol. Cell. Biol. 15: 3864-3869). Woodchuck hepatitis virus (WHV), a close relative of HBV, also harbors a PRE (hereinafter referred to as WPRE; see U.S. Pat. Nos. 6,136,597 and 6,287,814). The WPRE has been shown to be significantly more active than its HBV counterpart, correlating to the presence of additional cis-acting sequences not found in the HBV PRE. Insertion of the WPRE in lentiviral vectors resulted in significant stimulation of expression of reporter genes such as luciferase and green fluorescent protein (GFP) in a variety of cells spanning different species (Zufferey, R. et al, (1999) J. Virol 73: 2886-2892). Stimulation was irrespective of the cycling status of transduced cells.

The WPRE contains three cis-acting sequences important for its function in enhancing expression levels. However, in addition, it contains a fragment of approximately 180 base pairs (bp), comprising the 5′ end of the WHV X protein open reading frame, together with its associated promoter. The full-length X protein has been implicated in tumorigenesis (Flajolet, M. et al, (1998) J. Virol. 72: 6175-6180). Cis-activation of myc family oncogenes due to the insertion of viral DNA into the host genome is known to be a key mechanism of WHV-mediated carcinogenesis (Buendia, M. A. (1994) In C. Bréchot (ed.), Primary liver cancer: etiological and progression factors, p. 211-224; CRC Press, Boca Raton, Fla.; Fourel, G. (1994) In F. Tronche and M. Yaniv (ed.), Liver gene expression, p. 297-343; R.G. Landes Company, Austin, Tex.).

The present inventors have now shown that mutation of a region of the WPRE corresponding to the X protein ORF ablates the tumorigenic activity of the X protein, thereby allowing the WPRE to be used safely in retroviral and lentiviral expression vectors to enhance expression levels of heterologous genes or nucleotides of interest. Moreover, the modified WPRE can be used to identify genes involved in tumorigenesis by identifying its integration site in the chromosomal DNA of cells of interest.

According to a first aspect of the present invention, there is provided an isolated nucleic acid molecule comprising a woodchuck post-transcriptional regulatory element (WPRE) containing an X region, wherein the WPRE has a mutation in the X region, whereby expression of a functional X protein is prevented. In a preferred embodiment, the isolated nucleic acid molecule comprises the sequence of SEQ ID NO:1.

In a preferred embodiment, the X region comprises a sufficient number of base pair changes such that reversion to the wild type WPRE sequence is substantially prevented. In particularly preferred embodiments, the X region sequence comprises at least six, seven, eight, nine, ten, eleven, twelve or thirteen base pair changes, relative to the wild type WPRE sequence. Preferably, the X region of the invention comprises the sequence:

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