Method for selecting improved vectors

A method is provided for selecting an improved retroviral genome having an improved packaging efficiency which method comprises: a) introducing one or more random mutations into a retroviral genome comprising a packaging signal; b) introducing the mutagenised retroviral genome into a host cell expressing viral polypeptides required for packaging the retroviral genome; c) determining whether retroviral packaging efficiency in the cell is improved as compared with a retroviral genome comprising a non-mutated packaging signal; d) selecting a viral genome which has improved packaging efficiency.

EXAMPLES 
 Example 1 
 Effect of Each Viral Component on Viral Titres We have investigated effects of the stoichiometry of viral components on retroviral production using a transient transfection system. The murine leukaemia virus (MLV) genome was segregated into three different plasmids: one containing the gag/gag-pol, one containing the env and another containing the long terminal repeats, packaging signal and the lacZ marker (genome construct). Firstly, we determined the conditions under which none of the three viral components are saturating. The results shown in FIG. 1 indicate that since none of the viral components are saturating at 0.1 &mgr;g of each plasmid, then 0.1 &mgr;g would be a suitable starting point from which the amount of each component could then be raised. Raising the amount of one plasmid with respect to the other two, we then measured the viral titres and compared them to the titres produced when equal amounts of all three plasmids were used. The number of infectious particles was determined by X-gal staining of transduced NIH3T3 cells, while the total number of viral particles was measured by reverse transcriptase assay. The results shown in Table 1 indicate that genome is limiting and that 10-fold more genome increased titres. It was also found that similar titres could be achieved using 10 times less of the gag/gag-pol component compared to using equal amounts of all three components (Table 1). However, the former viral stock had a lower reverse transcriptase activity compared to the latter ( FIG. 2 —lanes 5 and 6 ), suggesting that the viral stock produced using less gag/gag-pol contained a larger infectious-particles-to-total-particles ratio. These results showed that it is possible to produce a viral stock in which all the particles are infectious by ensuring that they are all filled with genomic RNA. 1 TABLE 1 Effect of each component on viral titres. Amounts of plasmids used in transfection (&mgr;g) a pHIT60 pHIT111 pHIT456 (gag/gag-pol (genome) (env) Titres (I.f.u./ml) b 0.1 0.1 0.1 6.5 ± 0.9 × 10 3 1 0.1 0.1 1.6 ± 0 × 10 3 0.1 1 0.1 4.1 ± 0.1 × 10 4 0.1 0.1 1 1.9 ± 0.4 × 10 4 1 1 1 3.5 ± 0.5 × 10 5 0.1 1 1 1.6 ± 0.6 × 10 5 a Different amounts of plasmids were used to transfect 293T cells in 6 cm dishes using FuGene6 transfection reagent (Boebringer Mannheim). b Viral titres were measured as the number of lacZ forming units (l.f.u.) per ml as observed by X-gal staining. 
 Example 2 
 In Vivo Strategy to Select a Vector Genome with Improved Packaging Efficiency. We have also developed an in vivo strategy to select a vector genome with improved packaging efficiency which involves shuttling a retroviral vector between a mutagenic strain of Epicurian coli, where random mutations are introduced in its sequence, and mammalian cells, where selection is made for better packaging efficiency ( FIG. 3 ). Selection is effected by competition for packaging by vectors containing the existing packaging signal. A shuttle retroviral vector is constructed by cloning the bacterial ColE 1 origin of replication into the multiple cloning site of pLXSN. To create a vector that confers kanamycin resistance in bacteria a bacterial promoter is inserted upstream of the neomycin resistance gene by replacing the Sfi I-Rsr II fragment with the Sfi I-Rsr II fragment from pEGFPN1 (Clontech). The resulting vector can replicate in E. coli cells under kanamycin selection. When transduced into mammalian cells expressing the SV40 large-T antigen, it replicates extrachromosomally under neomycin selection. The Mammalian cell— E. coli -LTR containing vector is designated pMEL ( FIG. 4 ) and has the following features: (1) Kanamycin/Neomycin resistance: allows for selection on kanamycin in E. coli and G418 in eucaryotic cells. (2) ColE 1 Origin of replication: allows for high copy number replication in E. coli (3) SV40 Origin of replication: allows for extrachromosomal replication in eukaryotic cells expressing SV40 large T antigen e.g. COS7 cells (Cepko et al., 1984) pMEL is transformed into Epicurian Coli XL1-Red cells (Stratagene) to introduce random mutations in its sequence. The pool of mutants is extracted using standard large-scale plasmid isolation protocols. A competition for packaging is set up by co-transfecting 293T cells with equal amounts of the gag-pol expression plasmid, env expression plasmid, Neo-minus pMEL and ten times less of mutagenised pMEL. Vector genomes that are packaged are used to transduce COS cells. They are then isolated from neomycin resistant cells and used to transform XL1-Red cells. The process is repeated to obtain vector genomes with higher packaging efficiencies. 2 TABLE 2 Titres Amount of plasmid used for transfection (&mgr;g) (G418 resistant Gag/gag-pol env pMEL pLXSCD8 a pSA91 b colonies per ml) 1 1 0.1 1 — 2.0 ± 1.5 × 10 3 1 1 0.1 — 1 1.0 ± 0.5 × 10 4 a pLXSN derived vector containing CD8 marker in place of neomycin resistance gene. b Mammalian expression vector similar in size to pLXSCD8. An initial round of selection with the shuttle vector gave the results shown above in Table2. Thus, competition of pMEL for packaging was successfully set up as observed by the decrease in titres in the presence pLXSCD8. Vector genomes emerging from this selection and competition process are packaged more efficiently. The new high efficiency packaging sites can be engineered into any vector genome of the same viral origin using standard recombinant DNA procedures to produce vector systems generating higher titres. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims. References Adam, M. A. and A. D. Miller (1988). “Identification of a signal in a murine retrovirus that is sufficient for packaging of nonretroviral RNA into virions.” J Virol 62(10): 3802-6. Allen, P., B. Collins, D. Brown, Z. Hostomsky and L. Gold (1996). “A specific RNA structural motiff mediates high affinity binding by the HIV-1 nucleocapsid protein (NCp7).” Virology 225: 306-315. Beaudry, A. A. and G. F. Joyce (1992). “Directed evolution of an RNA enzyme.” Science 257: 635-641. Berglund. J. A., B. Charpentier and M. Rosbash (1997). “A high affinity binding site for the HIV-nucleocapsid protein.” Nucleic acids research 25(5): 1042-1049. Bornscheuer, U. T., M. M. Enzelberger, J. Altenbuchner and H. H. Meyer (1998). “Using XL1-Red mutator strain to generate esterase variants.” Strategies 11(1): 16-17. Cepko, C. L., B. E. Roberts and R. C. Mulligan. (1984). Cell. 37(3): 1053-62 Coffin et al “Retroviruses” 1997 Cold Spring Harbour Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmus pp 758-763. Joyce, G. F. (1992). “Directed Molecular Evolution.” Scientific American December: 48-55. Miller, A. D. (1997). Development and applications of retroviral vectors. Retroviruses. J. M. Coffin, S. H. Hughes and H. E. Varmus, Cold Spring Harbor Press: 437-473. Soneoka, Y., P. M. Cannon, E. E. Ramsdale, J. C. Griffiths, G. Romano. S. M. Kingsman and A. J. Kingsman (1995). “A transient three-plasmid expression system for the production of high titer retroviral vectors.” Nucleic Acids Res 23(4): 628-33. Sonstegard, T. S. and P. B. Hackett (1996). “Autogenous regulation of RNA translation and packaging by Rous Sarcoma virus Pr76gag.” J Virol. 70: 6642-6652. Stemmer. W. P. C. (1994). “Rapid evolution of a protein in vitro by DNA shuffling.” Nature 370: 389-391. Taplitz, R. A. and J. M. Coffin (1997). “Selection of an avian retrovirus mutant with extended receptor usage.” J Virol 71(10): 7814-9. Then invention will now be further described by the following numbered paragraphs: 1. A method for selecting an improved retroviral genome having an improved packaging efficiency which method comprises: (a) introducing one or more random mutations into a retroviral genome comprising a packaging signal; (b) introducing the mutagenised retroviral genome into a host cell expressing viral polypeptides required for packaging the retroviral genome; (c) determining whether retroviral packaging efficiency in the cell is improved as compared with a retroviral genome comprising a non-mutated packaging signal; (d) selecting a viral genome which has improved packaging efficiency. 2. A method according to paragraph 1 wherein an additional step (e) of determining the sequence of all or part of the viral genome to identify the sequence of the packaging signal. 3. A method according to paragraph 1 or 2 wherein step (a) is carried out in a bacterial strain which introduces random mutations into the retroviral genome. 4. A method according to paragraph 3 wherein the bacterial strain is a mutagenic strain of Epicurian coli. 5. A method according to any one of the preceding paragraphs wherein the host cell is a mammalian cell. 6. A method according to any one of the preceding paragraphs wherein the host cell comprises at least: (i) a first nucleotide sequence encoding a retroviral gag-pol polypeptide; (ii) a second nucleotide sequence encoding a retroviral envelope polypeptide; and (iii) a third nucleotide encoding a retroviral genome comprising a non-mutated packaging signal, wherein the third nucleotide is present as part of a nucleic acid vector which lacks a selectable marker; the mutagenised retroviral genome is present as part of a nucleic acid vector which contains the selectable marker; and the ratio of the vector comprising the third nucleotide to the vector comprising the mutagenised retroviral genome is greater than 2:1, preferably greater than 5:1. 7. A method according to any one of the preceding paragraphs wherein the packaging efficiency, measured as the proportion of the number of infectious retroviral particles to the total number of retroviral particles produced is greater than 25%. 8. A retroviral genome obtained by the method of any one of the preceding paragraphs. 9. A retroviral genome according to paragraph 8 having a packaging efficiency, measured as the proportion of the number of infectious retroviral particles to the total number of retroviral particles produced is greater than 25%. 10. A retroviral packaging signal obtainable from a retroviral genome according to paragraph 8 or 9. 11. A nucleic acid comprising a retroviral packing signal according to paragraph 10. 12. A retroviral vector comprising a retroviral packing signal according to paragraph 10. 13. A retroviral vector according to paragraph 12 for use in producing infectious retroviral particles. 14. A retroviral vector according to paragraph 12 or 13, which is a lentiviral vector. 15. A producer cell comprising a retroviral genome according to paragraph 8 or 9, a retroviral packaging signal according to paragraph 10, or a retroviral vector according to paragraph 12, 13 or 14. 16. A method for enhancing the efficiency of retroviral packaging which method comprises expressing in a producer cell at least a first nucleotide sequence encoding a retroviral gag-pol polypeptide, a second nucleotide sequence encoding a retroviral envelope polypeptide and a third nucleotide sequence encoding a retroviral genome wherein the ratio of the first nucleotide sequence to the second nucleotide sequence and the ratio of the first nucleotide sequence to the third nucleotide sequence is x:y and x:z, respectively wherein x is less than 1 and y and z are 1. 17. A method according to paragraph 16 wherein x is less than 0.5. 18. A method according to paragraph 16 or 17 wherein the packaging efficiency, measured as the proportion of the number of infectious retroviral particles to the total number of retroviral particles produced is greater than 25%. 19. A composition comprising infectious retroviral particles produced according to the method of any one of paragraphs 16 to 18. 20. A composition according to paragraph 19 for use in therapy. 21. A producer cell which expresses at least a first nucleotide sequence encoding a retroviral gag-pol polypeptide, a second nucleotide sequence encoding a retroviral envelope polypeptide and a third nucleotide sequence encoding a retroviral genome wherein the ratio of the first nucleotide sequence to the second nucleotide sequence and the ratio of the first nucleotide sequence to the third nucleotide sequence is x:y and x:z, respectively wherein x is less than 1 and y and z are 1.