Chemistry: molecular biology and microbiology – Vector – per se
Patent
1995-01-24
2000-12-12
Priebe, Scott D.
Chemistry: molecular biology and microbiology
Vector, per se
424 932, 424 936, 435375, 435455, 435456, 435471, 435472, A01N 6300, C12N 700, C12N 701
Patent
active
061597287
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a protein-based delivery system and is particularly directed to the delivery of encapsidated foreign moieties, especially to targeted sites in the human or animal body.
There is increasing interest in the targeting of foreign moieties to the sites in the body where their activity is required. Thus it is important that drugs, particularly those having undesirable side effects, are delivered to the site where they are to act. Many other molecular species require to be delivered in a site specific manner, often to particular cells, for example polynucleotides (anti-sense or ribozymes), metabolic co-factors or imaging agents. One such system has been described by Wu et al., J. Biol. Chem., 263, 14621-14624 and WO-A-9206180, in which a nucleic acid useful for gene therapy is complexed with polylysine linked to galactose which is recognised by the asialoglycoprotein receptors on the surface of cells to be targeted. However, there are many occasions, such as in the delivery of a cytotoxic drug, when it would not be satisfactory to use a delivery system in which the moiety to be delivered is so exposed. There is therefore a need to develop alternative delivery systems which have the flexibility to target a wide range of biologically active foreign moieties.
Co-pending UK patent applications no. 9114003.8 and 9201372.1 describe the modification of the coat protein of phage MS-2 as a presentation system for epitopic species, which may be included in a modified coat protein sequence or attached to the coat protein via a cysteine residue and optional further spacer. These applications relied on the ability of the coat protein of MS-2 and similar phages to be cloned and expressed in a bacterial host such as E. coli as largely RNA-free empty phage particles. Romaniuk et al., (1987), Biochemistry 26, 1563-1568 have studied the relationship between the MS-2 coat protein and the RNA genome. It is apparent that, although RNA-free coat protein assemblies can be produced in E. coli, capsid formation in natural infections is triggered by coat protein interaction with a 19 base stem-loop (translational operator) in the RNA genome sequence. Talbot et al., 1990, Nucleic Acids Research 18, No. 12, 3521-3528 have synthesised the 19 base sequence and variations of this sequence and investigated the recognition and binding by the coat protein. It has been found that not only does the translational operator RNA signal exist as the stem-loop structure within the larger genomic RNA but that it is also recognised as the short fragment of just 19 bases. This fragment has the ability to cause recombinant coat protein to bind specifically and self-assemble around it, resulting in recombinant capsids containing multiple copies of the RNA fragment.
According to the present invention there is provided a delivery system comprising capsids of the coat protein amino acid sequence of phage MS-2 or related phage, or a modification thereof which retains capsid-forming capability, or sufficient of said sequence or modification to retain capsid-forming capability, at least some of said capsids enclosing a moiety foreign to the genome of MS-2 or related phage.
The foreign moiety is suitably attached to a portion of the RNA genome sequence of MS-2 or related phage capable of functioning as a translational operator for capsid formation, or a variant thereof retaining the translational operator function. The RNA genome sequence was first defined by Fiers, Nature, 1976, 260, 500-517, and we have found that the 19-base stem loop (bases -15 to +4 relative to the start of the replicase gene) SEQ ID NO. 1 or a variant thereof, especially the variant where cytidine is substituted at the -5 position, is the minimum requirement for function as the translational operator (see Talbot et al., 1990, Nucleic Acids Research 18, No. 12, 3521-3528). The foreign moiety may be attached directly to the operator sequence or via a spacer moiety, for example a series of uridine residues (suitably 6) to ensure that the foreign moiety does not interfere
REFERENCES:
patent: 5298244 (1994-03-01), Redman et al.
Beckett et al., "Roles of operator and non-operator RNA sequences in bacteriophage R17 capsid assembly", J. Mol. Biol. 204: 939-947, Dec. 1988.
Gorden et al,Gene Therapy using Retroviral Vectors,Current Opinion in Biotechnology5:611-16, 1994.
Etienne-Julan et al, The efficiency of cell targeting by recombinant retroviruses depends on the nature of the receptor and the composition of the artificial cell-virus linker, Journal of General Virology, 73:3251-3255, 1992.
Rodriguiz et al, Vectors: A Survey of molecular cloning Vectors and their use, p495-97, 1987.
S.J. Talbot, et al. "Use of synthetic oligoribonucleotides . . . " Nucleic Acids Research, vol. 18 No. 12 (Jun. 25, 1990), pp. 3521-3528.
Mastico Robert Allan
Stockley Peter George
BTG International Limited
Priebe Scott D.
LandOfFree
RNA bacteriophage-based delivery system does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with RNA bacteriophage-based delivery system, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and RNA bacteriophage-based delivery system will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-215654