Title:  RNA bacteriophage-based delivery system

United States Patent:  6,159,728

Assignee:  BTG International Limited (London, GB)

Filed:  January 24, 1995

PCT NO:  PCT/GB93/01338

102(e) Date:  January 24, 1995

PCT PUB. Date:  January 6, 1994

Foreign Application Priority Data:  Jun 26, 1992[GB] (9213601)

A delivery system, especially for delivery to targeted sites in the human or animal body, comprises capsids of the coat protein amino acid sequence of phage MS-2 or related phage, or a modification thereof which retains capsid-forming capability, and at least some of the capsids enclosing a moiety foreign to the genome of MS-2 or related phage.

Abstract of Description of the Invention

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 with the operator function.

According to a preferred form of the invention the coat protein amino acid sequence has been modified to provide a site suitable for attachment thereto of a targeting moiety. The invention includes capsids having such a site for subsequent attachment of a targeting moiety and capsids to which the targeting moiety has already been attached.

The coat protein amino acid sequence is preferably that derived from phage MS-2, but it may also be derived from related RNA-phages capable of replication in E. coli, such as phages R17, fr, GA, Q.beta. and SP. Such RNA-phages of physical structure similar to that of MS-2 will contain some chemical variation in the amino acid residues of the coat protein and are thus conservatively modified variants of MS-2 coat protein. While it is believed at present that substantially the entire coat protein may be required for capsid assembly, deletions and/or insertions are also possible whilst still retaining capsid-forming capability. Proteins having such modified sequences are included within the scope of the invention.

The three-dimensional structure of the MS-2 phage particle has been published by Valegard et al., (Nature, 1990, 345, 36-41). The published data show that, firstly, the structure of the coat protein is not related to the eight-stranded .beta.-barrel motif found in all other spherical RNA virus subunits whose structures are known at the present time. Secondly, although the coat protein exhibits quasi-equivalent inter-subunit contacts, there are no other devices, such as extended arms of polypeptide, helping to secure each protein conformer. The coat protein structure can be viewed in terms of three separate regions. These are not domains in the usual sense but could represent independent folding units. These regions are residues 1-20, which form the .beta.-hairpin structure which protrudes from the surface of the phage forming the most distal radial feature. This region is followed by residues 21-94 which form five .beta.-strands including the "FG-loop" which is the site of the only major conformational change between quasi-equivalent conformers. These .beta.-strands are then followed by two .alpha.-helices, residues 95-125, which interdigitate to secure dimers of the coat protein sub-units. Valegard et al. are concerned solely with the physical structure of the MS-2 virus and do not attempt to elucidate the mode of action of the virus.

Co-pending UK patent application No. 9114003.8 describes the introduction of a cysteine residue into the N-terminal protruberant .beta.-hairpin of the coat protein (with removal of the cysteine residues present externally of the N-terminal protruberant .beta.-hairpin). Such a cysteine residue provides a preferred site for attachment thereto of a targeting moiety. The resultant coat protein has therefore been so modified in the region of amino acid residues 1 to 20, such numbering being with reference to the entire coat protein sequence of MS-2 as published by Fiers, Nature, 1976, 260, 500-507. Preferably the modification to introduce the cysteine residue is towards or at the middle of the hairpin region. It is preferred to introduce the cysteine in the region of the glycine 13 and 14 residues of the coat protein. The cysteine residues to be removed which are external of the .beta.-hairpin are found at positions 46 and 101. They may be removed by any convenient conventional genetic engineering technique, suitably by site-specific mutagenesis.

In a preferred method of removing the unwanted cysteine residues, two mutants of the MS-2 coat protein, one singly mutated at cys 46 and one singly mutated at cys 101 may be obtained by standard commercially available techniques for site specific mutagenesis and the corresponding cDNA sequences introduced into standard expression vectors, which vectors are subjected to digestion with restriction enzymes to obtain separately the DNA fragment containing the mutated cys 46 site and the corresponding fragment containing the mutated cys 101 site, the fragments being subsequently ligated to give a doubly-mutated coat protein cDNA. The doubly-mutated cDNA may then be subjected to site-directed mutagenesis using standard methods to introduce a cysteine residue in the .beta.-hairpin region.

Alternative modification of the coat protein which enables targeting of the encapsidated moiety may include insertion of peptide sequences in the protruberant .beta.-hairpin of the MS-2 coat protein as described in co-pending UK patent application no. 9201372.1.

The cysteine residue, or alternative modification site, can be further linked to a targeting moiety with or without interposition of a further spacer moiety. An example of such a targeting moiety is a galactose residue which can be used to direct the capsids to interact with specific cell surface receptors and thus carry the foreign moiety within the capsids to and/or into specific cells. Other possible targeting moieties are other cell surface receptor ligands or monoclonal antibodies. Suitable receptors for the targeting moieties are the asialoglycoprotein receptor and the receptor for melanocyte stimulating hormone.

Suitable spacer moieties, if employed, are selected from known commercially available heterobifunctional crosslinking reagents which couple with the exposed cysteine thiol group. Examples of such cross-linkers are m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, N-succinimidyl-(4-iodoacetyl)aminobenzoate and N-succinimidyl-3-(2-pyridyldithio)propionate. The choice of crosslinker will depend on the targeting moiety and its size. Thus larger molecular species may require longer crosslinking moieties to minimise steric hindrance. The crosslinker may be linked first to the cysteine residue or first to the targeting moiety.

Alternatively the thiol function (or other derivatisable group) can be introduced into wild type, empty capsids of MS2 coat protein using suitable heterobifunctional chemical reagents such as N-succinimidyl S-acetylthioacetate (SATA).

The foreign moiety held within the capsids can vary widely and include genes and gene fragments, ribozymes, anti-sense messages or cytotoxic and chemotherapeutic agents intended for such purposes as anti-sense gene therapy or selective killing of target cells.

The form in which the foreign moiety is held within the capsids will depend on the release properties required. For release at the targeted site it will be important to ensure that the right conditions prevail, for example to permit cell localisation and internalisation via receptor mediated endocytosis.

The capsids may suitably be obtained by first obtaining empty MS-2 capsids, for example by expression of vectors containing coat protein cDNA in E. coli as described in co-pending UK application No. 9201372.1. The MS-2 capsids may be of wild type MS-2 coat protein or have been modified, for example to introduce a cysteine site as described in co-pending UK application No. 9114003.8. The capsids are then disassembled, for example, at acid pH (e.g. using acetic acid), before reassembly suitably at raised pH, e.g. pH 7. In the presence of the desired foreign moiety linked to an RNA sequence capable of functioning as the translational operator in the reassembly of the coat protein around the RNA sequence and foreign moiety. Other methods of disassembly may be used, for example in the presence of urea. It is also contemplated that the capsids enclosing the foreign moiety may be obtained by random incorporation of the moiety in the capsids.

The RNA sequence may be obtained by biochemical methods from the complete MS-2 RNA genome. Alternatively, the RNA sequence is obtained by chemical synthesis, for example as described by Usman et al., (1987), J. Am. Chem. Soc., 109, 7845-7854. Chemical synthesis is preferred as it enables ready addition of any spacer moiety and linking to the foreign moiety to be delivered.

It will be apparent that there are several advantages in using MS-2 and related phages as a presentation system. Thus the empty coat protein capsids can be readily expressed in comparatively high yield in E. coli and the product is easily purified (see R. A. Mastico et al. J. Gen. Virol. (1993) 74, 541-548 the contents of which are herein incorporated by reference). It has been found that the assembled capsids show considerable stability with respect to a range of temperatures, pH and ionic strength.

Claim 1 of 16 Claims

What is claimed is:

1. A delivery system comprising a capsid formed from a coat protein of a bacteriophage selected from the group consisting of MS-2, R17, fr, GA, Q.beta., and SP and a foreign moiety enclosed in the capsid, wherein the foreign moiety is of a size sufficiently small to be enclosed in the capsid and wherein the foreign moiety is linked to a RNA sequence comprising a translational operator of the bacteriophage, which translational operator binds to the coat protein during formation of the capsid.

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