WO2002098909A1

WO2002098909A1 - Vaccines

Info

Publication number: WO2002098909A1
Application number: PCT/GB2002/002526
Authority: WO
Inventors: Alice Marie Bennett; Stuart David Perkins
Original assignee: The Secretary Of State For Defence
Priority date: 2001-06-05
Filing date: 2002-05-31
Publication date: 2002-12-12
Also published as: GB0113552D0

Abstract

A construct comprising a translocation protein linked to a nucleic acid, which encodes an antigen capable of generating a protective immune response in an animal to which it is administered. Such constructs are particularly useful in the formulation of naked DNA vaccines which use plasmid DNA. Various routes for preparing these constructs are described.

Description

VACCINES

The present invention relates to novel vaccines, and in particular to modified nucleic acid vaccines which are useful in producing a prophylactic or therapeutic immune response in a host to which they are applied_/ which response is protective against a particular pathogen. The invention further relates to pharmaceutical compositions containing the modified nucleic acid vaccines, and to methods of treatment using these.

Nucleic acid vaccines (NAVs) are known in the art and they represent a powerful approach to the generatipn of vaccines. In particular, they represent a radical change in the way that antigens are delivered, involving the introduction of plasmid DNA encoding an antigenic protein, which is then expressed in the cells of the vaccinated host. Nucleic acid vaccines have a number of advantages over conventional vaccines such as live attenuated, killed whole, or subunit vaccines. Although synthesised in the cells of the host like live-attenuated vaccines, nucleic acid vaccines are subunit vaccines which encode selected components of a pathogen, rather than the entire pathogen. Therefore, nucleic acid vaccines place the vaccinated host at no risk of infection.

Unlike most subunit vaccines however, nucleic acid vaccines can raise both humoral and cellular immune responses. This is due to the ability of proteins that are synthesised within cells to access pathways for presentation by both class I and class II major histocompatibility antigens. Nucleic acid vaccines are potentially simple and inexpensive to manufacture and produce. In addition, DNA is heat stable which would aid the transportation and subsequent storage of nucleic acid vaccines.

A major disadvantage of nucleic acid vaccination however, is that immune responses can be slow to develop and are of a lower order than those induced by conventional vaccines. This is thought to be due to poor uptake of the DNA by the target cells and the inability of DNA to spread from cell to cell after the original transfection has been performed. Gene gun technology gives improved DNA delivery compared with direct injection, but the DNA still only enters the bombarded cells, resulting in sub- optimal immune responses .

Certain proteins (termed translocation proteins) have the ability to translocate across cell membranes and so enter cells in an efficient manner. These proteins may also be. able to spread from cell to cell.

Examples of such proteins include the tat protein from HIV, the homeodomain of antennapedia, or a functional fragment thereof, or the herpes simplex virus type I tegument protein VP22 or a functional fragment or homologue thereof.

A particular example of a translocation protein is the herpes simplex virus type I tegument protein VP22 which exhibits the remarkable property of efficient intracellular transport (Elliott and O'Hare, 1997 Cell . 88: 223-233). It is exported from the cells in which it is synthesised by a golgi-independent mechanism termed nonclassical secretion, despite, lacking a signal sequence. VP22 is then able to re-enter surrounding cells with very high efficiency. It has been shown that the protein VP22 can spread between different cell types and can also deliver a heterologous peptide into cells, following either endogenous synthesis and transport, or by application to the media and subsequent uptake. In addition, a 27kDa protein has been delivered into the nuclei of adjacent cells following endogenous synthesis.

VP22 has been used in the successful delivery of functional proteins. The gene p53, which is mutated in a wide range of human malignancies, has been fused to VP22 and the chimeric proteins of approximately 90kDa have been used to establish intercellular transport (Phelan et al, Nat. Biotech. 1998 16(5): 440-443) . The function of p53 was determined using a p53- negative cell line. These findings have been further substantiated with work on VP22-GFP fusions (Elliott and O'Hare, Gene Therapy. 1999 6: 149-151) and a fusion between VP22 and the frequently-used enzyme in the enzy e-prodrug therapy of cancer, thymidine kinase. It has been found that the translocation function of VP22 resides in the C-terminal domain and in particular in the 142 amino acids located at the C-terminal region. Thus a preferred fragment of VP22 for use in the constructs of the invention is the C-terminal region, comprising 142 amino acids. VP22 is 301 amino acids in length and the preferred fragment corresponds to amino acids residue from 160- 301, in the published sequence.

The use of VP22 in vaccines, where it is fused to a protein antigen is described in W098/55145. Other types of fusion protein including VP22 are described in WO 98/32866.

The applicants have found that these proteins can be used for enhancing the effectiveness of NAVs by increasing their delivery into and between target cells.

According to the present invention, there is provided a construct comprising a translocation protein linked to a nucleic acid, which encodes an antigen capable of generating a protective immune response in an animal to which it is administered.

The term "construct" as used herein includes complexes and conjugates, which include ionic or covalent bonds as well as other forms of interaction.

By harnessing translocation proteins to nucleic acid vaccines, improved delivery of the nucleic acid vaccine into target cells can be achieved.

Suitably, the translocation protein is linked to the nucleic acid in such a way that neither component loses its full functional ability.

Suitable translocation proteins for use in the constructs of the invention include the tat protein from HIV, the homeodomain of antennapedia, or a functional fragment thereof, or the herpes simplex virus type I tegument protein VP22 or a functional ^' fragment thereof.

In particular, the translocation protein is the herpes simplex virus type I tegument protein VP22 or homologue thereof; or a functional fragment of either of these.

The term "VP22" as used herein refers to the protein VP22 of Herpes simplex virus (HSV) such as HSVl . The invention includes homologues thereof and fragments of any of these which are functionally active.

The VP22 protein has been fully characterised, for example as described in WO 97/05265 and WO 98/32866, the content of which is incorporated herein by reference.

The term "homologues" as used herein refers to sequences which share some level of sequence similarity as well as retaining translocation functionality. In particular, homologues will have at least 60% identity, preferably 80% identity and more preferably 90% identity with VP22. Homology is suitably assessed using one of the known algorithms, for example the similarity of a particular sequence to the VP22 sequence may be assessed using the multiple alignment method described by Lipman and Pearson, (Lipman, D.J. & Pearson, W.R. (1985) Rapid and

Sensitive Protein Similarity Searches, Science, vol 227, ppl435- 1441) . The "optimised" percentage score should be calculated with the following parameters for the Lipman-Pearson algorithm: ktup =1, gap penalty =4 and gap penalty length =12. The sequences for which similarity is to be assessed should be used as the "test sequence" which means that the base sequence for the comparison, such as the sequence of VP22 should be entered first into the algorithm.

Particular homologues are derived from other herpes viruses including varicella zoster virus (VZV) , equine herpes virus (EHV) and bovine herpes virus (BHV) .

Suitable fragments of HSV VP22 protein with transport activity include polypeptides corresponding to aminoacids 60-301 and 159- 301 of the full HSV1 VP22 sequence (1-301) .

Fusion polypeptides of one or more such fragments such as those described in W098/32886 may be used as the translocation protein in the constructs of the present invention.

The C-terminal domain of 142 amino acids is termed VP24 and is a particularly preferred translocation protein.

Various methods can be used in forming the constructs of the invention.

In one embodiment, translocation protein is first prepared in pure form, and then mixed with plasmid DNA (NAV) in a suitable ratio and under suitable pH conditions, that a complex is formed. The sort of conditions at which this will occur will vary depending upon the type of plasmid DNA involved as well as the nature of the translocation protein used. However, in general pH conditions in the range of from 5 to 9 might be useful. These can be optimised using routine methods. Preferably, however, the pH conditions are in the range of 2 to 11, for example from 2.6 to 10.6. Most preferably it is carried out at pH conditions in the range of from 2 to 3 and preferably at about 2.6.

The reaction is suitably effected in a buffer to maintain the desired pH. Examples of suitable buffers are well known in the art and include those buffers illustrated hereinafter The ratio of NAV to translocation protein again will be dependent upon the particular components employed. In general, however, an excess of translocation protein will be required, so that typical ratios of translocation protein to NAV will be from 1:2 to 1:500. Preferably, the preferred ratio of translocation protein to NAV will be from 1:1 to 1:10. It is most preferably a ratio of 1: 10. ,

The process will suitably be effected in a physiological buffer such as phosphate buffered saline.

Preferably, the translocation protein, such as VP22, is buffer exchanged with the appropriate buffer, for instance on an ion exchange column, and desalted, prior to mixing within the DNA.

The reaction is preferably effected in the presence of urea which assists in the binding.

Complex may be separated from the mixture after a suitable incubation period, for example of from 20-120 minutes and suitably at about 30 minutes. Separation may be effected using conventional methods Such as gel electrophoresis.

Alternatively, a DNA binding sequence is conjugated to the translocation protein. Conjugation may be effected using any conventional methods, but preferably a nucleic acid encoding a fusion protein comprising the translocation protein and a DNA binding sequence is prepared. This may then be included in an expression vector using conventional methods. Transformation of an expression host, which may be a prokaryotic or eukaryotic cell, and in particular will be a prokaryotic cell such as E. coll. with the expression vector and culture of the cell using conventional DNA technology. The translocation protein may be formed into a sphere with the nucleic acid entrapped within it. The sphere is preferably formed by mixing the translocation protein with an oligonucleotide. Fusion proteins of this type, nucleic acids encoding these fusion proteins, as well as vectors containing them and cells transformed with them form further aspects of the invention.

Suitable DNA binding sequences for use in the conjugates described above include the sequence KTPKKAK P (SEQ ID NO 1) which corresponds to residues 152-160 of human histone HI. This sequence has been shown in monomeric, di eric and trimeric forms to both interact with plasmid DNA and also to enhance transfection mediated by cationic lipids and PEI, both in vivo and in vitro (Schwartz et al, 1999 Gene Therapy 6: 282-292) .

There are other short peptide sequences of natural origin that can be used as DNA binding sequence and these include:

a) The nucleolin-derived sequence (ATPAKKAA -SEQ ID NO 2) which has been shown to bind linear DNA without promoting any alteration in DNA conformation (Erard et al, Eur J. Biochem 1990 191: 19-26) .

b) Protamine which is a small, naturally-occurring, arginine- rich DNA binding protein and can be used to avidly bind to DNA at physiological ionic conditions. It has been shown that a transferrin-protamine-DNA complex, formed by mixing of the transferrin-protamine conjugate and plasmid DNA, can bind an expression plasmid encoding the firefly luciferase gene (Wagner et al, Proc . Na tl . Acad. Sci , USA. 1990 87: 3410-3414). The positive charges of the protamine strongly interact with the negative charges of the phosphate backbone of DNA, resulting in neutral and stable NAV-protamine-VP24 complexes. Further to this, prota ine-derived peptides SRSRYYRQRQRSRRRRRR(SEQ ID NO 3) from Human protamine 1 (aa 11-28) and RRRLHRIHRRQHRSCRRRKRR (SEQ ID NO 4) from Human protamine 2 (aa 15-35) are also able to bind DNA in the way that the full length protamine is able to (Chen et al, Gene therapy. 1995 2: 116-123) .

c) GAL4 is a transcriptional activator of genes required for galactose catabolism in the yeast Sacchromyces cerevisiae and the full length protein is 881 amino acid in size. Within this there is a well-characterised GAL4 DNA-binding and dimerisation domain extending from amino acids 1-147 which has been expressed successfully and purified from E. coli, in functional form. This DNA-binding domain also harbours a natural nuclear localisation domain (Paul et al, 1997 Human Gene Therapy. 8: 1253-1262) . This GAL4 DNA-binding domain is expressed as a fusion with VP24 and purified. Specific binding of the GAL4 fusion to the NAV is controlled by the incorporation of appropriate DNA dyad sequences in the target DNA, i.e. the NAV. The GAL4 target oligomer which may incorporated in the NAV is 5'-TCGACGGAGTACTGTCCTCCGC-3' (SEQ ID NO 5).

The above examples of naturally occurring short peptide sequences, which are expressed and purified as either N-terminal or C-terminal fusions with VP24, can be used in conjunction with each other in any combination and at either terminus . A linker protein can be used to aid conformation.

This fusion is mixed in varying proportions with a NAV in order to cross-link the fusion protein and NAV. As before, suitable conditions of pH and/or salt can be determined in order to create the construct.

In a preferred embodiment, the construct of the invention further comprises a sequence which functions as a nuclear accumulation signal and so may direct transport of proteins to the nucleus of infected cells. Examples of such such sequences are found in the 10K proteins of Herpesvirus simiae (B virus) , Herpes simplex virus type 1, Pseudorabies virus, and Varicella- Zoster virus.

Specific examples of such sequences are KIPIK (SEQ ID NO 6) from the Mat α2 of Yeast; PKKKRKV (SEQ ID NO 7) from the large T antigen of SV40; AAFEDLRVLS (SEQ ID NO 8) from the nucleoprotein of Influenza virus; RKKRRQRRR (SEQ ID NO 9) from the Tat protein of HIV-1; RRRRRQTR (SEQ ID NO 10) from the 10K protein of B virus; RRRRRR (SEQ ID NO 11) from the 10K protein of HSV-1; RRRRR from the UK protein of PRZ and RKK from the UK protein of VZV.

These sequences are preferably formed as part of the fusion protein described above. Thus, in a preferred embodiment, a nucleic acid encoding a fusion protein including a translocation protein, a DNA binding sequence and a nuclear accumulation signal sequence is prepared and expressed in a suitable expression host using conventional methods.

In yet a further embodiment, the translocation protein is formed into a covalently bound conjugate with the nucleic acid using a coupling agent. An example of a suitable coupling agent is 1- ethyl-3- (3-dimmethylaminopropyl) carbodiimide (Pierce).

This form of linking has been demonstrated using an asialoglycoprotein and an expression vector expressing the full- length human methylmalonyl CoA mutase cDNA (Stankovics et al . _r Human gene Therapy 5:1994 1095-1104. 1994).

Suitably, the nucleic acid used in the construct of the invention is plasmid DNA which encodes an antigen. It is envisaged that any NAV can be used in the construct, for example :

1. NAV encoding the protective H_c domain of Botulinum neurotoxin type A, (Shyu et al., (2000) J. Biomed. Sci. 1_, 51-57); 2. NAV encoding P. falciparium circumsporozoite antigen (i.e. a malaria antigen), ( e et al., (2000) Vaccine 18_, 1893-1901);

3. NAVs encoding protective antigens from Yersinia pestis and

Venezuelan equine encephalitis virus, (Bennett et al., (2000)

Vaccine 18, 588-596) ; 4. NAV encoding protective antigens from St Louis encephalitis virus, (Phillpotts et al., (1996) Arch. Virol. 141, 743-749);

5. NAV encoding Hepatitis B virus surface antigen, (Tacket et al . , ( 1999 ) Vaccine 17 , 2826-2829) ;

6. NAV encoding influenza haemogglutinin, (Ulmer et al., (2000)

Vaccine 18, 18-28) .

In each of the above embodiments, the purified translocation protein is preferably buffer exchanged prior to mixing with the nucleic acid of the present invention. Binding in this way is based on charge or electrostatic interactions . Suitably, the protein will be buffer exchanged into either citrate phosphate buffer, carbonate-bicarbonate buffer or PBS or citrate phosphate buffer, carbonate-bicarbonate buffer and PBS each containing 8M urea. The protein is then desalted out. This protein, for example the herpes simplex virus type I tegument protein VP22, or a functional fragment or homologue thereof, is then used to mix with a DNA vaccine to form complexes.

Constructs of the invention are suitably administered in the form of pharmaceutical compositions in which they are combined with pharmaceutically acceptable carriers. Examples of such carriers are liquid or solids, and preferably liquids such as saline. The compositions of the invention are preferably formulated for parente,ral administration although other means of application are possible as described in the pharmaceutical literature, for example administration using a Gene Gun, (Bennett et al., (2000) Vaccine _18, 1893-1901).

The invention will now be particularly described by way of the following examples with reference to Figure 1, which shows the results of the VP22 and DNA binding reactions discussed in Example 8 below.

Example 1

DNA encoding the VP24 protein is cloned into a suitable expression vector (e.g. pET vector from Promega) . The VP24 projtein is expressed in E. coli and purified. Purified VP24 is mixed with plasmid DNA (NAV) in an optimised ratio, under optimised conditions of pH to form a VP24/NAV complex which can be delivered to target cells .

Example 2

DNA encoding the sequence KTPKKAKKP (SEQ ID NO 1) is fused to the N-terminus or the C-terminus of the VP24 gene in a suitable expression system (e.g. pET vector from Promega) to produce a peptide/VP24 fusion protein.

This fusion is mixed in varying proportions with a NAV in order to cross-link the fusion protein and NAV. Suitable conditions of pH and/or salt are used.

Example 3

VP24 can be coupled to polylysine by the addition of l-ethyl-3- (3-dimmethylaminopropyl) carbodiimide resulting in a covalently bound conjugate. The negatively charged plasmid DNA (NAV) noncovalently binds to the positively-charged polylysine in the presence of NaCl. This produces soluble complexes of VP24/ polylysine/NAV.

The interactions of the protein and the NAV can be studied by the techniques of Capillary gel electrophoresis and gel retardation. The translocation patterns of the NAV and VP24 protein can be studied in vitro and tracked using specific antibodies and conjugation of the protein with AP or HRP.

Example 4

Binding of Recombinant VP22 and a DNA Vaccine Purified, recombinant VP22 was bound to a DNA vaccine based on charge or electrostatic interactions. VP22 protein was buffer exchanged into either citrate phosphate buffer, carbonate- bicarbonate buffer or PBS or citrate phosphate buffer, carbonate-bicarbonate buffer and PBS, each containing 8M urea, and desalted out. This protein was then mixed with a DNA vaccine to form complexes. When electrophoresed through an agarose gel, complexes of VP22 remained in the well of the gel. . Materials and Methods :

Citrate Acid monohydrate C₆H₈0₇.H₂0 (BDH 100813M) Sodium Phosphate Na₂HP0₄.2H₂0 (BDH 30157) Sodium Carbonate anhydrous Na₂C0₃ (BDH 102404H) Sodium Bicarbonate Buffer NaHC0₃ (Sigma S-6297) Urea (Sigma ϋ-0631) PBS (Gibco 14190-136) Molecular Weight Markers IV (Roche 1418009 )

1. Preparation of buffers a) 0.1M Citrate Acid and 0.2M Sodium Phosphate were prepared.

These were used to form citrate phosphate buffer by addition of 891ml and 109ml respectively per litre. The pH was 2.6. b) 0.1M Sodium Carbonate and 0.1M Sodium Bicarbonate were prepared. These were used to form carbonate-bicarbonate buffer by addition of 900ml and 100ml respectively per litre. The pH was 10.6. c) Stocks of citrate phosphate buffer, carbonate-bicarbonate buffer and PBS were prepared, each containing 8M, 4M, 2M and 0M Urea.

2. Buffer exchange of recombinant VP22 protein a) 3χ 0.5ml of VP22 were buffer exchanged in PBS (Batch 003, VP6) 0.438mg/ml using NAP5 columns (Pharmacia 17-08553-02).

It was then eluted in 1ml of either citrate phosphate buffer, carbonate-bicarbonate buffer or PBS, each containing 8M Urea. b) Each 1ml sample was dialysed in a litre of either citrate phosphate buffer, carbonate-bicarbonate buffer and PBS, each containing 4M Urea for 2 hours c) Each 1ml sample was dialysed in a litre of either citrate phosphate buffer, carbonate-bicarbonate buffer and PBS, each containing 2M Urea for 2 hours d) Each 1ml sample was dialysed in a litre of either citrate phosphate buffer, carbonate-bicarbonate buffer and PBS, each containing 0M Urea overnight e) Each sample was removed for quantification f) 3x 0.5ml of VP22 was buffer exchanged in PBS (Batch 003, VP6) 0.438mg/ml using NAP5 columns (Pharmacia 17-08553-02) It was then eluted in 1ml of either citrate phosphate buffer, carbonate-bicarbonate buffer or PBS and quantify.

3. Quantification of VP22 a) The concentration of protein in mg/ml was calculated using the following equation: Cone. = (1,55 x A₂₈o) - (0.76 x A₂₆₀)as shown in the following Table.

4. Mixing of recombinant VP22 and DNA vaccine

Plasmid DNA was used at concentrations of 200ng and 400ng. The protein was mixed in 1:1, 1:2, 1:5 and 1:10 DNA:VP22 ratios as shown in the following Table.

200ng of plasmid DNA = 2μl 00ng of plasmid DNA = 4μl

The amount of VP22 in various buffers is given below, assuming an approximate concentration of 0.2 μg/μl:

200ng of VP22 = lμl 400ng of VP22 = 2μl 800ng of VP22 = 4μl lOOOng of VP22 = 5μl 2200ng of VP22 = lOμl 4000ng of VP22 = 20μl

The schedule of reactions is given in the Table below (DNA and VP22 units are μl) :

The reactions were incubated at room temperature for 30 minutes 6X sample buffer was added and electrophoresis was carried out on a 1% agarose gel.

Results:

The results are given in Figure 1.

Conclusions

1. It was seen that VP22 and DNA bind preferentially in citrate phosphate buffer at pH 2.6

2. The addition of urea to the protein improves binding slightly. Example 9

The method outlined above (without urea treatment) is used to see the effect these complexes have in mammalian cells. In this regard, the uptake of complexes of recombinant protein and DNA is studied in mammalian cells using tissue culture techniques i.e. it is an in vitro model. The mammalian cells used are African Green Monkey Kidney Cells (COS-7) .

Firstly, the presence of VP22 protein within the cells is studied using standard im unofluorescence techniques . The protein is detected using a specific antibody. A secondary antibody specific to the first and conjugated with FITC will allow detection of the protein by UV light via a confocal microscope. This allows determination of the uptake and localisation of the VP22 protein but does not give an indication of whether the DNA remains complexed.

Secondly, complexes of VP22:DNA are added to cells and the DNA labelled with a dye.' This allows determination of uptake and localisation of the DNA.

Finally, a time course of expression is carried out, where the DNA vaccine complexed to the VP22 expresses green fluorescent protein (GFP) . The GFP of DNA vaccine is taken up by the cell and expressed, is visible using the confocal microscope.

All references mentioned in the above specification are herein incorporated by reference. Other modifications of the present 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 the 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 the art, are intended to be within the scope of the following claims.

Claims

1. A construct comprising a translocation protein linked to a nucleic acid which encodes an antigen capable of generating a protective immune response in an animal to which it is administered.

2. A construct according to claim 1 wherein the translocation protein comprises the tat protein from HIV, the homeodomain of antennapedia, or a functional fragment thereof, or the herpes simplex virus type I tegument protein VP22 or a functional fragment thereof.

3. A construct according to claim 2 wherein the translocation protein is the herpes simplex virus type I tegument protein VP22 or a functional fragment thereof.

4. A construct according to claim 3 wherein the translocation protein comprised the C-terminal domain of VP22.

5. A construct according to any one of the preceding claims comprising a fusion protein of a translocation protein and a DNA binding sequence .

6. A construct according to any one of the preceding claims wherein the translocation protein is formed into a sphere and the nucleic acid is entrapped within said sphere.

7. A construct according to claim 6 wherein the translocation protein is formed into a sphere by mixing with an oligonucleotide .

8. A construct according to any one of the preceding claims which further comprises a nuclear accumulation signal, sequence.

9. A construct according to claim 8 wherein said nuclear accumulation signal sequence is fused to said translocation sequence and/or a DNA binding sequence.

10. A construct according to any one of the preceding claims wherein the nucleic acid is plasmid DNA.

11. A construct according to claim 10 wherein the plasmid DNA comprises a nucleic acid vaccine.

12. A nucleic acid which encodes a fusion protein according to claim 5 or claim 9.

13. A vector comprising a nucleic acid according to claim 12.

14. A cell transformed with a vector according to claim 13.

15. A pharmaceutical composition comprising a construct according to any one of the preceding claims in combination with a pharmaceutically acceptable carrier.

16. A method of forming a construct according to claim 1, said method comprising one of:

(a) mixing together a translocation protein and a nucleic acid which encodes an antigen capable of generating a protective immune response in an animal to which it is administered, under conditions in which a complex is formed; or

(b) obtaining a fusion protein comprising a translocation protein linked to a DNA binding sequence, and mixing said fusion protein with nucleic acid which encodes an antigen capable of generating a protective immune response in an animal to which it is administered under conditions where said DNA binding sequence binds said nucleic acid; or

(c) mixing together a translocation protein and a nucleic acid which encodes an antigen capable of generating a protective immune response in an animal to which it is administered, in the presence of a covalent coupling agent so that a covalently linked construct is formed.

17. A method of forming a construct according to claim 16 wherein the translocation protein is buffer exchanged prior to mixing with the nucleic acid.

18. A construct, a nucleic acid, vector, cell or composition substantially as hereinbefore defined with reference to the examples.

19. A method of forming a construct according to claim 1 substantially as hereinbefore described with reference to the Examples.