GB2359812A - Delivery method - Google Patents

Description

<U>Novel Method</U> The present invention generally relates to the delivery of a desired material, such as nucleic acid, into liposomes, organelles or cells, particularly eukaryotic cells. The methods are therefore particularly useful in producing transiently transfectedltransformed or stably transformed cells, as well as to cells transfected or transformed thereby. The invention relates to the fields of molecular biology, cell biology, biophysics, pharmacology, gene therapy and agriculture.

Several methods have been developed for introducing exogenous DNA molecules into eukaryotic cells in order to take advantage of the widespread benefits arising from the application of recombinant DNA technology to the production of transiently transfected/transformed cells as well as to transgenic cells and organisms generated from such cells. These methods include physical, chemical and biological techniques. Synthetic DNA delivery systems have recently been reviewed by Luo and Saltzman ((2000) Nature Biotechnology 18:33-37).

For example, recombinant DNA has been introduced into plant tissue by direct DNA uptake (Paszkowski et al. (1984) EMBO J. 3:2717), electroporation (Fromm et al. (1985) Proc. Natl. Acad. Sci. USA 82:5824), microinjection (Crossway et al. (1986) Mol. Gen. Genet. 202:179), microprojectile bombardment ("biolistics"), and via T-DNA mediated transfer from Agrobacterium tumefaciens (for a general overview, see chapters 2 and 3 of "Plant Genetic Transformation and Gene Expression: A Laboratory Manual", ed. by Draper et al., publ. by Blackwell Scientific Publications (1988); see also Potrykus et al., "Direct Gene Transfer: State of the Art and Future Potential", Plant Mol. Biol. Rep. 3:117-128 (1985)). T-DNA- mediated transformation of monocots (Hiei et al. (1994) Plant J. 6:271-282), gymnosperm (Dandekar <I>et al.</I> (1987) Biotechnology 5:587) and algae (Ausich, R., EPO application 108,580) has been reported.

The Agrobacterium plant transformation system is widely used for the stable transformation of higher plants. In this system, genes to be transferred are carried by the T-DNA, a well defined region of the Agrobacterium Ti plasmid. The Ti plasmid also contains a virulence (vir) region, which encodes proteins involved in the transformation via Agrobacterium of plant cells. At least one of these proteins, VirD2 is involved in DNA targeting to the plant nucleus and integration into the plant genome (Tinland et al. (1992) Proc. Natl. Acad. Sci. USA 89:7442; Tinland et al. (1995) EMBO J. 14:3588-3595). WO 95/05471, discloses a method for producing stably transformed plant material, including phenotypically normal looking and fertile plants, which does not involve Agrobacterium transformation. In particular, a specifically adapted nucleic acid/protein complex comprising a chimeric recombinant nucleic acid is introduced into cells using standard non-biological delivery techniques and allows for ordered integration of the delivered nucleic acid into the cell genome. The nucleic acid/protein complex preferably comprises an expressible DNA or an oligonucleotide operably linked to suitable plant expression sequences and covalently associated with VirD2, and, optionally, VirE2 protein units. See also W097/12046.

Similar methods have been used in mammalian cells. For example, see USPN 5,831,020 and Ziemienowicz et al. ((1999) Proc. Natl. Acad. Sci. USA 96:3729-3733). In addition, nucleic acids have been introduced into animal cells by electroporation, microinjection, calcium phosphate, or polyethylene glycol (PEG) mediated DNA uptake or cell fusion, and using liposomes (Gao et al. (1996) Biochemistry 35:1027) or viral vectors (Crystal (1995) Science 270:404). For example, retroviral vectors have been demonstrated to be quite effective in transferring genes into different cell types. Modified retroviruses developed to address concerns that retroviral vectors could recombine<I>in vivo</I> to generate wild-type virus have been used to transfer genes into a variety of established mammalian cell lines, as well as into certain primary cells. However, their use is dependent on gene transfer into dividing cells. Other viruses have also been used to generate recombinant viral vectors for gene transfer studies, including adenovirus, adeno-associated virus, and<I>herpes simplex</I> virus. Nevertheless, the inflammatory host defences against viruses may be a risk for the recipient and hinder the efficacy of repeat administration of vectors. Liposome delivery has been extensively used as well, especially the use of liposomes containing cationic lipids. However, the cationic lipids used in liposomes have also exhibited toxic effects (Belting and Petersson (1999) Biochem. J. 342:281).

There exists a continuous need for further techniques which are useful for the introduction of nucleic acids into animal and plant cells, e.g. oligonucleotides for antisense- or antigene- approaches, or for the permanent transformation of eukaryotic cells. The object of the present invention is therefore to provide a new method for introducing desired molecules, including nucleic acids, into cells, in particular eukaryotic cells. The present invention provides a method for delivering desired materials across membranes, in particular into cells or organelles. The method comprises introducing Agrobacferium VirE2, a fragment or homologue thereof into a membrane, whereby the VirE2, fragment or homologue thereof forms a channel through the membrane; contacting the membrane with a molecule desired to be transferred across the membrane; and allowing the molecule to cross the membrane through the channel.

For example, the present invention particularly provides a method for delivering nucleic acids across membranes, where the nucleic acid can be DNA, RNA, or combinations thereof, or comprise modified residues. The nucleic acid can be single-stranded or double-stranded, including anti-sense and sense oligonucleotides, ribozymes, chimeric oligonucleotides and. expressible nucleic acids, such as exogenous DNA for stably transforming or transiently transfectingltransforming plant or animal, preferably mammalian cells. The nucleic acid can optionally be provided in a complex with protein to achieve a desired property. For example, a nucleic acid comprising at least one T-DNA border sequence or functional part thereof complexed to Agrobacterium VirD2 can be delivered to a cell using the method of the invention to achieve nuclear targeting. VirE2 itself is also known to protect single-stranded DNA from nuclease attack.

Delivery across a membrane is not limited to delivery into a cell but includes liposome loading, for example. However, the method of the invention is particularly useful to deliver materials into a cell or organelle, optionally using VirE2 liposomes, where the cell can be prokaryotic or eukaryotic, such as a yeast cell, an insect cell, a plant cell or an animal cell, preferably a mammalian, most preferably a human cell. Transfer of material to an organelle, such as to the nucleus, mitochondria or plastids, in particular chloroplasts, is also . envisioned.

In another aspect of the invention, animal or plant cells or tissues stably transformed with a discrete DNA fragment delivered by the methods described herein are regenerated to produce transgenic animal organs or whole animals or plants that stably express a desired homologous or heterologous nucleic acid and, in the latter case, pass it on to progeny in which stable expression of the transgene is inherited as a Mendelian trait. Furthermore, the present invention provides novel means for the<I>in vivo, ex</I> vivo <I>or in vitro</I> transformation and integration, or transient transfectionltransformation of exogenous nucleic acids desired to be expressed within host cells, using the delivery methods of the invention. The present invention is based on the novel finding that the VirE2 protein, previously shown to be a soluble, single stranded (ss) DNA binding protein, can insert into membranes and form channels. These channels have been demonstrated to be anion selective, thereby allowing molecules such as nucleic acids to be transported across membranes, although the transport of uncharged or cationic material is also envisioned in the methods of the invention. The opening and closure (gating) of the VirE2 channels can be regulated by various parameters, including, without limitation, the transmembrane potential.

Thus, in its broadest aspect, the present invention provides a method for delivering a molecule of interest, preferably charged molecules, more preferably anions, such as nucleic acids, across membranes by (a) introducing Agrobacterium VirE2, a fragment or homologue thereof into a membrane, whereby the VirE2, fragment or homologue thereof forms a channel through the membrane; (b) contacting the membrane with the molecule of interest and (c) allowing the molecule to cross the membrane through the channel.

The term "membrane" as used herein refers to a lipid monolayer, bilayer or combinations thereof, as well as a layer (or layers) of molecules (in particular polymers) which mimic lipid layers. The membrane may or may not contain additional substances, such as proteins, sterols, lipid derivatives and ceramides. Thus, the term "membrane" is used to encompass man-made materials such as liposomes, as well as naturally occurring materials, including without limitation, organelle membranes, such as nuclear, mitochondria) or plastid membranes, plasma membranes, cells, in particular eukaryotic cells, including plant cells or protoplasts, yeast cells, insect cells, or animal cells, in particular human cells. In certain embodiments, the use of VirE2 to transport molecules into bacteria, in particular Escherichia coli, as well as nematode and fungal cells is also envisioned.

The present invention relies on the introduction of Agrobacterium VirE2, preferably Agrobacterium tumefaciens VirE2, a fragment or homologue thereof into a membrane. VirE2 can be purified by methods known in the art such as those described in Christie<I>et al.,</I> ((1988) J. Bacteriol. 170(6):2659-2667) or below in Example 1. Alternatively, the entire nucleotide and amino acid sequences of the VirE2 protein of Agrobacterium have been published by Hirooka et al. ((1987) Bacteriol. 169(4):1529-36) allowing recombinant VirE2 production.

The term "fragment or homologue thereof" as used herein refers to modified or unmodified peptides or polypeptides derived from VirE2, which retain the characteristic, alone or in combination, to form a channel across a membrane. Unless otherwise stated or clear from the context, all reference to VirE2 herein encompasses VirE2, its functional fragments or its homologues, where the term "homologue" encompasses deletions, substitutions and additions to the VirE2 sequence referred to above, preferably where any substitution is a conservative amino acid substitution. Thus, also encompassed by the term homologue are fusion proteins that comprise additional coding sequences, optionally separated from the VirE2 sequences by a linker sequence, such as an epitope tag, an affinity tag, or a reporter molecule or enzyme (e.g., green fluorescent protein or -galactosidase), as exemplified below (see Example 1). VirE2 homologues with amino acid substitutions relative to native VirE2 are described in U.S. Patent No. 5,831,020, for example. Also encompassed are naturally occurring functional VirE2 homologues, i.e. VirE2 homologues that form channels in membranes allowing passage of desired molecules across the membrane. Although the Examples below exemplify the invention using VirE2 derived from Agrobacterium tumefaciens, a protein identified in Agrobacterium rhizogenes has been shown to complement a VirE2 defective strain for virulence, although the protein shows no sequence homology to A. tumefaciens VirE2 (Hirayama and Oka (1990) Bulletin of the Institute of Chemical Research, Kyoto Univ, 67, No. 5-6). Thus, functional VirE2 homologues need not necessarily have sequence homology to Agrobacterium tumefaciens VirE2 but can be identified using functional assays. Preferably, VirE2 homologues will comprise sequences with at least 80% identity to<I>A.</I> tumefaciens VirE2, more preferably at least 90% identity, most preferably at least 95% identity.

VirE2 can be introduced into the membrane by various methods as is apparent to one of skill in the art. For example, the peptide or protein can simply be mixed with appropriate lipids (e.g., POPO-Et or other commercially available lipids) to form VirE2-lipid vesicles or liposomes. Liposomes prepared in this manner can optionally comprise additional biologically active molecules, such as cell- or tissue4argeting molecules, or molecules that regulate opening of the VirE2 channels. U.S. Pat. No. 5,264,618 describes a number of techniques for using lipid carriers, including the preparation of liposomes, the teachings of which can easily be applied to the present invention. Methods to introduce VirE2 channels into cells or naturally-occurring membranes are likely to depend on the cell or membrane type, plant cells requiring slightly different treatment to animal cells, for example. In any event, purified VirE2 may be added directly to cells (or protoplasts) in culture, or to organelles or to membrane preparations<I>in vitro,</I> in an amount sufficient for integration of VirE2 in the membrane. Methods for preparing organelles and cellular membranes are well known in the art. For example, traditional cell fractionation methods such as density sedimentation can be used. In this aspect of the invention, the membrane preparation comprising the integrated VirE2 can be optionally rinsed with an appropriate solution maintaining physiological conditions, for example, prior to addition of the material to be transferred across the membrane. Alternatively, expression of recombinant VirE2 in a cell will result in its integration in the cellular membranes, in particular the plasma membrane (see Example 1, below).

The amount of VirE2 integrated in the membrane should be sufficient to obtain the desired effect (e.g., loading of liposomes or delivery of a desired material to a cell) without undue toxicity to the cell, if and when cells are used. Transient toxic effects of an open VirE2 channel to a cell are possible. However, as is apparent to one of skill in the art in light of this disclosure, routine laboratory procedures will allow determination of conditions promoting stability of the cell and/or regulation of the transfer of a desired molecule across a membrane by VirE2. For example, it is known in the art that channels can be gated (open and closed) by pH, salt concentration, lipids, hydrostatic pressure, peptides, polyamines and lipopolysaccharides (Todt and McGroarty (1992) Biochem. Biophys. Res. Commun. 189:1498-502; Simon and Blobel (1992) Cell 69:677-84; Ishii and Nabac (1995) FEBS 320:251-255). Thus conditions for each cell type used, or other criterium can be easily optimized using one or more of these parameters, The transfer of nucleic acid (or other desired material) across the membrane can be easily detected. For example, loading of liposomes with nucleic acid can be followed by vesicle swelling assays as described in Example 1 below. Conditions for transfer of a desired molecule through a VirE2 channel in a particular system (e.g., using a particular lipid compositions, cell type or cellular membrane) can initially be determined by following the transfer of an easily detected molecule, such as a labelled molecule (for example, a radiolabelled or fluorescently labelled nucleic acid, or a radiolabelled sugar), through the VirE2 channel. As nucleic acids of various lengths and sequences were transferred across membranes using similar loading times (see Example 1), it is expected that conditions determined in this manner will be broadly applicable, at least for further optimization. Such methods are also useful in determining functional fragments and homologues of VirE2, and comparing their functional activity (transfer of a desired molecule across a membrane) to VirE2 (full length, unmutated VirE2 protein).

The materials that will be transported across the membrane will typically be molecules with a specific biological function, for example in bacteria, insects, animals or plants. The materials include molecules of therapeutic and diagnostic value in animal or human systems, as well as molecules effective in in vitro or ex vivo systems. For example, liposomes have been used effectively as biological carriers in a number of pharmaceutical and other biological situations, particularly to introduce drugs, radiotherapeutic agents, enzymes, viruses, nucleic acids, transcriptional factors and other cellular vectors into a variety of cultured cell lines and animals. See, for example, U.S. Pat. No. 5,264,618, which describes a number of techniques for using lipid carriers, including the preparation of liposomes and pharmaceutical compositions and the use of such compositions in clinical situations. Similarly, the method of the invention can be used to load liposomes having VirE2 channels with any of these desired molecules or to introduce the same materials into cells or organelles.

As the VirE2 channel has been demonstrated to be an anion-selective channel, the material will preferably be anionic material, although it will be apparent to the artisan that conditions may be altered to allow transport of cationic or uncharged molecules across a VirE2 channel (for example, by altering the gating properties of the channel or modifying VirE2 sequences). The term "anion" or "anionic material" as used herein is meant to encompass any chemical compound with a negative charge, but will typically refer to a macromolecular anion, such as proteins or nucleic acids, including anti-sense oligonucleotides or exogenous DNA for stably transforming cells, for example.

"Nucleic acid(s)" according to the present invention may be any type of nucleic acid, such as single- or double-stranded nucleic acid, sense or anti-sense, DNA or RNA, modified or unmodified, wherein DNA is the preferred form, and single stranded DNA most preferred. Modified nucleic acids (mimetics) may comprise one or more modified (i.e., synthetic or non- naturally occurring) nucleotides or similarly functional moieties. Usually, nucleotide monomers in a nucleic acid are linked by phosphodiester bonds or analogues thereof. Analogues of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, peptide, and the like linkages.

For example, PNA is an oligonucleotide with peptide bonds instead of phosphodiester bonds in its backbone. Because PNA has no charge, PNA has a higher binding affinity than deoxyribonucleic acid and therefore may offer particular advantages for use in anti-sense or sense technolgies. Similarly, other linkages have been shown to be particularly resistant to nuclease attack. Those of skill in the art will recognize that the reagents employed to prepare modified nucleic acids are commercially available and, in the case of oligonucleotides, can be prepared using commercially available instrumentation.

The method of the present invention is particularly useful for delivering nucleic acid to a cell, where the nucleic acid may be, without limitation, a nucleic acid designed for<I>in situ</I> hybridization, an anti-sense or sense nucleic acid, or a specifically designed exogenous nucleic acid desired to be expressed in a cell targeted for transformation. In this context, the terms "expressed" or "expressible" used throughout the specification shall mean that a given nucleic acid can at least serve as a target for transcription within the nucleus of a cell to be transiently or permanently transfected or transformed. As used herein, the term "exogenous" refers to material initially separate from the host cell. Thus, the term "exogenous" DNA or nucleic acid may be of homologous or heterologous origin with respect to the cell type involved, it may be of synthetic or recombinant origin, or a combination thereof.

For example, antisense or sense oligonucleotides can be designed which, once delivered to the cell, can modify native cellular DNA sequences to alter specific codons (for example, to take into account preferred codon usage by a particular polymerase, to mutate or correct a mutation in a sequence), to improve expression efficiency, etc. As used herein, the terms "sense" and "antisense" are not limited to coding regions of a gene but encompass oligonucleotides that may hybridize specifically to non-coding regions. Thus, oligonucleotides can be used corresponding in sequence to a cellular sequence to be targeted, either corresponding to the coding strand (sense oligonucleotides) or complementary to the coding strand (antisense oligonucleotides), and optionally carrying a mutation. In this respect, oligonucleotides can be delivered by the methods of the invention to various cell types for<I>in</I> <I>situ</I> hybridization allowing, for example, diagnosis of a genetic defect in animals or humans, or identification and/or quantification of a particular gene product in plants.

In the context of this invention, "hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. "Complementary," as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of<I>in</I> vivo assays or therapeutic treatment, and in the case of<I>in vitro</I> assays, under conditions in which the assays are performed.

Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use. The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.

While antisense oligonucleotides (RNA or DNA) are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds including but not limited to oligonucleotide mimetics such as are described above. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 30 nucleobases (i.e. from about 8 to about 30 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 25 nucleobases.

According to the present invention ribozymes or oligozymes are also provided, for example, for use within a given host cell, by delivering to the cell a nucleic acid complementary to the coding strand of a desired gene, which optionally can become part of the host cell's genome. Alternatively, the ribozymes or oligozymes can be delivered directly to the cell.

In a further aspect of the invention, chimeric oligonucleotides comprising deoxyribo- and ribo-nucleotides, which allow site-directed mutagenesis, are delivered across a membrane.. The chimeric oligonucleotides comprise a DNA "mutator" region complementary to the target except for the mutation, flanked by 2'-O-methyl-RNA bridges, also complementary to the target locus. The RNA bridges are linked to DNA sections complementary to the chimeric region flanked with non-complementary, loop-forming nucleotides. A strand break in the lower DNA strand allows topological interwinding of the chimeric oligonucleotide into the target DNA allowing the precise introduction of a nucleotide change into the targeted sequence (Proc. Natl. Acad. Sci. USA 96:8321-8323, 8768 et seq. and 8774<I>et</I> seq. (1999)). In a further embodiment, exogenous nucleic acid is delivered to a cell to achieve expression of a desired transgene by the cell. The exogenous nucleic acid will therefore comprise an expressible nucleic acid, in particular a structural gene, preferably a heterologous structural gene. Especially suitable for use in the process according to the invention are structural genes that upon expression produce proteins or polypeptides that are beneficial for the transformed cells, tissues or organisms, e.g. which compensate eventual mutations, which. have pharmacological properties and could be used as pharmaceutical agents in the treatment of diseases in animals or humans, or which provide insect or fungal resistance to plants. The encoded protein or enzyme is typically not being expressed in the host cell or is being expressed at very low levels. Apart from naturally occurring structural genes that code for a useful and desirable property or a pharmacological agent, the use of genes that have previously been modified using chemical or genetic engineering methods is also within the scope of this invention. Many examples of nucleic acid molecules for which it would be desirable to import the molecules into a host cell should be readily apparent to those skilled in the art.

Examples of structural genes for use in animal systems include those encoding hormones,. immunomodulators and other physiologically active substances, such as genes encoding insulin, somatostatin, interleukins (for example, IL-1, IL-2 and IL-10), interleukin-1 receptor antagonist protein (1L-1 ra), <I>herpes simplex</I> virus thymidine kinase (HSV-tk), clotting factor IX, clotting factor VIII, tissue plasminogen activator (t-PA), erythropoietin, brain-derived neurotrophic factor, apolipoprotein E (apo E), fumarylacetoacetate hydrolase (FAN), adenosine deaminase and cystic fibrosis transmembrane conductance regulator (CFTR) (associated diseases are described in USPN 5,831,020). As should be readily apparent from the above examples, many applications of the method of the subject invention could be in the area of gene therapy, where a protein or enzyme of interest can be expressed in a desired host cell.

Examples of structural genes for use in plant systems include those encoding pathogen resistance genes, genes improving crop quality or quantity, or genes involved in metabolic pathways. . The expressible nucleic acid will typically be operably linked to appropriate expression signals active in the recipient cell, such as an enhancer, promoter and transcription termination sequences, as well as, optionally, to further coding and/or non-coding sequences of the 5' and/or 3' region, such as e.g. signal sequence or nuclear targeting sequences. It is also often advantageous to incorporate a leader sequence between the promoter sequence and the adjacent coding DNA sequence, the length of the leader sequence being so selected that the distance between the promoter and the DNA sequence to be expressed is the optimum distance for expression of the associated structural gene. Furthermore, the exogenous nucleic acid may additionally comprise sequences encoding . one or more selectable markers useful in screening for positive transformants. In general, these markers are proteins necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection markers encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, hygromycin, kanamycin, neomycin, puromycin, methotrexate or tetracycline, complement auxotrophic deficiencies, sulfonomide, or supply critical nutrients not available from complex media. Further examples of genes that confer antibiotic resistance include those coding for the kanamycin resistance (NPT II) gene derived from Tn5 (Bevan et al. (1983) Nature 304:184- 187), and chloramphenicol acetyltransferase.

Suitable selectable markers for animal, particularly mammalian cells are those that enable the identification of cells competent to take up the nucleic acid encoding said selectable marker, such as dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase, or genes conferring resistance to G418 or hygromycin (see, for example, Blochlinger and Diggelmann (1984) Molecular and Cellular Biology 4:2929-2931; Robertson and Whalley (1988) Nucl. Acids Res. 16:11303-11317; O'Brian et al. (1997) Gene 184:115-120). The animal cell transformants are placed under selection pressure where only those transformants expressing the marker survive. In the case of a DHFR or glutamine synthase (GS) marker, selection pressure can be imposed by culturing the transformants under conditions in which the pressure is progressively increased, thereby leading to amplification (at its chromosomal integration site) of both the selection gene and the linked DNA that encodes the structural gene of interest. Increased quantities of desired protein are usually synthesised from thus amplified DNA.

For the purpose of screening transient expression of the desired gene introduced into a suitable host cell according to the invention the exogenous DNA may also comprise sequences encoding reporter molecules, such as (3-galactosidase, P-glucuronidase, green fluorescent protein (gfp) or different colour variants, or luciferase. Methods for the detection of the expression of said markers are well known in the art. Screening of cells and organisms derived from such cells for the presence of specific nucleic acid sequences may also be performed by Southern analysis (Southern (1975) J. Mol. Biol. 98:503). Details of this procedure are given in Maniatis et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. This screening may also be performed by the use of Polymerase Chain Reaction procedures (PCR). PCR procedures are described in detail in Mullis et al. (1987) Meth. Enzymol. 155:335-350 and Erlich (ed.), PCR Technology, Stockton Press, New York (1989).

The expression signals active in target cells usually comprise a promoter that is recognised by the host organism and is operably linked to the exogenous DNA to be expressed in the transformant. Such a promoter may be inducible or constitutive. The promoters are operably linked to the DNA by removing the promoter from a source DNA by restriction enzyme digestion and combining the isolated promoter sequence with the expressible DNA sequence. Both the native promoter sequence of the structural gene of interest and many heterologous promoters may be used to direct amplification and/or expression of the structural gene in the host cell. Promoters vary in their strength, i.e., ability to promote transcription. For the purpose of expressing the nucleic acid, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters can be used, such as, the lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda, and others, including but not limited to, T7, IaCUV5, ompF, bla, Ipp promoters and the nopaline synthase (nos), the 1', 2', the cauliflower mosaic virus 35S promoters, and promoters isolated from plant genes, including the small subunit ribulose bisphosphate carboxylase, the small subunit chlorophyll A/B binding polypeptide and the Pto promoters (Vallejos <I>et al.</I> 1986), the latter directing high levels of transcription of adjacent DNA segments. Suitable promoters for animal and in particular mammalian hosts are those derived from the genomes of viruses such as polyoma virus, adenovirus, fowipox virus, bovine papilloma virus, avian sarcoma virus, Rouse sarcoma virus (RSV), cytomegalovirus (CMV) and Simian Virus 40 (SV40), and heterologous mammalian promoters such as the [3-actin promoter or a very strong promoter, e.g. a ribosomal protein promoter, and the promoter normally associated with structural gene sequence to be expressed, provided such promoters are compatible with the host cell's system.

The transcription of an exogenous DNA encoding the desired structural gene can be increased by inserting an enhancer sequence into the DNA. Enhancers are eukaryotic DNA elements that appear to increase transcriptional efficiency in a relatively orientation and position independent manner. Many enhancer sequences are known from mammalian genes (e.g. elastase and globin). However, typically one will employ an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer (bp 100-270) and the CMV early promoter enhancer. The enhancer may be spliced into the recombinant chimeric sequence at a position 5' or 3' to the coding DNA sequence, but is preferably located at a site 5' from the promoter.

Preferably, the nucleic acid described herein is targeted into the nucleus of a host cell, where the nucleic acid molecule is to be expressed. Since the nuclear-localized nucleic acid will eventually be degraded, it may be desirable for long term expression of the DNA molecule in the nucleus of the host cell to integrate the DNA into the genome of the host cell. In such an embodiment, the nucleic acid of the subject invention further includes a molecule to direct integration of a DNA molecule into the genome of the host cell. Such integration sequences are known in the art, and include, for example, the inverted terminal repeats (ITRs) of adeno-associated virus, retroviral long terminal repeats (LTRs), and other viral sequences shown to promote incorporation or integration of the viral genome into the host cell genome. It may also be desirable to include a bacterial origin of replication in the nucleic acid (such as on C for replication in Escherichia coli, or the origin of replication of Bacillus subtilis, or the origin of replication of Pseudomonas aeruginosa, etc.) so that the nucleic acid can be maintained and replicated in a bacterial host.

In a further aspect of the invention, the nucleic acid is delivered complexed with protein, such as recombination promoting proteins (e.g., RecA) or nuclear targeting proteins, such as Agrobacterium Vir proteins. For example, transformation of plant and animal cells using the DNA/protein complex disclosed in WO 95I05471 and Ziemienowicz <I>et al.</I> ((1999) Proc. Natl. Acad. Sci. USA 96:3729-3733), as well as similar complexes in which the nucleic acid component is in the form of an oligonucleotide allowing antisense-, sense- and oligozyme- approaches is envisioned. Briefly, the nucleic acid/protein complex disclosed in WO 95l05471 may be obtained by first providing a recombinant nucleic acid construct that comprises in operable linkage to the elements already mentioned above at least one T-DNA border sequence or functional part thereof as a substrate in the VirD2 cleavage reaction together with at least one Agrobacterium-derived protein that promotes the integration of the exogenous DNA into the host cell genome, preferably VirD2. The purification of VirD2 is described by Pansegrau et al. ((1993) Proc. Natl. Acad. Sci. USA 90:11538). The term "T- DNA border(s) or functional part(s) thereof' encompasses the whole T-DNA border sequence(s) of Agrobacterium as well as those parts thereof which have functional consensus or cleavage site or binding domain sequence(s) necessary for a desired protein to interact with the nucleic acid. If the nucleic acid comprises only part of the T-DNA border sequence, the partial sequence still comprises those parts of the T-DNA border sequence that encompass the recognition and cleavage site of the VirD2 protein. Nevertheless, in other embodiments the methods of the present invention do not require any Agrobacterium- derived material other than VirE2, its homologues or fragments and therefore the use of exogenous DNA lacking T-DNA borders is contemplated, as well as the absence of VirD2 and/or VirD1 proteins.

The nucleic acid molecule delivered to a host cell (or organelle) is most likely to express a useful product in animal, mammalian, plant, or insect host cells. Whether a nucleic acid or other desired material, the material can be delivered directly to the cell of interest by providing the cell with VirE2 channels or indirectly using liposomes having VirE2 channels. The latter can fuse with cellular membranes<I>in vitro, ex vivo</I> or<I>in vivo,</I> thereby delivering their contents to the desired recipient cell or organelle. In this embodiment, plant protoplasts rather than plant cells are more likely to be targeted, as fusion of the liposome with cellular membranes is more easily achieved in the absence of a cell wall. Direct delivery to plant cells is more easily achieved using VirE2 expressing transgenic plants as described in Example 6, below. In contrast, delivery of materials to animal cells <I>in vivo</I> will typically involve <I>in vitro</I> culturing of recipient cells (to allow introduction of VirE2 for subsequent delivery of material) or VirE2-liposome administration.

The method according to the invention can, therefore, be advantageously used for effective nucleic acid (or other material) transfer into eukaryotic cells. For example, the use of VirE2 proteoliposomes may be an elegant alternative to circumvent problems, such as toxicity or host defenses, obtained with presently used delivery methods. The lipid composition of the proteoliposomes can easily be varied to avoid toxicity or an immune response, for example, and liposome preparation procedures allow the use of other molecules, such as proteins, to be co-administered to favour targeting and gene delivery regulation. In addition, by obtaining efficient DNA delivery, the transformation efficiency can be improved compared to conventional techniques. The quality of the delivered nucleic acid can also be improved resulting from the absence of extraneous sequences (e.g., viral or vector sequences) thus avoiding possible deleterious rearrangements. Thus, the method is useful in the treatment of cancer cells, relying on a new non-viral system without LTR and the possible hazards connected with them.

Suitable host cells are any cells into which a molecule can be delivered to obtain a desired effect. For example, and referring to the many possible uses of the subject invention discussed above with respect to nucleic acids, the host cell may be a pulmonary epithelial cell where gene therapy of cystic fibrosis lung disease is being treated and/or prevented. Vascular cells may be a suitable host cell where tPA is desired to be expressed. Plant cells, such as of various crop plants including potato, tomato, cereals, etc., are suitable host cells where plant disease resistance genes are desired to be expressed. Many other suitable host cells should be readily apparent, as the invention has broad applicability to various host cells and various molecules to be delivered thereto, The importation of a nucleic acid to a host cell may also be desirable in vitro, using various cell lines or strains known in the art.

In recent years propagation of cells, whether plant tissue, protoplasts or vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful plant cells are tobacco BY-2, maize BMS or Arabidopsis cell suspensions. Examples of useful vertebrate host cell lines are epithelial or fibroblastic cell lines such as Chinese hamster ovary (CHO) cells, COS1 cells (monkey kidney cells transformed with SV40 T-antigen), CV1 cells (parent line of the former), Rat1 (rat fibroblast) cells, NIH 3T3 cells, HeLa cells, LLC-Pk1 (pig kidney epithelial) cells or 293T cells, although cells may also be obtained from the host (e.g., stem cells). Examples of insect cell lines are Drosophila <I>Schneider,</I> Drosophila Ksub.C, and Sf9. The host cells referred to in this disclosure comprise cells in<I>in</I> vitro%x <I>vivo</I> culture as well as cells that are within a host organism.

Techniques are known for the<I>in vitro</I> culture of plant tissue, and in a number of cases, for regeneration into whole plants (Hansen and Chilton, "Lessons in Gene Transfer to Plants by a Gifted Microbe" in Current Topics in Microbiology and Immunology , vol. 240, pub[. Springer Verlag, Heidelberg, Berlin, New York (1999), pp 22-57. Similarly, techniques for producing transgenic animals, in particular transgenic rodents, as well as for<I>ex</I> vivo human therapy, are also well known in the art. Thus, the present invention also concerns the preparation of transgenic cells, including oocytes, spermatocytes and zygotes etc., transgenic organs, and transgenic animals and plants, as well as the cells and organisms obtained by use of a method according to the invention. Once introduced into the plant or animal, the expression of the structural gene (or presence of the desired molecule) may be assayed by any means known to the art. For example, expression may be measured as mRNA transcribed or as protein synthesized.

The invention is further described, for the purposes of illustration only, in the following examples.

<U>Examples</U> Example 1: DNA transfer into VirE2-lipid vesicles T-DNA transfer from Agrobacterium into plant cells is the only known example for interkingdom DNA transfer. The bacterial virulence protein VirE2 has previously been shown to play an important role in this process by binding to the imported single-stranded (or ss) DNA. This example describes for the first time that VirE2 can additionally insert in membranes and form anion specific channels. Planar lipid bilayer experiments and vesicle swelling assays show that the opening of anion specific channels can be regulated by the membrane potential, for example, allowing high affinity transport of nucleic acids across membranes.

(a) Cellular localization of VirE2 A VirE2-GUS fusion construct has previously been used to show a primarily nuclear localisation of VirE2 in the cell (Citovsky et aL(1992) Science 256:1802). As the localization studies relied on light microscopy and a diffusable GUS substrate, VirE2 cellular localization is visualized here at higher resolution and using methodology that lowers background staining. VirE2 flanked at its 3' end with Kozak and (hemagglutinine; HA) epitope sequences is obtained by PCR using 5'-TCATGGATCCACCACCATGGATCTTTCTGGCAATGAGAAA-3' (SEQ ID NO: 1) and 5'-ACTCTCTAGATCAAGCGTAATCTGGAACATCGTATGGGTA AAAG CTGTTGACGCTTTGGCT-3' (SEQ ID NO: 2) as primers and pVCK225 (Knauf and Nester (1982) Plasmid 8:45-54) as template, and cloned in pCR2.1 (Invitrogen). The BamHl-PStI fragment containing the VirE2-HA gene is cut out and inserted into pCAMBIA (Hajdukiewicz et al (1994) Plant Molecular Biology 25:989-994; Hiei et al. (1994) The Plant Journal 6:271- 282; www.cambia.org/mainlr et camvec.htm) using the same restriction enzymes. The resulting construct is used to transfect tobacco BY-2 cells in culture by bombardment. The VirE2 fusion protein is visualized in the cells using monoclonal antibodies specific for the HA tag (commercially available), thus abolishing background staining. Immunofluorescence shows staining of vesicle-like structures throughout the cell, including the nucleus, and areas adjacent to the cellular membrane. Because fluorescence microscopy has a resolution of just 400 nm, electron microscopic analysis is carried out to study the localisation of VirE2 more precisely. Electron microscopy studies of VirE2 inside the plant cells shows that the majority of Vir E2 molecules localize near the plasma membrane.

(b) VirE2 integration into lipid monolayers To study if VirE2 interacts with lipids, Langmuirtrough experiments (Schwarz and Taylor (1995) Langmuir 11:4341-6) are performed using VirE2 fused to a His6 tag (Janknecht et al. (1991) Proc. Natl. Acad. Sci. USA 88:8972-8976) for ease of purification. Briefly, a Stul- Smal fragment from plasmid pSW108 (Winans et al. (1987) Nucl. Acids Res. 15(2):825-37) containing VirE1 and VirE2 genes of the VirE operon is inserted in the Hincll site of pUC18. (ATCC 37253; commercially available) resulting in plasmid pUCSS. An Ndel restriction site is introduced at the level of the first ATG of VirE2 by PCR using primer p5 (5'-ATCGTAGCC TGCAGAGTCATATGGATCT TTCTGGCAATGAGAAATCC-3'; SEQ ID NO: 3), primer p6 (5'-GTTTGATAAAAGATCTCTGTGCC-3'; SEQ ID NO: 4) and plasmid pSW108 as a template. The amplified PCR product is cut with Pstl and Bglll and inserted into pUCSS previously cut with the same restriction enzymes resulting in plasmid pUCE2. An Ndel- BamHl fragment from plasmid pUCE2 is inserted in plasmid pET3a (Studier et al. (1990) Methods Enzymol. 185:60-89) previously digested with the same enzymes, resulting in plasmid pETE2. Sequence encoding six histidines followed by a stop codon is introduced at the C-terminal of VirE2 by PCR using oligonucleotide A (5'-AAGACGTCCTCAGTGATGGT GATGTGATG-3'; SEQ ID NO: 5) and B (5'-TATCTGGAATCCTGGGAACG-3'; SEQ ID NO: 6) and plasmid pETE2 as a template. The PCR is performed at 50 C with Taq polymerase (Qiagen) for 10 cycles with 300 ng of plasmid and 4 minutes of elongation. The amplified PCR product is cut with Sacl and Pstl and inserted into pETE2 previously cut with the same restriction enzymes resulting in pETE2-His6. pETE2-His6 is used to transform E. coli cells (BL21 (DE3) strain) by electroporation. Protein expression is induced with 1 mM IPTG for 4 hours at 28 C. Cells ere harvested and then lysed in 50mM NaH2P04 pH8, 300mM NaCl, 20mM imidazole and 1 mg/ml lysozyme on ice for 30 minutes. After centrifugation at 10000g for 20 minutes at 4 C, the supernatant is loaded on a Ni-NTA-agarose (Qiagen) following the manufacturer's instructions. The column is washed with 50mM NaH2P04 pH8, 300mM NaCl, 50mM imidazole and the protein eluted in 50mM NaH2P04 pH8, 300mM NaCl, 250mM imidazole. The purified protein is finally dialyzed to 50mM NaH2P04, 300mM NaCl, pH8.0 and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) for purity.

The purified VirE2 is added with a calibrated microsyringe directly in the 1-palmitoyl-2-oleoyl- phosphatidy1choline (POPC) subphase composed of a 50mM NaH2P04, 300 mM NaCl, pH 8.0 buffer, essentially as described by Schwarz and Taylor (1995) and measurements started 30 minutes after injection. The subphase is stirred and experiments are carried out at room temperature. The resulting compression-isotherm curves of 1-palmitoyl-2-oleoyl- phosphatidylcholine (POPC) monolayers are recorded in the absence or presence of different amounts (2nM, 5.9nM or 15.7nM) of purified His-tagged VirE2 in the subphase. Surprisingly, the addition of VirE2 in the subphase of the monolayer film induces a consequent shift towards larger area/molecule in the compression-isotherm curves. This can be either explained by insertion of VirE2 into the monolayer or by an absorption process modifying the lipid organization at the air/water interface.

(c) VirE2 channel formation in lipid bilayers To test for possible channel activity planar lipid bilayer experiments are performed (Van Gelder et al. (2000) Eur. J. Biochem. 267:79-84). Planar lipid bilayer experiments are carried out in 10mM Tris, 1 mM CaCl2, 1 M KCI, pH 7.4 buffer at 20 C. After formation of a lipid bilayer, 3-5ug of purified VirE2 are added to the cis chamber (0.5pglml final concentration) and an electrical potential difference of 100mV is applied across the membrane. Discrete conductance steps are observed indicating that VirE2 induced the formation of channels in the membrane. From the analysis of the conductance jumps, a single VirE2 channel is determined to have.a conductance of 1.5nS +l- 0.2nS (n=56). This value is 10 times larger than that measured for the folded protein translocation YopB-YopD channel of Yersinia (Tardy et al. (1999) EMBO J. 18:6793-9). Planar lipid bilayer and patch-clamp experiments demonstrate that most membrane channels are not permanently open but can be closed upon application of a membrane potential above a certain threshold value (Delcour (1997) FEMS Microbiol. Left. 151:115-23); Morgan et al. (1990) Biochim Biophys Acta 1021:175-81). This phenomenon, known as voltage gating, is also observed with the VirE2 channels.

The current/voltage relationship of two VirE2 channels in a phospholipid bilayer is assessed by gradually changing the applied membrane voltage from 0 to +150 mV and 0 to -150 mV using a voltage ramp. All VirE2 channels are closed above 120 mV transmembrane potential. Thus as the plasma membrane potential of plant cells is in a range of 100-250 mV (Shabala et al. (1998) Plants 204:146-52); Pouliquin et al. (1999) Biophys J. 76:360-73), VirE2 channels are likely to be typically in a closed state in plants, unless activated.

Regulation of channel opening at the molecular level presents an attractive possibility both for ssDNA transfer and for the survival of the plant cell. Indeed, a variety of factors such as pH, hydrostatic pressure, lipids or peptides have been shown to modulate the opening/closing of channels (Todt and McGroarty (1992) Biochem. Biophys. Res. Commun. 189:1498-502; Simon and Blobel (1992) Cell 69:677-84; Ishii and Nakae (1993) FEBS Lett. 320:251-5; Le Dain et al. (1996) EMBO J. 15:3524-8).

(d) Selectivity of VirE2 channels Planar lipid membrane measurements performed under asymmetrical conditions (0.1 M and 1 M of KCI on the cis and<I>trans</I> side of the membrane, respectively) reveal an anion selectivity of the channels. The transmembrane voltage difference ()V) is given for e.g. a NaCl and KCI solution by the Goldman-Hodgkin-Katz equation:

Here, R is the molar gas constant, F the Faraday constant, T the temperature, P, the respective permeability and [X],, the ion concentration in the respective compartment. Permeability (PC, IPK+) calculated according to the Goldman-Hodkin-Katz equation shows a ratio of anion over cation influx of 9. Both the large diameter of the channel and its anion selectivity suggests that it can interact with or transport anionic molecules such as DNA or RNA. Verification of this hypothesis can be obtained by the addition of a 19-mer oligonucleotide (5'-AGTTGATGAACCGGGTTGC-3'; SEQ ID N0: 7) to one side of the planar lipid bilayer. The ssDNA partially blocks the channel in a concentration dependent manner, decreasing the ion flux across the membrane by approximately 20%. A plot of the relative conductance inhibition (Gmax-G)/Gmax, as a function of the 19-mer ssDNA concentration in the aqueous phase can be used to determine the affinity, where Gmax corresponds to the conductance measured in the absence of ssDNA and G, the conductance obtained in the presence of different amounts of ssDNA (0-0.25:M in steps of 0.05:M). The voltage applied across the membrane is 20mV.

All channels are saturated at a 0.2pM ssDNA concentration, revealing a Kd of 0.075pM for the 19-mer ssDNA. Experiments using other oligonucleotides (the 26-mer, 5'-CCGGAAGCT GCAGCGCCATGGAAATC-3', SEQ ID NO: 8; the 18-mer, 5'-TAGGGGGATCCACCGGCC- 3', SEQ ID NO: 9; the 32-mer, 5'-CCCACTAGTGCTTTACTTGTACAGCTCGTCCA-3', SEQ ID NO: 10; the 19-mer, 5'-CTCACATGACGGTCGCGGG-3', SEQ ID NO: 11) or boiled salmon sperm DNA produce similar results, demonstrating that binding to the channel does not depend on the sequence or the length of the ssDNA.

(e) Osmotic swelling assay The previous experiments clearly demonstrate the interaction between ssDNA and the VirE2 channel. They do not, however, address whether ssDNA can actually be transferred through the channel. In order to investigate this possibility an osmotic swelling assay was performed (Luckey and Nikaido (1980) Proc. Natl. Acad. Sci. USA 77:167-71). Vesicles reconstituted in the presence or in the absence of VirE2 proteins are added to a ssDNA solution. The protocol is a modification of that described by Luckey and Nikaido ((1980) Proc. Natl. Acad. Sci. USA 77:167-71). Briefly, 2pmol soybean phospholipids (commercially available from Sigma) are dried as a thin film at the bottom of a tube and resuspended by vortex mixing in 600p.i 10mM Tris, 150mM NaCl pH 7.4 containing 5pg purified MrE2. Samples are then left for 1 hour at 4 C ("relaxed"). Control vesicles are prepared without addition of VirE2. Liposome swelling is followed by measuring optical density at 500nm. When 30<B>1.11</B> of VirE2 proteoliposomes are injected into 600p1 of 10mM Tris, 150mM NaCl buffer pH 7.4 containing (or lacking in controls) 2pM of the 19-mer nucleotide ssDNA solution (sequence provided . above), a decrease in the OD5oo is observed, revealing a swelling of the vesicles. Control experiments where liposomes or proteoliposomes are injected into buffer alone does not induce any change in vesicle state. The results demonstrate that the observed vesicle swelling is induced by the transport of ssDNA into the liposome via the VirE2 protein.

In summary, the experiments described above demonstrate a novel VirE2 function as a transmembrane channel, allowing nucleic acid transport across membranes and having applications in gene delivery systems.

Example 2: VirE2-mediated DNA transfer into animal cells To demonstrate VirE2-mediated DNA transfer into animal cells, VirE2 (30 mg) is added to a 50% confluent 3.5 cm petri dish containing HeLa cells and the cells are observed under a light microscope over one hour. If cell shrinkage is observed (suggesting loss of cell contents), conditions can be chosen to regulate the VirE2 channel opening, or the amount of VirE2 protein in the membrane can be decreased, to allow DNA delivery without toxic effect to the cell.

Fluorescently labeled DNA (Ziemienowicz et al. (1999) Proc. IN atl. Acad. Sci. USA 96:3729- 3733) is then added to the cells and transfer of the fluorescent label (and therefore DNA) can be followed by fluorescence microscopy. Analysis of the specificity of nucleic acid transport (e.g., by altering sequence, length, ssDNA, dsDNA or RNA) or other material of interest into a specific cell can similarly be carried out.

Example 3: VirE2 homologue-mediated DNA transfer into animal cells To screen for VirE2 homologues useful in DNA transport, native VirE2 can be mutagenized by PCR mutagenesis (inducing mainly point mutations), cloning in pCAMBIA (see Example 1), and transformation of non induced Agrobacterium tumefaciens. Under these conditions, the only bacteria growing are mutants in the VirE2 gene. These mutants can be affected in the amino acids associated with the membrane, or in the gating properties. Sequencing of the resulting mutants allows the mutation to be localized to a particular nucleotide. If desired, Western blotting can be used to verify that the protein is full length and/or appropriately tagged (e.g., His tag). The mutant VirE2 can be expressed in E.coli by cloning in the commercially available pEt vector, for example (see also Example 1). The presence of a His tag allows purification of the mutant VirE2 by Ni resin based procedures. The purified mutated protein can then be tested in lipid monolayers for the mutant's ability to associate with the membrane (see Example 1). Lipid bilayer experiments (see Example 1) can also be carried out to determine the gating properties of those mutants still able to associate with membranes. This approach also provides information on the amino acids of VirE2 involved in membrane association as well as those involved in the gating, which can then be specifically targeted for modification using recombinant or chemical techniques.

Once an effectively modified VirE2 homologue is identified, DNA transport (or other molecule transport) can be tested as described in Example 2.

Example 4: VirE2-mediated DNA transfer into plant cells VirE2 (30 g) is added to tobacco BY-2 cells 3 days after dilution (1.5:80) and the cells are observed under a light microscope over one hour. If cell shrinkage is observed, conditions can be altered to regulate the VirE2 channel opening or the amount of protein in the membrane as described in Example 2. Fluorescently labeled DNA (Ziemienowicz et al. (1999) Proc. Natl. Acad. Sci. USA 96:3729-3733) is then added to the cells and transfer of the fluorescent label (and therefore DNA) can be followed by fluorescence microscopy. Analysis of the specificity of nucleic acid transport into a specific cell (e.g., sequence, length, ssDNA, dsDNA or RNA) or other material of interest can similarly be carried out. In addition, modified VirE2 proteins useful for nucleic acid (or other molecule) transport into plant cells using the techniques described in Example 3 can be identified.

<U>Example 5:</U> VirE2-mediated DNA-VirD2 <U>transfer into cells</U> VirD2 has previously been shown to direct nuclear localization of nucleic acids complexed to VirD2. Improving nuclear localization of nucleic acids introduced into a cell is advantageous when expression of the nucleic acid is desired, for example. To demonstrate delivery of protein-nucleic acid complexes into a cell through a VirE2 channel, a nuclear localization protein-DNA complex has been designed containing single-stranded DNA comprising a sequence of interest linked to the right border sequence of T-DNA and VirD2. The complex is introduced into the desired host cell essentially as described above. Localization of the transferred nucleic acid to the nucleus can be visualized by detecting expression of the nucleic acid or any other means known in the art, for example, by using a labeled nucleic acid of interest, essentially as described above.

As is apparent to the artisan, double-stranded DNA may be similarly used, together with VirD2 and VirD1. The purification of the VirD1 protein can be achieved according to the method disclosed in WO 95l05471.

Example 6: DNA transfer into VirE2 expressing cells Tobacco seedlings expressing VirE2 (Citovsky et al. (1992) Science 256(5065):1802-5) are grown for 8 days on sterile, wet Whatman paper in a growth chamber at 25 C with 16 hours light/day. Five micrograms of ssDNA coding for the beta-glucuronidase gene, operably linked to the CaMV 35S promoter and terminator is cut by the VirD2 protein (10 micrograms) for 1 hour at 37 C in TNM buffer (20mM Tris.HCl, pH8.0, 50mM NaCl, 2 mM MgCl2). The VirD2 DNA complex is added to 100 seedlings in 10 MM MgS04. The mixture is exposed to reduced pressure (0.15 atm) in a sterile vacuum chamber for 5 minutes. The seedlings are placed on MS plates (1 % agar) and further cocultivated with the VirD2 DNA complex for 3 days in a growth chamber (25 C, 16 hours light/day). The plantlets are washed in sterile 10mM MgS04 and blotted dry on sterile Whatman paper. The plantlets are then stained by histochemical X-Gluc assay as reported by Rossi<I>et al.</I> ((1993) Plant Molecular Biology Reporter 11(3):220-229). Blue spots are visualized on the leaves, The disclosures of each publication referred to herein are hereby incorporated by reference in their entireties.

SEQUENCE LISTING < 110> Novartis Research Foundation < 120> Novel Method < 130> < 140> < 141> < 160> 11 < 170> PatentIn Ver. 2.1 < 210> 1 < 211> 40 < 212> DNA < 213> Agrobacterium tumefaciens < 400> 1 tcatggatcc accaccatgg atctttctgg caatgagaaa 40 < 210> 2 < 211> 61 < 212> DNA < 213> Agrobacterium tumefaciens < 400> 2 actctctaga tcaagcgtaa tctggaacat cgtatgggta aaagctgttg acgctttggc 60 t 61 < 210> 3 < 211> 47 < 212> DNA < 213> Agrobacterium tumefaciens < 400> 3 atcgtagcct gcagagtcat atggatcttt ctggcaatga gaaatcc 47 < 210> 4 < 211> 23 < 212> DNA < 213> Agrobacterium tumefaciens < 400> 4 gtttgataaa agatctctgt gcc 23 < 210> 5 < 211> 29 < 212> DNA < 213> Agrobacterium tumefaciens < 400> 5 aagacgtcct cagtgatggt gatgtgatg 29

< 210> 6 < 211> 20 < 212> DNA < 213> Agrobacterium tumefaciens < 400> 6 tatctggaat cctgggaacg 20 < 210> 7 < 211> 19 < 212> DNA < 213> Agrobacterium tumefaciens < 400> 7 agttgatgaa ccgggttgc 19 < 210> 8 < 211> 26 < 212> DNA < 213> Agrobacterium tumefaciens < 400> 8 ccggaagctg cagcgccatg gaaatc 26 < 210> 9 < 211> 18 < 212> DNA < 213> Agrobacterium tumefaciens < 400> 9 tagggggatc caccggcc 18 < 210> 10 < 211> 32 < 212> DNA < 213> Agrobacterium tumefaciens < 400> 10 cccactagtg ctttacttgt acagctcgtc ca 32 < 210> 11 < 211> 19 < 212> DNA < 213> Agrobacterium tumefaciens < 400> 11 ctcacatgac ggtcgcggg 19

Claims (16)

1. WHAT IS CLAIMED IS: 1. A method for delivering material across a membrane, said method comprising: (a) introducing Agrobacterium VirE2, a fragment or homologue thereof into a membrane, whereby said VirE2, fragment or homologue thereof forms a channel through said membrane; (b) contacting said membrane with a molecule desired to be transferred across said membrane; and (c) allowing said molecule to cross said membrane through said channel.
2. 2. The method according to claim 1, wherein said membrane is a cell membrane.
3. 3. The method according to claim 1, wherein said cell membrane is part of a prokaryotic cell.
4. 4. The method according to claim 1, wherein said cell membrane is part of a eukaryotic cell.
5. 5. The method according to claim 4, wherein said eukaryotic cell is selected from the group consisting of a yeast cell, an insect cell, a plant cell and an animal cell.
6. 6. The method according to claim 5, wherein said eukaryotic cell is a human cell.
7. 7. The method according to claim 1, wherein said membrane is an organelle membrane.
8. 8. The method according to claim 7, wherein said organelle membrane is selected from the group consisting of nuclear, mitochondria) and plastid membranes.
9. 9. The method according to claim 1, wherein said membrane is a liposome.
10. 10. The method according to claims 1-9, wherein Agrobacterium tumefaciens VirE2 is introduced into said membrane.
11. 11. The method of claims 1-10, wherein said molecule is a nucleic acid.
12. 12. The method of claim 11, wherein said nucleic acid is DNA.
13. 13. The method of claim 12, wherein said DNA is single-stranded.
14. 14. The method of claim 11, wherein said nucleic acid is an anti-sense oligonucleotide, a sense oligonucleotide, a ribozyme, a chimeric oligonucleotide or an expressible nucleic acid.
15. 15. The method of claim 11, wherein said nucleic acid comprises at least one T-DNA border sequence or functional part thereof and is complexed to Agrobacterium VirD2.
16. 16. A transformed cell produced using the method of claims 1-8 and 10-13.