BACKGROUND The delivery of polypeptides to specific tissues is desirable typically to ensure that a sufficiently high dose of a given agent is delivered to a selected tissue. Moreover, it is often the case that the polypeptide, although advantageously having beneficial therapeutic effects on the diseased tissue, may have undesirable side effects on tissues that are not diseased. For example, in the treatment of certain types of disorders, such as cancer, it is necessary to use a high enough dose of a drug to kill the cancer cells without killing an unacceptably high number of normal cells. Thus, one of the major challenges of disease treatment is to identify ways of exploiting cellular drug delivery vehicles to incorporate and to selectively release agents at a desired target site.

It has been suggested that red blood cells (RBCs) may be exploited as active agent/drug delivery vehicles (DeLoach & Sprandel 1985, Bibliotheca Haematologica ; Publ. Karger, Munich) as it is possible to incorporate agents into human RBCs using a variety of loading techniques. An example of such a loading technique is electroporation which involves short pulses of high electric fields, making red blood cell membranes transiently porous and allowing the agents of interest enter the cells. The electroporation process is advantageous as high loading indices can be achieved within a very short time period (Flynn et al., 1994, Cancer Letts., 82,225-229). Osmotic shock and modifications

thereof such as hypotonic shock and subsequent recovery of isotonicity and reverse hypotonic dialysis (Luque & Pinilla, 1993, Ind. Farmac. 8,53-59) may also be used.

Our International Patent Application PCT/GBOO/02848 discloses the use of an electric field for sensitising a red blood cell to ultrasound. Red blood cells are provided by taking a blood sample from a donor, isolated by centrifugation, and washed. The red blood cells are then loaded with an agent to be delivered, introduced into a recipient animal, and subsequently disrupted by administration of ultrasound. Lysis of the red blood cell results in delivery of agent to the bloodstream or tissue. Electrosensitised red blood cells may be selectively disrupted using diagnostic ultrasound, therapeutic ultrasound or a combination of diagnostic and therapeutic ultrasound.

Production of large quantities of recombinant therapeutic molecules has long been a goal of modern biotechnology. Bacteria have been exploited for the production of useful recombinant therapeutic molecules, as have yeast. However, the production of therapeutically useful polypeptides in higher eukaryotic systems is often preferred, since the post-translational processing (e. g., glycosylation) of the recombinant products more closely resembles the processing that occurs in human cells. Many efforts to date have focused on the generation of transgenic mammals that secrete the transgene products in their milk. There have been difficulties in obtaining high level expression of recombinant polypeptides in the milk of transgenic animals, and the approach also has the disadvantages that only female animals are useful, and they must generally reach sexual maturity and bear offspring before the transgene product may be obtained in quantity.

There is a need in the art for improved methods of production of therapeutically useful recombinant products.

SUMMARY We have now discovered that, instead of obtaining red blood cells from animals and loading these with an agent to be delivered, it is possible to make use of an animal which has been engineered to express a polypeptide and package it into its red blood cells.

Such transgenic animals provide a source of ready loaded red blood cells. Use of such red blood cells relieves or eliminates the need for mechanical loading with agent. We have surprisingly found that such red blood cells may be sensitised by various means in the same way as non-transgenic red blood cells, and that the sensitised red blood cells may be disrupted by the application of an external stimulus such as ultrasound.

According to a first aspect of the present invention, we provide a method of producing a red blood cell suitable for delivery of a polypeptide to a vertebrate, the method comprising the steps of : providing a transgenic animal carrying and expressing a transgene encoding the polypeptide; obtaining a red blood cell containing the polypeptide from the animal; and sensitising the red blood cell to render it susceptible to disruption by an energy source.

There is provided, according to a second aspect of the present invention, method of producing a red blood cell suitable for delivery of a polypeptide to a vertebrate, the method comprising the steps of : providing a red blood cell containing a polypeptide, the red blood cell being derived from a transgenic animal carrying and expressing a transgene encoding the polypeptide; and sensitising the red blood cell to render it susceptible to disruption by an energy source.

We provide, according to a third aspect of the present invention, a method for the delivery of a polypeptide to a vertebrate, the method comprising the steps of : providing a transgenic animal carrying and expressing a transgene encoding the polypeptide; obtaining a red blood cell containing the polypeptide from the animal; sensitising the red blood cell to render it susceptible to disruption by an energy source; introducing the sensitised red blood cell to a vertebrate; and exposing the vertebrate, or a part of it, to an energy source at a level sufficient to disrupt the sensitised red blood cell.

As a fourth aspect of the present invention, there is provided a method of producing a polypeptide, the method comprising the steps of : isolating a red blood cell from a transgenic animal carrying and expressing a transgene encoding the polypeptide;

sensitising the red blood cell to render it susceptible to disruption by an energy source; exposing the red blood cells to an energy source sufficient to disrupt the sensitized red blood cell; and isolating the polypeptide.

Preferably, the transgenic animal is selected from the group consisting of : mouse, rat, rabbit, sheep, goat, cow, and pig. In a highly preferred embodiment, the transgenic animal is a pig.

The polypeptide may be expressed under the control of any suitable promoter; preferably the polypeptide is expressed under the control of a (3-globin promoter or enhancer. Most preferably, the polypeptide is expressed under the control of a-globin Locus Control Region (LCR).

Preferably, sensitisation renders the red blood cell more susceptible to disruption than a red blood cell which is not sensitised. Any suitable sensitisation means may be used as known in the art. Preferably, sensitisation comprises the step of applying an electric field to a red blood cell (electrosensitisation). Preferably, the electric field is from about 0. 1kVolts/cm to about 10 kVolts/cm under in vitro conditions. More preferably, the electric field is applied for between lus and 100 milliseconds. The electric field may be applied in the form of a pulse.

Application of energy disrupts the sensitised red blood cells. Preferably, the energy used to disrupt the sensitized RBCs comprises ultrasound energy. The ultrasound may be selected from the group consisting of diagnostic ultrasound, therapeutic ultrasound and a combination of diagnostic and therapeutic ultrasound. Preferably, the ultrasound is applied at a power level of from about 0. 05W/cm2 to about 100W/cm2.

In a preferred embodiment, the transgenic animal is null for one or more blood group determinant antigens. The blood group determinant antigen may comprise an antigen involved in the ABO blood group system, or a Rhesus antigen, preferably a Rhesus D antigen. In a highly preferred embodiment, the methods described above

comprise a further step of treating the red blood cell to reduce reticuloendothelial system (RES) mediated clearance from an animal into which the red blood cell is introduced. The red blood cell may be treated with any suitable agent, preferably polyethylene glycol (PEG).

We provide, according to a fifth aspect of the present invention, a sensitised red blood cell produced by a method according to the first aspect of the invention, or the second aspect of the invention.

The present invention, in a sixth aspect, provides a red blood cell carrier suitable for delivery of a polypeptide to a vertebrate, in which the red blood cell contains a polypeptide, is derived from a transgenic animal carrying and expressing a transgene encoding the polypeptide, and is sensitised to render it more susceptible to disruption by exposure to a stimulus than a red blood cell which is not sensitised.

In a seventh aspect of the present invention, there is provided a polypeptide produced by a method according to the fourth aspect of the invention.

According to an eighth aspect of the present invention, we provide for the use of a transgenic animal as a source of red blood cells suitable for delivery of a polypeptide to a vertebrate, in which the red blood cells are sensitised such that they are more susceptible to disruption by exposure to a stimulus than red blood cells which are not sensitised.

We provide, according to a ninth aspect of the invention, use of a transgenic red blood cell in therapy.

There is provided, in accordance with a tenth aspect of the present invention, a red blood cell derived from a transgenic animal for use as a medicament.

As an eleventh aspect of the invention, we provide use of a red blood cell in the manufacture of a medicament for delivery of a therapeutic agent to a vertebrate, the red blood cell being derived from a transgenic animal.

In further aspects, we provide methods and compositions for assaying delivery of an agent to a location of an organism.

We provide, according to a twelfth aspect of the invention, a method of assaying delivery of an agent to a cell, the method comprising the steps of : (a) providing a cell comprising a first nucleotide sequence encoding a reporter, in which the cell expresses a first polypeptide capable of interacting directly or indirectly with the first nucleotide sequence to modulate expression of the reporter; (b) contacting the cell with an modulator molecule capable of modulating the interaction between the first polypeptide and the first nucleotide sequence; and (c) detecting the reporter.

We provide, according to a thirteenth aspect of the invention, a method of assaying delivery of an agent to a cell, the method comprising the steps of : (a) providing a cell comprising a first nucleotide sequence encoding a reporter ; (b) providing a first polypeptide capable of interacting directly or indirectly with the first nucleotide sequence to modulate expression of the reporter; (c) contacting the cell with the first polypeptide in the presence, preferably intracellularly, of a modulator molecule capable of modulating the interaction between the first polypeptide and the first nucleotide sequence; and (d) detecting the reporter.

Preferably, the first polypeptide interacts with the first nucleotide sequence to promote expression of the reporter. Alternatively or in addition, the first polypeptide interacts with the first nucleotide sequence to inhibit expression of the reporter. Preferably, the first polypeptide interacts with an operator sequence in the first nucleotide sequence.

In a preferred embodiment, the modulator molecule is capable of inhibiting the interaction between the first polypeptide and the first nucleotide sequence.

The modulator molecule may modulate the interaction between the first polypeptide and the first nucleotide sequence in a number of ways, for example by (a) modulating the transcription of an mRNA encoding the first polypeptide; (b) modulating the transport, processing, splicing, stability, turnover or degradation of a mRNA encoding the first polypeptide; (c) modulating the translation of an mRNA encoding the first polypeptide; (d) modulating the transport, processing, post-translational modification, stability, turnover or degradation of the first polypeptide; or (e) sterically hindering the interaction between the first polypeptide and the first nucleotide sequence.

Preferably, the modulator molecule comprises an antisense nucleic acid which binds to a nucleotide sequence encoding the first polypeptide and thereby inhibits its expression. Preferably, the antisense nucleic acid comprises an antisense RNA capable of binding to a messenger RNA encoding the first polypeptide.

The first nucleotide sequence may comprise a sequence capable of directing tissue specific expression of the reporter. Preferably, (a) the first polypeptide comprises a Tet repressor (TetR), and the first nucleotide sequence comprises a Tet-responsive element (TRE); (b) the first polypeptide comprises an oestrogen receptor, and the first nucleotide sequence comprises an oestrogen responsive element (ORE); or (c) the first polypeptide comprises an ecdysone receptor, and the first nucleotide sequence comprises an ecdysone responsive element (EcRE).

The first polypeptide may comprise a transcriptional activator domain, preferably selected from a VP16, a VP64, a maize C1, and a PI domain, or a transcriptional repressor domain, preferably selected from a KRAB-A domain, an engrailed domain or a snag domain. The first nucleotide sequence may encode a reporter selected from the group consisting of : a fluorescent protein, luciferase, p-galactosidase, or chloramphenicol acetyl transferase (CAT). Preferably, the reporter comprises a fluorescent protein selected from the group consisting of : a Green Fluorescent Protein, a Cyan Fluorescent Protein, a Yellow Fluorescent Protein, a Blue Fluorescent Protein and a Red Fluorescent Protein. In a

preferred embodiment, the reporter is detected by detecting fluorescent resonance energy transfer (FRET).

Preferably, the agent to be assayed is provided as an agent-MTS (membrane translocation sequence) conjugate, the agent to be assayed is provided as a virus or a virus- like particle comprising the agent. Preferably, the agent is loaded into a red blood cell for delivery to the cell. Preferably, the red blood cell is sensitised. Preferably, the agent is released from the red blood cell by application of ultrasound energy.

In a highly preferred embodiment, the cell is provided from or comprised in a transgenic animal carrying and expressing a transgene encoding the first polypeptide and a transgene comprising the first nucleotide sequence. The transgenic animal is preferably selected from the group consisting of : mouse, rat, rabbit, sheep, goat, cow, and pig.

Preferably, the cell forms part of an animal and the reporter is detected in situ in the animal. Alternatively or in addition, the cell may form part of a tissue mass grafted onto a host animal.

In a preferred embodiment, the reporter is expressed under the control of a tissue specific promoter or enhancer, preferably a Locus Control Region (LCR). Expression of the reporter may substantially be restricted to vascular endothelial cells.

In a further aspect of the invention, we provide a method of assaying delivery of an agent to a cell, the method comprising the steps of : (a) providing a cell comprising a first nucleotide sequence encoding a reporter; (b) providing a first polypeptide capable of interacting directly or indirectly with the first nucleotide sequence to modulate expression of the reporter; (c) providing a molecule capable of modulating the interaction between the first polypeptide and the first nucleotide sequence; (d) contacting the cell with the first polypeptide in the presence of the molecule; and detecting the reporter, in which detection of the reporter indicates that the first polypeptide is delivered to the cell; or (e) contacting the cell with the molecule in the presence of the first polypeptide, in which detection of the reporter indicates that the molecule is delivered to the cell.

Preferably, the assays described here are used for identifying a modulator molecule, a first polypeptide, or a combination of a modulator molecule and a first polypeptide, capable of intracellular entry.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a graph showing laser-mediated cell lysis () and transgene product galactocidase enzyme) (A) release following treatment of photosensitised transgenic murine erythrocytes with light. The X-axis shows treatment time, the right hand Y-axis shows percentage enzyme release (solid triangles) and the left hand Y-axis shows percentage lysis (solid squares).

Figure 2 is a graph showing ultrasound-mediated cell lysis (N) and transgene product (p-galactocidase enzyme) release (41) from electrosensitised transgenic murine erythrocytes. Cell lysis (A) and transgene product release () is also monitored following treatment of control transgenic erythrocyte populations. The X-axis shows power density, the right hand Y-axis shows percentage enzyme release and the left hand Y-axis shows percentage cell lysis. Solid squares represent lysis of electrosensitised cells, solid triangles show lysis of control cells, solid circles represent enzyme release from electrosensitised cells, and solid diamonds show enzyme release from control cells.

Figure 3 is a graph showing cell lysis (N) and transgene product (ß-galactocidase enzyme) release (A) following treatment of transgenic murine erythrocytes with ultrasound in a tissue mimicking system. The X-axis shows power density in W/cm2, the right hand Y-axis shows percentage enzyme release and the left hand Y-axis shows percentage cell lysis. Solid squares represent percentage lysis and solid triangles show percentage enzyme release.

Figure 4 is a graph showing ultrasound-mediated lysis of sensitised (A) and normal (N) porcine erythrocytes following treatment with ultrasound in a tissue mimicking system. The X-axis shows power density in W/cm2, while the left hand Y-axis

shows percentage cell lysis. Solid squares represent lysis of control cells, while solid triangles show lysis of electrosensitised cells.

Figure 5 is a graph showing ß-galactocidase enzyme release and cell lysis of transgenic murine erythrocytes over a range of electrosensitisation voltages. The X-axis shows voltage, the left hand Y-axis shows the percentage of total enzyme released, and the right hand Y-axis shows the percentage of cells lysed. Solid squares (N) show percentage release by ultrasound, solid triangles (A) show percentage enzyme release by electrosensitisation (inverted triangles show percentage cell lysis).

DETAILED DESCRIPTION The methods and compositions described here are based upon the demonstration that red blood cells (RBCs) containing a polypeptide may be generated in animals transgenic for a gene encoding that polypeptide. Use of such transgenic animals enables the isolation of red blood cells which are comprise a polypeptide of interest. Such red blood cells are"ready loaded", without the need for a separate loading step to introduce the polypeptide into the red blood cell.

Our methods therefore provide for the delivery of a biological effector molecule to a target site in vivo.

RBCs containing the polypeptide by virtue of its expression from a transgene may be sensitised, for example, by exposure to an electric field or by the incorporation of a compound such as a porphyrin, administered to a mammal and subjected to energy sufficient to break them using, for example, ultrasound or laser, thereby delivering the polypeptide within the mammal. The methods described here allow for the targeted delivery of an agent to a tissue of interest in a vertebrate using sensitized RBCs which have no particular affinity for the target tissue. This is of particular importance where the target tissue is of a type which is widely distributed throughout the body (for example,

skeletal muscle). We also provide methods of producing a recombinant polypeptide by isolating it from RBCs harvested from animals transgenic for the polypeptide.

The polypeptide agents to be delivered may be fused, conjugated, coupled to, or otherwise joined to one or more polypeptide sequences capable of membrane translocation activity. This facilitates delivery of agent into the intracellular environment, as described in further detail below. Furthermore, the polypeptide agents may comprise pro-drugs, as described in further detail below. The polypeptides may comprise activators of pro-drugs, such as pro-drug converting enzymes. Second agents may be loaded into the transgenic red blood cells for co-delivery to a target cell or cells. Such second agents may themselves comprise membrane translocation sequences for intracellular localisation. The second agents may comprise pro-drugs, which are converted by the transgenic polypeptides into active agents.

Furthermore, the loaded transgenic red blood cells may be mixed with other cells such as red blood cells, which may optionally themselves be loaded any number of agents as described here, prior to delivery into the patient.

Sensitisation and release by, for example, ultrasound, as described in further detail below and also in our International Patent Application Nos. PCT/GBOO/02848 and PCT/GBOO/03056, allows targeted local release of the expressed polypeptide and optionally second and further agents for effective localised action.

Furthermore, the methods and compositions described here encompass the use of translocation means, which enable the passage of a polypeptide or other agent across a cellular membrane (such as a plasma membrane, nuclear membrane, an organelle membrane, a chloroplast membrane, mitochondrial inner and/or outer membrane, Golgi membrane, etc). Suitably, a transgenic animal may be generated which expresses a transgene encoding a membrane translocation sequence (MTS), as described below.

We also describe assays for delivery of an agent to a target cell. The assays described here are useful for determining the presence, extent or quantity of agent delivered to any target site. Using the methods and compositions described here, delivery to any site, for example, a cell, a tissue, an organ, a system, an intracellular location such as an organelle (for example, the nucleus, mitochondria, vacuole, chloroplast, etc) may be assayed. Successful delivery or targeting of agent can readily be determined by use of our assays.

These and other aspects of the methods and compositions disclosed here are described in detail below. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning : A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; C7urrent Protocols in Molecular Biology, ch. 9,13, and 16, John Wiley & Sons, New York, N. Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing : Essential Techniques, John Wiley & Sons J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization : Principles and Practice ; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis : A Practical Approach, Irl Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology : DNA Structure Part A : Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference.

POLYPEPTIDES According to one aspect, polypeptides which are the product of a transgene carried and expressed by the animal from which the RBCs are isolated may be delivered to a vertebrate. Such polypeptides may include therapeutic polypeptides and biological effector molecules or agents, etc.

Agents useful according to the methods and compositions described here include, but are not limited to a protein, polypeptide or peptide, or RNA, including, but not limited to, a structural protein, an enzyme, a receptor, a ligand, a regulatory factor, a cytokine (such as an interferon and/or an interleukin) an antibiotic, a polyclonal or monoclonal antibody, or an effective part thereof, such as an Fv fragment, which antibody or part thereof may be natural, synthetic or humanized, a peptide hormone, and a signaling molecule. Included within the term"immunoglobulin"are intact immunoglobulins as well as antibody fragments such as Fv, a single chain Fv (scFv), a Fab or a F (ab') 2.

Preferred immunoglobulins, antibodies, Fv fragments, etc are those which are capable of binding to antigens in an intracellular environment, known as"intrabodies"or "intracellular antibodies". An"intracellular antibody"or an"intrabody"is an antibody which is capable of binding to its target or cognate antigen within the environment of a cell, or in an environment which mimics an environment within the cell.

Selection methods for directly identifying such"intrabodies"have been proposed, such as an in vivo two-hybrid system for selecting antibodies with binding capability inside mammalian cells. Such methods are described in International Patent Application number PCT/GB00/00876, hereby incorporated by reference. Techniques for producing intracellular antibodies, such as anti-ß-galactosidase scFvs, have also been described in Martineau, et al., 1998, JMol Biol 280, 117-127 and Visintin, et al., 1999, Proc. Natl.

Acad. Sci. USA 96,11723-11728.

A therapeutic polypeptide is intended to include a lipoprotein, a glycoprotein, a phosphoprotein, or an apolipoprotein. Therapeutic polypeptides also include nuclear proteins, cytoplasmic proteins, mitochondrial proteins, secreted proteins, plasmalemma- associated proteins, viral antigens, bacterial antigens, protozoal antigens, parasitic antigens, clotting factors, neuropeptides and tumor antigens. Therapeutic polypeptides also include any polypeptide encoded by a therapeutic nucleic acid as described here.

A therapeutic agent may also be a nucleic acid, including, but not limited to an RNA molecule, which includes, but is not limited to an RNA aptamer (an RNA molecule capable of binding to another molecular species, see for example U. S. Pat. No. 5,792,613; 5,688,670), an antisense RNA or a ribozyme. Ribozymes of the hammerhead class are the smallest known, and lend themselves both to in vitro synthesis and delivery to cells (summarized by Sullivan, 1994, J Invest. Dermatol., 103: 85S-98S ; Usman et al., 1996, Curr. Opin. Struct. Biol., 6: 527-533). Therapeutic nucleic acid sequences also include sequences encoding proteins, lipoproteins, glycoproteins, phosphoproteins and nucleic acids (e. g., RNAs such as ribozymes or antisense nucleic acids).

The therapeutic compounds which can be expressed in a RBC of a transgenic animal are only limited by the availability of the nucleic acid sequence encoding a given protein or polypeptide. Nucleic acid sequences encoding a vast number of polypeptides are available in databases such as GenBank or the EMBL database. These databases are constantly updated as new sequences become available. One skilled in the art will readily recognize that as more proteins and polypeptides become identified, their corresponding genes can be cloned and used to generate transgenic animals expressing the protein, ribozyme or polypeptide, etc in RBCs.

Particularly useful classes of therapeutic agents include, but are not limited to, antibiotics, anti-inflammatory drugs, angiogenic or vasoactive agents, growth factors and cytotoxic agents (e. g., anti-tumor agents or tumor growth-suppressing agents). Cytotoxic agents of use in here include, but are not limited to diphtheria toxin, Pseudomonas exotoxin, cholera toxin, and pertussis toxin. Cytotoxic agents useful in the methods and compositions described here also include the group of polypeptide effector molecules (i. e., enzymes) known to activate a pro-drug.

A pro-drug is essentially non-cytotoxic until activated by an enzymatic activity.

For example, Herpes Simplex Virus thymidine kinase (HSV-TK, encoded by Genbank Accession No. J02224)) activates the cytotoxic effect of gancyclovir. Cytosine deaminase (GenBank Accession No. S56903) activates the cytotoxic effect of 5-fluorocytosine, and nitroreductase (U. S. Patent No. 5,780,585 andGenbankAccessionNo. AR018125)

activates the cytotoxic effect of the pro-drug CB 1954, as does DT Diaphorase (GenBank Accession no. AH005427). Other pro-drug activators include, but are not limited to carboxypeptidase A (encoded by Genbank Accession No. M27717), I-galactosidase (encoded by Genbank Accession No. M13571), J-glucuronidase (encoded by Genbank Accession No. M15182), alkaline phosphatase (encoded by Genbank Accession No.

J03252 J03512), or cytochrome P-450 (encoded by Genbank Accession No. D00003 N00003), plasmin, carboxypeptidase G2, glucose oxidase, xanthine oxidase, J-glucosidase, K-gutamyl transferase, J-lactamase, and penicillin amidase.

Therapeutic agents also include those agents useful for imaging of tissues in vivo or ex vivo. For example, imaging agents, such as antibodies or antigen-binding portions thereof (e. g., Fv fragments or single chain antibodies) which are specific for defined molecules, tissues or cells in an organism, may be used to image specific parts of the body by releasing them at a desired location using an energy source such as ultrasound. This allows imaging agents which are not completely specific for the desired target, and which might otherwise lead to more general imaging throughout the organism, to be used to image defined tissues or structures. For example, an antibody which is capable of imaging endothelial tissue may be used to image liver vasculature by releasing the antibody selectively in the liver by applying an energy source such as ultrasound thereto.

EXPRESSION OF TRANSGENE In order to be useful in the methods and compositions described here, it is necessary that the polypeptide be expressed in RBCs. Preferably, the red blood cell is a mammalian red blood cell. In order to obtain high level expression of a transgene product in RBCs, the transgene is preferably driven by or operably linked to a promoter that is specific for the erythroid cell lineage, most preferably, in reticulocytes.

Reticulocytes are immature RBCs which have extruded their nucleus, but retain a large amount of RNA, and thus display a grainy basophilic staining pattern in hematoxylin and eosin stained preparations. Circulating reticulocytes, which make up approximately

1% of circulating blood cells are transient blood cells; after leaving the bone marrow, reticulocytes retain their RNA and thus their protein synthetic ability for approximately 24 hours, before full maturation into essentially mRNA-free erythrocytes. During its life cycle in circulating blood, reticulocytes, by virtue of their RNA content, continue to produce haemoglobin and thus continue to translate mRNAs, endogenous or recombinant, derived from genes which possess erythrocyte-specific promoters. Therefore, the polypeptides described above, driven by the erythrocyte promoters described below, will be expressed in virtually all circulating RBCs by virtue of transgene synthesis in reticulocytes prior to their maturation to mature RBCs.

Any promoter known to be active in cells of the erythrocytic lineage may be used to direct the expression of a polypeptide in the methods described here. However, examples of promoters that direct high level expression of erythroid-specific genes include the globin gene promoters. Haemoglobin is expressed in a tissue-specific manner in RBCs, where it accounts for about 95% of total cellular protein. Globin gene promoters include those for the I, J ( (3 globin), L, M and N globin genes. Particularly preferred among these is the human ß globin promoter, which is most active in adults. g, YG,'', S AND D GLOBIN GENES Human globin (also known as J globin) genes are found in a cluster on chromosome 11, comprising about 50 kb of DNA that also includes one embryonic gene encoding s globin (also known as M globin), two fetal genes encoding K globins YG, YA (also known as G and A globins), and two adult genes encoding 5 and P globin (also known as L and J globin), in that order (Fritsch et al., 1980, Cell 19 : 959-972). It has been found that DNA sequences both upstream and downstream of the P globin translation initiation site are involved in the regulation of p globin gene expression (Wright et al., 1984, Cell 38: 263). In particular, a series of four DNAse I super hypersensitive sites (now referred to as the locus control region, or LCR) located about 50 kilobases upstream of the human ß globin gene are extremely important in eliciting properly regulated (3 globin- locus expression (Tuan et al., 1985, Proc. Natl. Acad. Sci. U. S. A. 83: 1359-1363 ; PCT

Patent Application WO 8901517 by Grosveld; Behringer et al., 1989, Science 245: 971- 973; Enver et al., 1989, Proc. Natl. Acad. Sci. U. SA. 86 : 7033-7037; Hanscombe et al., 1989, Genes Dev. 3: 1572-1581 ; Van Assendelft et al., 1989, Cell 56: 967-977; Grosveld et al., 1987, Cell 51 : 975-985). Thus, in a highly preferred embodiment, the transgene is operably linked to or its expression is regulated from a globin LCR.

Expression systems, including expression vectors, useful for erythroid expression are described in detail in US Patent No. 5,538,885 and GB 2251622. The vectors described in this document comprise a promoter, a DNA sequence which codes for a desired polypeptide and preferably a dominant control region. Preferably, the dominant control region comprises a micro locus which comprises a 6.5 kb fragment obtained by ligating the fragments: 2.1 kb Xbal-XbaI ; 1.9 kb Hindlll-Hindlll ; 1.5 kb KpnI-BgIII ; and 1.1 kb partial SacI ; from the p-globin gene. As used herein the term"dominant control region" (or"DCR") means a sequence of DNA capable of conferring upon a linked gene expression system the property of host cell-type restricted, integration site independent, copy number dependent expression when integrated into the genome of a host compatible with the dominant control region. The dominant control region retains this property when fully reconstituted within the chromosome of the host cell; and the ability to direct efficient host cell-type restricted expression is retained even when fully reformed in a heterologous background such as a different part of the homologous chromosome or even a different chromosome.

A method for making a desired peptide in transgenic animals is described in US Patent No 5,627,268. A transgenic animal is engineered to comprise an artificial gene, which is controlled by globin locus control region (LCR) and which encodes a fusion protein. In the fusion protein, the desired peptide is linked via a cleavable peptide bond to a globin polypeptide. The erythrocytes of the transgenic animal express the fusion protein which is incorporated into hemoglobin produced by the host cell. The desired peptide can be obtained from a hemolysate of the red cells of the transgenic animals by cleavage of the linking bond and separation of the peptide away from globin portions. Production of recombinant haemoglobin is described in US Patent No 5,821,351.

Other promoters useful in the method as described here include the promoter of the Erythroid-specific GATA-1 transcription factor gene or a heterologous construct comprising the enhancer from the GATA-1 transcription factor gene (Grande et al, 1999, Blood 93: 3276). Other alternatives include but are not limited to the NF-E2 proximal 1B promoter (Moroni et al. 2000, JBC 275: 10567) and the B 19 p6 promoter with or without an erythrocyte-specific enhancer element (Kurpad et al, 1999, J. Hematother. Stem. Cell.

Res. 8: 585). The skilled person will appreciate that any suitable promoter may be used, so long as it directs expression of the desired polypeptide at an appropriate level in the red blood cell.

TRANSGENIC ANIMALS A transgenic animal is an animal containing at least one foreign gene, called a transgene, in its genetic material. Preferably, the transgenic animal is a non-human animal, preferably a mammal. Preferably, the transgene is contained in the animal's germ line such that it can be transmitted to the animal's offspring. Transgenic animals are useful for producing RBCs comprising polypeptides, in particular therapeutic polypeptides.

We therefore provide a transgenic animal, carrying and expressing a transgene encoding a polypeptide, in which a red blood cell derived from the transgenic animal comprises the polypeptide. preferably, the polypeptide is located in or on the red blood cell, preferably within the red blood cell membrane. Thus, preferably, the red blood cell is loaded with the polypeptide by virtue of the transgenic animal carrying and expressing the transgene. The transgenic animal is preferably a mammal, more preferably a non-human animal or mammal, most preferably a mouse, rat, rabbit, sheep, goat, cow, or a pig.

Preferably, the polypeptide does not comprise or consist of (3-galactocidase, or any portion or fragment of-galactocidase. More preferably, the polypeptide comprises a therapeutic polypeptide, i. e., a polypeptide having a desired therapeutic activity. Most preferably, the polypeptide comprises a polypeptide selected from the polypeptides disclosed in the section above.

Preferably, the polypeptide is expressed under the control of a p-globin promoter or enhancer. More preferably, the polypeptide is expressed under the control of a-globin Locus Control Region (LCR). Preferably, a red blood cell derived from the transgenic animal is capable of being sensitised so that it is rendered method according to any preceding claim, in which sensitisation renders the red blood cell more susceptible to disruption than a red blood cell which is not sensitised. Preferably, the red blood cell is suitable for electrosensitisation by applying an electric field to a red blood cell. electrosensitisation is described in a separate section in this document, and it should be understood that the embodiments described there will be equally applicable here.

According to the methods and compositions described here, a transgenic animal is constructed which expresses a suitable transgene, preferably a transgene encoding a therapeutic polypeptide, in its red blood cells. The red blood cells may be isolated from the animal, and sensitised according to the methods described here. The sensitised red blood cells are then introduced into a human or animal, and disrupted locally or generally by application of a stimulus, for example, ultrasound. The disrupted red blood cells release their payload, i. e., the polyptide of interest in the organism.

CONSTRUCTION OF TRANSGENIC ANIMALS Transgenic animals for use in the methods and compositions described here may be constructed by various means known in the art.

A number of techniques may be used to introduce the transgene into an animal's genetic material, including, but not limited to, microinjection of the transgene into pronuclei of fertilized eggs and manipulation of embryonic stem cells (U. S. Pat. No.

4,873,191 by Wagner and Hoppe; Palmiter and Brinster, 1986, Ann. Rev. Genet. 20: 465- 499; French Patent Application 2593827 published Aug. 7,1987). Transgenic animals may carry the transgene in all their cells or may be genetically mosaic.

According to the method of conventional transgenesis, additional copies of normal or modified genes are injected into the male pronucleus of the zygote and become integrated into the genomic DNA of the recipient mouse. The transgene is transmitted in a Mendelian manner in established transgenic strains.

Constructs useful for creating transgenic animals comprise genes encoding therapeutic molecules, preferably under the control of nucleic acid sequences directing their expression in cells of the erythroid lineage. Alternatively, therapeutic molecule encoding constructs may be under the control of their native promoters, or inducibly regulated. A transgenic animal expressing one transgene can be crossed to a second transgenic animal expressing second transgene such that their offspring will carry both transgenes.

Although the majority of studies have involved transgenic mice, other species of transgenic animal have also been produced, such as rabbits, sheep, pigs (Hammer et al., 1985, Nature 315: 680-683-; Kurnar, et al., U. S. 05922854; Seebach, et al., U. S. 06030833) and chickens (Salter et al., 1987, Virology 157: 236-240). While the transgenic animals described here are not limited to swine, the description which follows details the methodology for transgene expression in larger animals, such as swine, but may be adapted for smaller animals as need requires. Transgenic animals are currently being developed to serve as bioreactors for the production of useful pharmaceutical compounds (Van Brunt, 1988, BiolTechnology 6: 1149-1154; Wilmut et al., 1988, New Scientist (July 7 issue) pp. 56-59).

Methods of expressing recombinant protein via transgenic livestock have an important theoretical advantage over protein production in recombinant bacteria and yeast; namely, the ability to produce large, complex proteins in which post-translational modifications, including glycosylation, phosphorylation, subunit assembly, etc. are critical for the activity of the molecule.

In particular, we provide recombinant swine RBCs expressing polypeptides. RBCs containing the polypeptide may be prepared by introducing a recombinant nucleic acid molecule which encodes said polypeptide into a tissue, such as bone marrow cells, using known transformation techniques. These transformation techniques include transfection and infection by retroviruses carrying either a marker gene or a drug resistance gene. See for example, Current Protocols in Molecular Biology, Ausubel et al. eds., John Wiley and Sons, New York (1987) and Friedmann (1989) Science 244: 1275-1281. A tissue containing a recombinant nucleic acid molecule may then be reintroduced into an animal using reconstitution techniques (See for example, Dick et al. (1985) Cell 42: 71).

The recombinant constructs described here may be used to produce a transgenic animal by any method known in the art, including, but not limited to, microinjection, embryonic stem (ES) cell manipulation, electroporation, cell gun, transfection, transduction, retroviral infection, etc. Transgenic animals can be produced by introducing transgenes into the germline of the animal, particularly into the genome of bone marrow cells, e. g. hematopoietic cells. Embryonal target cells at various developmental stages can be used to introduce the human transgene construct. As is generally understood in the art, different methods are used to introduce the transgene depending on the stage of development of the embryonal target cell.

One technique for transgenically altering an animal is to microinject a recombinant nucleic acid molecule into the male pronucleus of a fertilized egg so as to cause 1 or more copies of the recombinant nucleic acid molecule to be retained in the cells of the developing animal. The recombinant nucleic acid molecule of interest is isolated in a linear form with most of the sequences used for replication in bacteria removed.

Linearization and removal of excess vector sequences results in a greater efficiency in production of transgenic mammals. See for example, Brinster et al. (1985) PNAS 82: 4438-4442.

In general, the zygote is the best target for micro-injection. In the swine, the male pronucleus reaches a size which allows reproducible injection of DNA solutions by standard microinjection techniques. Moreover, the use of zygotes as a target for gene

transfer has a major advantage in that, in most cases, the injected DNA will be incorporated into the host genome before the first cleavage. Usually up to 40 percent of the animals developing from the injected eggs contain at least 1 copy of the recombinant nucleic acid molecule in their tissues. These transgenic animals will generally transmit the gene through the germ line to the next generation. The progeny of the transgenically manipulated embryos may be tested for the presence of the construct by Southern blot analysis of a segment of tissue. Typically, a small part of the tail is used for this purpose.

The stable integration of the recombinant nucleic acid molecule into the genome of transgenic embryos allows permanent transgenic mammal lines carrying the recombinant nucleic acid molecule to be established. Alternative methods for producing a mammal containing a recombinant nucleic acid molecule include infection of fertilized eggs, embryo-derived stem cells, to potent embryonal carcinoma (EC) cells, or early cleavage embryos with viral expression vectors containing the recombinant nucleic acid molecule.

(See for example, Palmiter et al. (1986) Ann. Rev. Genet. 20: 465-499 and Capecchi (1989) Science 244 : 1288-1292.) Retroviral infection can also be used to introduce transgene into an animal, including swine. The developing embryo can be cultured in vitro to the blastocyst stage.

During this time, the blastomeres can be targets for retroviral infection (Jaenich (1976) PNAS 73: 1260-1264). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan et al. (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al. (1985) PNAS 82 : 6927-6931; Van der Putten et al.

(1985) PNAS 82: 6148-6152).

Transfection can be obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra ; Stewart et al. (1987) EMBO J 6: 383-388).

Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al. (1982) Nature 298: 623.628). Most of the founders will be mosaic for the transgene since incorporation typically occurs only in a

subset of the cells which formed the transgenic swine. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line, albeit with low efficiency, by intrauterine retroviral infection of the mid-gestation embryo (Jahner et al. (1982) supra).

A third approach, which may be useful in the construction of transgenic animals, would target transgene introduction into an embryonal stem cell (ES). ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292: 154-156; Bradley et al. (1984) Nature 309: 255-258; Gossler et al.

(1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature 322: 445-448). Transgenes might be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells could thereafter be combined with blastocysts from the same species. The ES cells could be used thereafter to colonize the embryo and contribute to the germ line of the resulting chimeric animal. For review, see Jaenisch (1988) Science 240 : 1468-1474.

Introduction of the recombinant gene at the fertilized oocyte stage ensures that the gene sequence will be present in all of the germ cells and somatic cells of the transgenic "founder"animal. As used herein, founder (abbreviated"F") means the animal into which the recombinant gene is introduced at the one cell embryo stage. The presence of the recombinant gene sequence in the germ cells of the transgenic founder animal in turn means that approximately half of the founder animal's descendants will carry the activated recombinant gene sequence in all of their germ cells and somatic cells. Introduction of the recombinant gene sequence at a later embryonic stage might result in the gene's absence from some somatic cells of the founder animal, but the descendants of such an animal that inherit the gene will carry the activated recombinant gene in all of their germ cells and somatic cells.

Microinjection of swine oocytes In preferred embodiments the transgenic animals, including but not limited to swine are produced by: i) microinjecting a recombinant nucleic acid molecule encoding a polypeptide into a fertilized egg to produce a genetically altered egg; ii) implanting the genetically altered egg into a host female animal of the same species; iii) maintaining the host female for a time period equal to a substantial portion of the gestation period of said animal fetus. iv) harvesting a transgenic animal having at least one cell that has developed from the genetically altered mammalian egg, which expresses a gene which encodes a polypeptide In general, the use of microinjection protocols in transgenic animal production is typically divided into four main phases: (a) preparation of the animals; (b) recovery and maintenance in vitro of one or two-celled embryos; (c) microinjection of the embryos and (d) reimplantation of embryos into recipient females. The methods used for producing transgenic livestock, particularly swine, do not differ in principle from those used to produce transgenic mice. Compare, for example, Gordon et al. (1983) Methods in Enzymology 101: 411, and Gordon et al. (1980) PNAS 77 : 7380 concerning, generally, transgenic mice with Hammer et al. (1985) Nature 315: 680, Hammer et al. (1986) JAnim Sci 63: 269-278, Wall et al. (1985) Biol Reprod. 32: 645-651, Pursel et al. (1989) Science 244: 1281-1288, Vize et al. (1988) JCell Science 90: 295-300, Muller et al. (1992) Gene 121 : 263-270, and Velander et al (1992) PNAS 89: 12003-12007, each of which teach techniques for generating transgenic swine. See also, PCT Publication WO 90/03432, and PCT Publication WO 92/22646 and references cited therein One step of the preparatory phase comprises synchronizing the estrus cycle of at least the donor females, and inducing superovulation in the donor females prior to mating.

Superovulation typically involves administering drugs at an appropriate stage of the estrus cycle to stimulate follicular development, followed by treatment with drugs to synchronize estrus and initiate ovulation. As described in the example below, a pregnant female animal's serum is typically used to mimic the follicle-stimulating hormone (FSH) in combination with human chorionic gonadotropin (hCG) to mimic luteinizing hormone

(LH). The efficient induction of superovulation depends, as is well known, on several variables including the age and weight of the females, and the dose and timing of the gonadotropin administration. See for example, Wall et al. (1985) Biol. Reprod. 32: 645 describing superovulation of pigs. Superovulation increases the likelihood that a large number of healthy embryos will be available after mating, and further allows the practitioner to control the timing of experiments After mating, one or two-cell fertilized eggs from the superovulated females are harvested for microinjection. A variety of protocols useful in collecting eggs from animals are known. For example, in one approach, oviducts of fertilized superovulated females can be surgically removed and isolated in a buffer solution/culture medium, and fertilized eggs expressed from the isolated oviductal tissues. See, Gordon et al. (1980) PNAS 77 : 7380 ; and Gordon et al. (1983) Methods inEnzymology 101 : 411. Alternatively, the oviducts can be cannulated and the fertilized eggs can be surgically collected from anesthetized animals by flushing with buffer solution/culture medium, thereby eliminating the need to sacrifice the animal. See Hammer et al. (1985) Nature 315: 600. The timing of the embryo harvest after mating of the superovulated females can depend on the length of the fertilization process and the time required for adequate enlargement of the pronuclei. This temporal waiting period can range from, for example, up to 48 hours for larger animal species.

Fertilized eggs appropriate for microinjection, such as one-cell ova containing pronuclei, or two-cell embryos, can be readily identified under a dissecting microscope The equipment and reagents needed for microinjection of the isolated embryos from larger animals are similar to that used for the mouse. See, for example, Gordon et al.

(1983) Methods in Enzymology 101 : 411 ; and Gordon et al. (1980) PNAS 77: 7380, describing equipment and reagents for microinjecting embryos. Briefly, fertilized eggs are positioned with an egg holder (fabricated from 1 mm glass tubing), which is attached to a micro-manipulator, which is in turn coordinated with a dissecting microscope optionally fitted with differential interference contrast optics. Where visualization of pronuclei is difficult because of optically dense cytoplasmic material, such as is generally the case with swine embryos, centrifugation of the embryos can be carried out without compromising

embryo viability. Wall et al. (1985) Biol. Reprod. 32: 645. Centrifugation will usually be necessary in this method.

A recombinant nucleic acid molecule is provided, typically in linearized form, by linearizing the recombinant nucleic acid molecule with at least 1 restriction endonuclease, with an end goal being removal of any prokaryotic sequences as well as any unnecessary flanking sequences. In addition, the recombinant nucleic acid molecule containing the tissue specific promoter and the human class I gene may be isolated from the vector sequences using 1 or more restriction endonucleases. Techniques for manipulating and linearizing recombinant nucleic acid molecules are well known and include the techniques described in Molecular Cloning : A Laboratory Manual, Second Edition. Maniatis et al. eds., Cold Spring Harbor, N. Y. (1989). The linearized recombinant nucleic acid molecule may be microinjected into an egg to produce a genetically altered mammalian egg using well known techniques.

Typically, the linearized nucleic acid molecule is microinjected directly into the pronuclei of the fertilized eggs as has been described by Gordon et al. (1980) PNAS 77: 7380-7384. This leads to the stable chromosomal integration of the recombinant nucleic acid molecule in a significant population of the surviving embryos. See for example, Brinster et al. (1985) PNAS 82: 4438-4442 and Hammer et al. (1985) Nature 315: 600-603. The microneedles used for injection, like the egg holder, can also be pulled from glass tubing. The tip of a microneedle is allowed to fill with plasmid suspension by capillary action. By microscopic visualization, the microneedle is then inserted into the pronucleus of a cell held by the egg holder, and plasmid suspension injected into the pronucleus. If injection is successful, the pronucleus will generally swell noticeably. The microneedle is then withdrawn, and cells which survive the microinjection (e. g. those which do not lyse) are subsequently used for implantation in a host female The genetically altered mammalian embryo is then transferred to the oviduct or uterine horns of the recipient. Microinjected embryos are collected in the implantation pipette, the pipette inserted into the surgically exposed oviduct of a recipient female, and the microinjected eggs expelled into the oviduct. After withdrawal of the implantation

pipette, any surgical incision can be closed, and the embryos allowed to continue gestation in the foster mother. See, for example, Gordon et al. (1983) Methods in Enzymology 101: 411 ; Gordon et al. (1980) PNAS 77 : 7390; Hammer et al. (1985) Nature 315: 600; and Wall et al. (1985) Biol. Reprod. 32: 645 The host female mammals containing the implanted genetically altered mammalian eggs are maintained for a sufficient time period to give birth to a transgenic mammal having at least 1 cell, e. g. a bone marrow cell, e. g. a hematopoietic cell, which expresses the recombinant nucleic acid molecule that has developed from the genetically altered mammalian egg At two-four weeks of age (post-natal), tissue samples are taken from the transgenic offspring and digested with Proteinase K. DNA from the samples is phenol-chloroform extracted, then digested with various restriction enzymes. The DNA digests are electrophoresed on a Tris-borate gel, blotted on nitrocellulose, and hybridized with a probe consisting of the at least a portion of the coding region of the recombinant cDNA of interest which had been labeled by extension of random hexamers. Under conditions of high stringency, this probe should not hybridize with the endogenous (non-transgene) genes, but should produce a hybridization signal in animals expressing the transgene, allowing for the identification of transgenic pigs According to a preferred specific embodiment, a transgenic pig may be produced by the methods as set forth in Example 1.

Production of Transgenic Animals by Cloning Transgenic animals for use in the methods and compositions described here may also be made by other methods, for example, by cloning. Cloning by nuclear transfer to enucleated cells is described in US Patent No. 6,147,276, and in numerous publications, including Campbell et al., 1996, Nature 380 64-66 ; Wilmut et al, 1997, Nature 385 810- 813; Schneike et al., 1997, Science 278 2130-2133 ; Ashworth et al., 1998, Nature 394

329; Sheils et al., 1999, Nature 399 316-317; and Evans et al., 1999, Nature Genetics 23 90-93.

For example, in order to clone an animal, the following technique may be used.

Unfertilised eggs are flushed out of a female animal, which may be induced to produce a larger than normal number of eggs. A sample of tissue is taken from a suitable part of a donor animal (for example, adult tissue such as udder tissue or embryonic tissue) and cultured in vitro. Cultured cells are then starved to send them into a resting or quiescent state by, for example, serum starvation) The donor cell is then fused or injected into the recipient cell. For example, a cell from the culture is placed beside the egg and an electric current used to fuse the couplet. The reconstructed embryo is put into culture and allowed to grow for a length of time (for example, seven days). The recipient cell is activated before, during or after nuclear transfer. Embryos which grow successfully are taken and transferred to a recipient animal which is at the same stage of the oestrus cycle as the egg.

The recipient animal becomes pregnant and produces a cloned animal after a suitable gestation period.

Direct microinjection of donor cell nuclei may also be used (the so-called "Honolulu technique"). Direct microinjection of a nucleus from an adult cell into an oocyte from which the nucleus has already been removed has been used to clone mice.

The eggs are then prevented from dividing and forming multicelled blastocysts for periods of time (for example, from one to six hours) and subsequently allowed to divide.

Cloning using nuclear transfer from established cell lines is described in Nature 380, 64-66, and also in International Patent Application Numbers PCT/GB96/02099, and PCT/GB96/02098. Transgenic lambs producing recombinant blood clotting factor IX have also been produced. Delayed activation of donor cells is described in UK Patent Numbers GB 2318792 and GB 2340493.

Knock-out Technology In addition to the addition of exogenous genes to RBCs, a further embodiment includes the potential for deletion of genes from RBCs, wherein the deletion provides a therapeutic advantage. For example, it may be advantageous to delete one or more cell surface blood group antigens or epitopes using gene knock out techniques in order to avoid or lessen a host immune response to administered RBCs. i. Standard Knock Out Animals Knock out animals are produced by the method of creating gene deletions with homologous recombination. This technique is based on the development of embryonic stem (ES) cells that are derived from embryos, are maintained in culture and have the capacity to participate in the development of every tissue in the animals when introduced into a host blastocyst. A knock out animal is produced by directing homologous recombination to a specific target gene in the ES cells, thereby producing a null allele of the gene, GCK/IRS1, IRS1/INSR, MC4R (Huszar et al., 1997, Cell, 88: 131) and BRS3 (Ohki-Hamazaki et al., 1997, Nature, 390: 165) ii. Tissue Specific Knock Out The method of targeted homologous recombination has been improved by the development of a system for site-specific recombination based on the bacteriophage PI site specific recombinase Cre. The Cre-loxP site-specific DNA recombinase from bacteriophage P1 is used in transgenic mouse assays in order to create gene knockouts restricted to defined tissues or developmental stages. Regionally restricted genetic deletion, as opposed to global gene knockout, has the advantage that a phenotype can be attributed to a particular cell/tissue (Marth, 1996, Clin. Invest. 97: 1999). In the Cre-loxP system one transgenic mouse strain is engineered such that loxP sites flank one or more exons of the gene of interest. Homozygotes for this so called'floxed gene'are crossed with a second transgenic mouse that expresses the Cre gene under control of a cell/tissue type transcriptional promoter. Cre protein then excises DNA between loxP recognition

sequences and effectively removes target gene function (Sauer, 1998, Methods, 14: 381).

There are now many in vivo examples of this method, including the inducible inactivation of mammary tissue specific genes (Wagner et al., 1997, Nucleic Acids Res., 25: 4323) iii. Bac Rescue of Knock Out Phenotype In order to verify that a particular genetic polymorphism/mutation is responsible for altered protein function in vivo one can"rescue"the altered protein function by introducing a wild-type copy of the gene in question. In vivo complementation with bacterial artificial chromosome (BAC) clones expressed in transgenic mice can be used for these purposes. This method has been used for the identification of the mouse circadian Clock gene (Antoch et al., 1997, Cell 89 : 655) SENSITISATION To facilitate release of loaded agents, the present method makes use of sensitised red blood cells. Red blood cells which are sensitised are more susceptible to disruption than red blood cells which are not so sensitised. Thus, application of an energy source to sensitised red blood cells allows the release of loaded agent, and delivery to the target tissue, etc.

Sensitisation may be effected through various means. For example, dye compounds such as porphyrins, phthalocyanines, BPDs, and related compounds may be incorporated into red blood cells to render them more susceptible to disruption by light treatment, such as laser light (photodynamic activation). Thus, photosensitisation may be used as a means of sensitisation of red blood cells, and disruption by application of light energy used to effect release of loaded agent. Methods of photodynamic activation, photosensitisation, and dyes useful for such purposes are disclosed in detail in US Patent Nos. 5,512,675,5,556,992,5,726,304, and 5,773,460.

In a highly preferred embodiment, sensitisation of red blood cells is achieved by administration of an electric field ; this is known as"electrosensitisation".

ELECTROSENSITISATION The present methods and compositions encompass the use of an electric field for sensitising a red blood cell to ultrasound ("electrosensitisation"), such a red blood cell being derived from a transgenic animal and containing a polypeptide. Such a sensitised red blood cell is suitable for delivering the polypeptide to a vertebrate, as it is possible to selectively disrupt the red blood cell by means of a stimulus such as ultrasound.

The term"electrosensitisation"encompasses the destabilisation of cells without causing fatal damage to the cells. According to this method, a momentary exposure of a cell to one or more pulses at high electric field strength results in membrane destabilisation. The strength of the electric field is adjusted up or down depending upon the resilience or fragility, respectively, of the cells being loaded and the ionic strength of the medium in which the cells are suspended Electrosensitisation typically occurs in the absence of the agent to be loaded into the cell. Electroporation, which facilitates passage of agents into the cell, occurs in the present of an exogenous agent to be loaded, and is well known in the art.

Electroporation has been used in both in vitro and in vivo procedures to introduce foreign material into living cells. With in vitro applications, a sample of live cells is first mixed with the agent of interest and placed between electrodes such as parallel plates.

Then, the electrodes apply an electrical field to the cell/implant mixture. Examples of systems that perform in vitro electroporation include the Electro Cell Manipulator ECM600 product, and the Electro Square Porator T820, both supplied by the BTX Division of Genetronics, Inc (see US Patent No 5,869,326).

These known electroporation techniques (both in vitro and in vivo) function by applying a brief high voltage pulse to electrodes positioned around the treatment region.

The electric field generated between the electrodes causes the cell membranes to temporarily become porous, whereupon molecules of the agent of interest enter the cells.

In known electroporation applications, this electric field comprises a single square wave pulse on the order of IkV/cm, of about 100 lls duration. Such a pulse may be generated, for example, in known applications of the Electro Square Porator T820.

Electrosensitisation may be performed in a manner substantially identical to the procedure followed for electroporation, with the exception that the electric field is delivered in the absence of an exogenous agent of interest, as set forth below, and may be carried out at different electric field strengths (and other parameters) from those required for electroporation. For example, lower field strengths may be used for electrosensitisation.

In a preferred aspect, the electric field has a strength of from about 0.1 kV/cm to about 10 kV/cm under in vitro conditions, more preferably from about 1.5 kV/cm to about 4.0 kV/cm under in vitro conditions. Most preferably, the electric field strength is about 3.625kV/cm under in vitro conditions.

Preferably the electric field has a strength of from about 0.1 kV/cm to about 10 kV/cm under in vivo conditions (see W097/49450). More preferably, the electric field strength is about 3.625kV/cm under in vitro conditions.

Preferably the application of the electric field is in the form of multiple pulses such as double pulses of the same strength and capacitance or sequential pulses of varying strength and/or capacitance. A preferred type of sequential pulsing comprises delivering a pulse of less than 1.5 kV/cm and a capacitance of greater than 5 suF, followed by a pulse of greater than 2.5 kV/cm and a capacitance of less than 2 u, F, followed by another pulse of less than 1.5 kV/cm and a capacitance of greater than 5 u. F. A particular example is 0.75 kV/cm, 10, uF ; 3.625 kV/cm, 1 I1F and 0.75 kV/cm, 10 I1F.

Preferably the electric pulse is delivered as a waveform selected from an exponential wave form, a square wave form and a modulated wave form.

As used herein, the term"electric pulse"includes one or more pulses at variable capacitance and voltage and including exponential and/or square wave and/or modulated wave forms.

Other electroporation procedures and methods employing electroporation devices are widely used in cell culture, and appropriate instrumentation, including the use of flow cell technology, is well known in the art. These procedures and methods may be adapted to perform electrosensitisation on a red blood cell.

In a particularly preferred embodiment, the following electrosensitisation protocol is used. Cells are suspended in PBS to yield concentrations of about 6-8x108 cells/ml and 0.8 ml aliquots are dispensed into sterile electroporation cuvettes (0.4 cm electrode gap) and retained on ice for 10 min. Cells are then exposed to an sensitisation strategy involving delivery of two electric pulses (field strength = 3.625 kV/cm at a capacitance of 1 jj. F) using a BioRad Gene Pulser apparatus. Cells are immediately washed with PBS cantaining MgCl2 (4-m-M)- (PBS/Mg) and retained at room temperature for at least 30min in the PBS/Mg buffer at a concentration of 7x108 cells/ml to facilitate re-sealing. Optionally, cells are subsequently washed and suspended at a concentration of 7x108 cells/ml in PBS/Mg containing 10 mM glucose (PBS/Mg/glucose) for at least 1 hour.

Sensitivity of the sensitised red blood cells to an external stimulus such as an energy source may be assayed by any conventional method. For example, cell lysis as judged by total cell counts may be determined after exposure to a stimulus such as ultrasound.

SELECTIVE RELEASE USING ULTRASOUND As noted above, ultrasound may be used as an energy source to disrupt sensitised red blood cells to effect release of the polypeptide.

As used here, the term"ultrasound"refers to a form of energy which consists of mechanical vibrations the frequencies of which are so high they are above the range of human hearing. The lower frequency limit of the ultrasonic spectrum may generally be taken as about 20 kHz. Most diagnostic applications of ultrasound employ frequencies in the range 1 and 15 MHz (from Ultrasonics in Clinical Diagnosis. Edited by PNT Wells, 2nd. Edition, Publ. Churchill Livingstone [Edinburgh, London & NY, 1977].

Ultrasound has been used in both diagnostic and therapeutic applications. When used as a diagnostic tool ("diagnostic ultrasound"), ultrasound is typically used in an energy density range of up to about 100 mW/cm2 (FDA recommendation), although energy densities of up to 750mW/cm2 have been used. In physiotherapy, ultrasound is typically used as an energy source in a range up to about 3 to 4 W/cm2 (WHO recommendation). In other therapeutic applications, higher intensities of ultrasound may be employed, for example, HIFU at 100W/cm2 up to IkW/cm2 (or even higher) for short periods of time. The term"ultrasound"as used in this specification is intended to encompass diagnostic, therapeutic and focused ultrasound.

Focused ultrasound (FUS) allows thermal energy to be delivered without an invasive probe (see Morocz et al., 1998 Journal ofMagnetic Resonace Irnaging Vol. 8, No. l, pp. 136-142. Another form of focused ultrasound is high intensity focused ultrasound (HIFU) which is reviewed by Moussatov et al. in Ultrasonics, 1998 Vol. 36, No. 8, pp. 893-900 and TranHuuHue et al. in Acustica, 1997, Vol. 83, No. 6, pp. l 103-1106.

LOADING AND DELIVERY OF SECOND AGENT Red blood cells derived from a transgenic animal, made by the methods described here, are already loaded with polypeptide product. These red blood cells may be subjected to sensitisation procedures as described here, and the resulting cells may be directly used as loaded delivery vehicles for introduction into a recipient animal. However, in some cases, it may be desired to load the red blood cells with one or more agents for delivery; this is referred to here as a"second agent". It will be understood, however, the methods

and compositions described here are not limited to loading of a second agent; third and subsequent agents may also be loaded in the same manner as described here.

Such a"second agent"may be anything that is capable of being loaded and delivered, for example, an atom or molecule, wherein a molecule may be inorganic or organic, a biological effector molecule and/or a nucleic acid encoding an agent such as a biological effector molecule, a protein, a polypeptide, a peptide, a nucleic acid, a peptide nucleic acid (PNA), a virus, a virus-like particle, a nucleotide, a ribonucleotide, a synthetic analogue of a nucleotide, a synthetic analogue of a ribonucleotide, a modified nucleotide, a modified ribonucleotide, an amino acid, an amino acid analogue, a modified amino acid, a modified amino acid analogue, a steroid, a proteoglycan, a lipid, a fatty acid and a carbohydrate. An agent may be in solution or in suspension (e. g., in crystalline, colloidal or other particulate form). The agent may be in the form of a monomer, dimer, oligomer, etc, or otherwise in a complex. The agent may be coated with one or more molecules, preferably macromoleucles, most preferably polymers such as PEG (polyethylene glycol).

Use ofaPEGylated agent increases the circulating lifetime of the agent once released.

For example, an imaging agent may be incorporated into the red blood cell to allow the progress and efficiency of introduction of the polypeptide to be monitored. The term "imaging agent"is intended to include an agent which may be detected, whether in vitro in the context of a tissue, organ or organism in which the agent is located. The imaging agent may emit a detectable signal, such as light or other electromagnetic radiation. The imaging agent may be a radio-isotope as known in the art, for example 32p or 35S or 99Tc, or a molecule such as a nucleic acid, polypeptide, or other molecule as explained below conjugated with such a radio-isotope. The imaging agent may be opaque to radiation, such as X-ray radiation, by (for example) having high electron density. The imaging agent may be detected by any means such as magnetic resonance imaging. The imaging agent may also comprise a targeting means by which it is directed to a particular cell, tissue, organ or other compartment within the body of an animal. For example, the agent may comprise a radiolabelled antibody specific for defined molecules, tissues or cells in an organism.

The imaging agent may be combined with, conjugated to, mixed with or combined with, any of the agents or polypeptides disclosed here.

It will be appreciated that it is not necessary for a single agent to be used, and that it is possible to load two or more agents for into the vehicle. Accordingly, the term "second agent"also includes mixtures, fusions, combinations and conjugates, of atoms, molecules etc as disclosed herein. For example, a second agent may include but is not limited to: a nucleic acid combined with a polypeptide; two or more polypeptides conjugated to each other ; a protein conjugated to a biologically active molecule (which may be a small molecule such as a prodrug); or a combination of a biologically active molecule with an imaging agent.

As used herein, the term"biological effector molecule"or"biologically active molecule"refers to an agent that has activity in a biological system, including, but not limited to, a protein, polypeptide or peptide including, but not limited to, a structural protein, an enzyme, a cytokine (such as an interferon and/or an interleukin) an antibiotic, a polyclonal or monoclonal antibody, or an effective part thereof, such as an Fv fragment, which antibody or part thereof may be natural, synthetic or humanised, a peptide hormone, a receptor, a signalling molecule or other protein; a nucleic acid, as defined below, including, but not limited to, an oligonucleotide or modified oligonucleotide, an antisense oligonucleotide or modified antisense oligonucleotide, cDNA, genomic DNA, an artificial or natural chromosome (e. g. a yeast artificial chromosome) or a part thereof, RNA, including mRNA, tRNA, rRNA or a ribozyme, or a peptide nucleic acid (PNA); a virus or virus-like particles; a nucleotide or ribonucleotide or synthetic analogue thereof, which may be modified or unmodified ; an amino acid or analogue thereof, which may be modified or unmodified; a non-peptide (e. g., steroid) hormone; a proteoglycan ; a lipid; or a carbohydrate. If the biological effector molecule is a polypeptide, it may be loaded directly into a red blood cell; alternatively, a nucleic acid molecule bearing a sequence encoding the polypeptide, which sequence is operatively linked to transcriptional and translational regulatory elements active in a cell at the target site, may be loaded. Small molecules, including inorganic and organic chemicals, are also of use here. In a

particularly preferred embodiment, the biologically active molecule is a pharmaceutically active agent, for example, an isotope.

A preferred embodiment comprises loading a ribozyme or an oligonucleotide such as an antisense oligonucleotide, which may optionally comprise a membrane translocation sequence (as described elsewhere in this document) into a red blood cell, which is sensitised, for delivery into a target cell or tissue.

Particularly useful classes of biological effector molecules include, but are not limited to, antibiotics, anti-inflammatory drugs, angiogenic or vasoactive agents, growth factors and cytotoxic agents (e. g., tumour suppressers). Cytotoxic agents of use include, but are not limited to, diptheria toxin, Pseudomonas exotoxin, cholera toxin, pertussis toxin, and the prodrugs peptidyl-p-phenylenediamine-mustard, benzoic acid mustard glutamates, ganciclovir, 6-methoxypurine arabinonucleoside (araM), 5-fluorocytosine, glucose, hypoxanthine, methotrexate-alanine, N- [4- (a-D-galactopyranosyl) benyloxyca. rbonyl]-daunorubicin, amygdalin,. azobenzene mustards, glutamyl p- phenylenediamine mustard, phenolmustard-glucuronide, epirubicin-glucuronide, vinca- cephalosporin, phenylenediamine mustard-cephalosporin, nitrogen-mustard-cephalosporin, phenolmustard phosphate, doxorubicin phosphate, mitomycin phosphate, etoposide phosphate, palytoxin-4-hydroxyphenyl-acetamide, doxorubicin-phenoxyacetamide, melphalan-phenoxyacetamide, cyclophosphamide, ifosfamide or analogues thereof. If a prodrug is loaded in inactive form, a second biological effector molecule may be loaded into the red blood cell. Such a second biological effector molecule is usefully an activating polypeptide which converts the inactive prodrug to active drug form, and which activating polypeptide is selected from the group that includes, but is not limited to, viral thymidine kinase (encoded by Genbank Accession No. J02224), carboxypeptidase A (encoded by Genbank Accession No. M27717), a-galactosidase (encoded by Genbank Accession No.

M13571), B-glucuronidase (encoded by Genbank Accession No. M15182), alkaline phosphatase (encoded by Genbank Accession No. J03252 J03512), or cytochrome P-450 (encoded by Genbank Accession No. D00003 N00003), plasmin, carboxypeptidase G2, cytosine deaminase, glucose oxidase, xanthine oxidase, B-glucosidase, azoreductase, t-

gutamyl transferase, B-lactamase, or penicillin amidase. Either the polypeptide or the gene encoding it may be loaded; if the latter, both the prodrug and the activating polypeptide may be encoded by genes on the same recombinant nucleic acid construct. Furthermore, either the prodrug or the activator of the prodrug may be transgenically expressed and already loaded into the red blood cell. The relevant activator or prodrug (as the case may be) is then loaded as a second agent according to the methods described here.

Preferably the biological effector molecule is selected from the group consisting of a protein, a polypeptide, a peptide, a nucleic acid, a virus, a virus-like particle, a nucleotide, a ribonucleotide, a synthetic analogue of a nucleotide, a synthetic analogue of a ribonucleotide, a modified nucleotide, a modified ribonucleotide, an amino acid, an amino acid analogue, a modified amino acid, a modified amino acid analogue, a steroid, a proteoglycan, a lipid and a carbohydrate or a combination thereof (e. g., chromosomal material comprising both protein and DNA components or a pair or set of effectors, wherein one or more convert another to active form, for example catalytically).

As described in detail in our International Patent Application PCT/GB00/03056 (incorporated by reference), pre-sensitisation of red blood cells by exposure to ultrasound or an electric field increases their loading capacity, so that they are able to take up larger amounts of the agent (s) to be loaded than otherwise. Pre-sensitisation may therefore be used to aid in the loading of a second agent into the transgenic red blood cell. Preferably, the red blood cells are pre-sensitised by exposure to ultrasound, more preferably, ultrasound that has an energy density in the therapeutic range. In a highly preferred embodiment, treatment is at 2.5W/cm2 for 5 min using a lMHz ultrasound head. This combination is however not intended to be limiting. Indeed, various combinations of frequency, energy density and exposure time may be used to pre-sensitise the red blood cells so that their loading efficiency is increased.

POLYMER THERAPEUTICS The agents may further be delivered attached to polymers. Polymer based therapeutics have been proposed to be effective delivery systems, and generally comprise one or more agents to be delivered attached to a polymeric molecule, which acts as a carrier. The agents are thus disposed on the polymer backbone, and are carried into the target cell together with the polymer.

The agents may be coupled, fused, mixed, combined, or otherwise joined to a polymer. The coupling, etc between the agent and the polymer may be permanent or transient, and may involve covalent or non-covalent interactions (including ionic interactions, hydrophobic forces, Van der Waals interactions, etc). The exact mode of coupling is not important, so long as the agent is taken into a target cell substantially together with the polymer. For simplicity, the entity comprising the agent attached to the polymer carrier is referred to here as a"polymer-agent conjugate".

Any suitable polymer, for example, a natural or synthetic polymer, may be used, preferably the carrier polymer is a synthetic polymer such as PEG. More preferably, the carrier polymer is a biologically inert molecule. Particular examples of polymers include polyethylene glycol (PEG), N- (2-hydroxypropyl) methacrylamide (HPMA) copolymers, polyamidoamine (PAMAM) dendrimers, HEMA, linear polyamidoamine polymers etc.

Any suitable linker for attaching the agent to the polymer may be used. Preferably, the linker is a biodegradable linker. Use of biodegradable linkers enables controlled release of the agent on exposure to the extracellular or intracellular environment. High molecular weight macromolecules are unable to diffuse passively into cells, and are instead engulfed as membrane-encircled vesicles. Once inside the vesicle, intracellular enzymes may act on the polymer-agent conjugate to effect release of the agent. Controlled intracellular release circumvents the toxic side effects associated with many drugs.

Furthermore, agents may be conjugated, attached etc by methods known in the art to any suitable polymer, and delivered. The agents may in particular comprise any of the molecules referred to as"second agents", such as polypeptides, nucleic acids, macromolecules, etc, as described in the section above. In particular, the agent may comprise a pro-drug as described elsewhere.

The ability to choose the starting polymer enables the engineering of polymer- agent conjugates for desirable properties. The molecular weight of the polymer (and thus the polymer-agent conjugate), as well as its charge and hydrophobicity properties, may be precisely tailored. Advantages of using polymer-agent conjugates include economy of manufacture, stability (longer shelf life) and reduction of immunogencity and side effects.

Furthermore, polymer-agent conjugates are especially useful for the targeting of tumour cells because of the enhanced permeability and retention (EPR) effect, in which growing tumours are more'leaky'to circulating macromolecules and large particules, allowing them easy access to the interior of the tumour. Increased accumulation and low toxicity (typically 10-20% of the toxicity of the free agent) are also observed. Use of hyperbranched dendrimers, for example, PAMAM dendrimers, is particularly advantageous in that they enable monodisperse compositions to be made and also flexibility of attachment sites (within the interior or the exterior of the dendrimer). The pH responsiveness of polymer-agent conjugates, for example, those conjugated to polyamindoamine polymers, may be tailored for particular intracellular environments.

This enables the drug to be released only when the polymer therapeutic encounters a particular pH or range of pH, i. e., within a particular intracellular compartment. The polymer agent conjugates may further comprise a targeting means, such as an immunoglobulin or antibody, which directs the polymer-agent conjugate to certain tissues, organs or cells comprising a target, for example, a particular antigen. Other targeting means are described elsewhere in this document, and are also known in the art.

Particular examples of polymer-agent conjugates include"Smancs", comprising a conjugate of styrene-co-maleic anhydride and the antitumour protein neocarzinostatin, and a conjugate of PEG (poly-ethylene glycol) with L-asparaginase for treatment of leukaemia; PK1 (a conjugate of a HPMA copolymer with the anticancer drug

doxorubicin); PK2 (similar to PK1, but furthermore including a galactose group for targeting primary and secondary liver cancer); a conjugate of HPMA copolymer with the anticancer agent captothecin; a conjugate of HPMA copolymer with the anticancer agent paclitaxel; HPMA copolymer-platinate, etc. Any of these polymer-agent conjugates are suitable for co-loading into the transgenic cells.

SENSITISATION/PRE-SENSITISATION Where the transgenic red blood cells are additionally loaded with a second agent (or agents) the cells are subject to at least two sensitisation steps, one of which must be performed prior to, or concomitant with, the loading of the second agent (s), preferably prior to the loading step. For this reason, the first sensitisation step is referred to herein as a pre-sensitisation step. The purpose of the pre-sensitisation step is to enhance the loading of the agent, although an increase in sensitivity to ultrasound mediated lysis may also be achieved. The additional sensitisation steps may be performed at any stage in the process after the pre-sensitisation step. The purpose of the additional sensitisation step or steps is to increase the sensitivity of the cells to ultrasound.

In two particular embodiments that are exemplified herein, a second sensitisation step is carried out either after the pre-sensitisation step but prior to dialysis loading, or after dialysis loading. Further sensitisation steps may be performed as required. Generally, the sensitisation steps and the loading step are temporally separated. For example, cells are typically allowed to rest in buffer, such as PBS/Mg/glucose buffer, for at least 30 mins, preferably at least 60 mins, after a pre-sensitisation step to allow the cells to recover prior to loading or further sensitisation steps. It may be desirable to allow cells to rest for several hours, such as overnight, after the loading step.

The pre-sensitisation step increases the efficiency of loading of an agent into a red blood cell, compared to a red blood cell which has not been subject to pre-sensitisation.

The pre-sensitisation may take the form of an electrosensitisation step, as described elsewhere in this document. Alternatively, or in addition, the pre-sensitisation may be

effected by the use of ultrasound, as described elsewhere in the documents. Other methods may be used to pre-sensitise cells and enhance loading efficiency. For example, electromagnetic radiation such as microwaves, radio waves, gamma rays and X-rays may be used. In addition, the use of chemical agents such as DMSO and pyrrolidinone may be envisaged. Furthermore, thermal energy may be imparted on the red blood cells to pre- sensitise them. This may be achieved by raising the temperature of the red blood cells by conventional means, by heat shock, or by the use of microwave irradiation. In general, any method which allows pores to be formed on the surface membrane of a red blood cell is a suitable candidate for use as a pre-sensitisation step.

TRANSLOCATION MEANS The polypeptide may comprise a translocation means to enable or assist intracellular delivery of the polypeptide. Suitable translocation means are those which facilitate passage through or across a cellular membrane (such as a plasma membrane, nuclear membrane, an organelle membrane, a chloroplast membrane, mitochondrial inner and/or outer membrane, Golgi membrane, etc). Highly preferred translocation means include membrane translocation sequences (MTS), described in further detail below.

Suitably, trangenic animals are engineered such that they express and produce red blood cells loaded with polypeptides comprising MTS, for example as a fusion protein.

Thus, we provide for a method of producing a red blood cell suitable for delivery of a polypeptide to a vertebrate, the method comprising the steps of : (a) providing a transgenic animal carrying and expressing a transgene encoding a fusion protein comprising the polypeptide and a membrane translocation sequence (MTS); (b) obtaining a red blood cell containing the fusion protein from the animal; and (c) sensitising the red blood cell sensitising the red blood cell to render it susceptible to disruption by an energy source.

Furthermore, we provide for a method of producing a red blood cell suitable for delivery of a polypeptide to a vertebrate, the method comprising the steps of : (a) providing

a red blood cell containing a polypeptide, the red blood cell being derived from a transgenic animal carrying and expressing a transgene encoding a fusion protein comprising the polypeptide and a membrane translocation sequence (MTS); and (b) sensitising the red blood cell to render it susceptible to disruption by an energy source.

Another aspect includes a method for the delivery of a polypeptide to a vertebrate, the method comprising the steps of : (a) providing a transgenic animal carrying and expressing a transgene encoding a fusion protein comprising the polypeptide and a membrane translocation sequence (MTS); (b) obtaining a red blood cell containing the fusion protein from the animal; (c) sensitising the red blood cell to render it susceptible to disruption by an energy source; (d) introducing the sensitised red blood cell to a vertebrate; and (e) exposing the vertebrate, or a part of it, to an energy source at a level sufficient to disrupt the sensitised red blood cell.

In another aspect, there is provided a method of producing a polypeptide agent- MTS conjugate, the method comprising the steps of : (a) isolating a red blood cell from a transgenic animal carrying and expressing a transgene encoding a fusion protein comprising the polypeptide and a membrane translocation sequence (MTS); (b) sensitising the red blood cell to render it susceptible to disruption by an energy source; (c) exposing the red blood cell to an energy source sufficient to disrupt the sensitized red blood cell; and (d) isolating the fusion protein to provide the polypeptide agent-MTS conjugate.

The transgenic animal is preferably selected from the group consisting of : mouse, rat, rabbit, sheep, goat, cow, and pig. More preferably, the polypeptide is expressed under the control of a p-globin promoter or enhancer. Most preferably, the polypeptide is expressed under the control of a p-globin Locus Control Region (LCR).

MEMBRANE TRANSLOCATION SEQUENCES The polypeptide to be delivered may be operatively linked to a membrane translocation sequence (MTS), for example conjugated with it as a fusion protein.

Suitably, a transgenic animal may be generated which expresses a transgene encoding a membrane translocation sequence (MTS), for example a transgene encoding fusion protein comprising a polypeptide of interest.

Membrane translocation sequences include polypeptide sequences or domains which are able to direct proteins, polypeptides, and other molecules across the cell membrane and into the cell. The use of fragments or variants of such sequences which comprise membrane translocational activity is also included, as are sequences, variants, fragments etc of polypeptides capable of directing localisation into subcellular compartments (such as the nucleus). Such sequences, and their fragments, are referred to here as"membrane translocation sequences"or MTS.

The presence of such sequences facilitates the intake of agent into a cell, and thus enables efficient intracellular delivery of agent. As explained below, one or more of these sequences may be coupled, fused, conjugated or otherwise joined to the agent to be delivered in order to effect intracellular delivery. In a highly preferred embodiment, polypeptides for delivery are expressed as fusion proteins with one or more membrane translocation sequences, the fusion proteins being expressed from a transgene in a transgenic animal.

As used here, the term'translocation'refers to transfer of an agent across a membrane such that the agent is internalised within a cell. Preferred membrane translocation sequences include the whole sequence or subsequences of the HIV-1-trans- activating protein (Tat), Drosophila Antennapedia homeodomain protein (Antp-HD), Herpes Simplex-1 virus VP22 protein (HSV-VP22), signal-sequence-based peptides, Transportan and Amphiphilic model peptide, among others. These membrane translocation sequences, as well as domains and sequences from them which are useful, are described in further detail below.

HIV-l-trans-activating protein (Tat) The Human Immunodeficiency Virus trans-activating protein (Tat) is a 86-102 amino acid long protein involved in HIV replication. Exogenously added Tat protein can translocate through the plasma membrane to reach the nucleus, where it transactivates the viral genome. Intraperitoneal injection of a fusion protein consisting of-galactosidase and Tat results in delivery of the biologically active fusion protein to all tissues in mice (Schwarze et al., (1999), Science 285,1569-72). Methods of delivering molecules such as proteins and nucleic acids into the nucleus of cells using Tat or Tat-derived polypeptides are described in detail in US Patent Numbers 5652122,5670617,5674980,5747641 and 5804604.

Vives et al. (1997), J BioL Chem. 272,16010-7 identified a sequence of amino acids 48-60 (CGRKKRRQRRRPPQC) from Tat important for translocation, nuclear localisation and trans-activation of cellular genes. This core sequence also includes a nuclear localisation sequence and has been found to exhibit translocational activity.

Accordingly, we provide for the use of polypeptides comprising the entire HIV-Tat sequence as well as polypeptides comprising the core sequence for translocating an agent into a cell. It will however be appreciated that variations about the core sequence, such as shorter or longer fragments (such as for example 47-58), may also possess translocational activity, and that these sequences may also be usefully employed.

To date, numerous Tat derived short membrane translocation domains and sequences have been identified that possess translocation activity; furthermore, translocation has been found to occur in various different cell types (Lindgren et al.

(2000), Trends Pharma. Sci. 21, 99-103). Examples of fragments which possess translocational activity include amino acids 37-72 (Fawell et al., (1994), Proc. Natl. Acad.

Sci. USA. 91,664-668), 37-62 (Anderson et al., (1993), Biochem. Biophys. Res. Commun.

194,876-884) and 49-58 (having the basic sequence RKKRRQRRR). Any of these fragments may be used alone or in combination with each other, and/or preferably with the core sequence, to enable translocation of an agent into a cell.

Internalisation of Tat is though to occur by endocytosis (Frankel & Pabo (1988), Cell 55, 1189-1193). Co-administration of basic peptides such as protamine or Tat fragments (amino acids 38-58) has been found to stimulate Tat uptake into cells.

Accordingly, we further provide the use of these and other agents which stimulate uptake ("translocation enhancers") to enhance the delivery of an agent into a cell. Use of such translocation enhancers need not necessarily be restricted to enhancing translocation of Tat conjugates/fusions-the methods and compositions described here encompass the use of such enhancers to enhance delivery of conjugates and/or fusions with other membrane translocation sequences (and/or fragments or domains of these), as described below. Thus, one or more translocation enhancers may be administered to the recipient before, after or at the same time as the loaded red blood cells are administered. Alternatively, the red blood cell may be loaded with the translocation enhancer (s) as well as the agent, preferably joined to a membrane translocation sequence, to be delivered. Disruption of the red blood cell at the point of delivery releases both the agent to be delivered and the translocation enhancer, thus stimulating uptake of the agent by the target cell or tissue, etc.

Tat-derived polypeptides lacking the cysteine rich region (22-36) and the carboxyl terminal domain (73-86) have been found to be particularly effective in tranlocation.

Absence of the cysteine rich region and the carboxy terminal domain prevents spurious trans-activiation and disulphide aggregation. In addition, the reduced size of the transport polypeptide minimises interference with the biological activity of the molecule being transported and increases uptake efficiency. Such polypeptides are used in the methods described in US Patent Numbers 5652122,5670617,5674980,5747641 and 5804604.

Accordingly, we disclose the use of such Tat-derived polypeptides lacking the carboxyl terminal domain and/or the cysteine rich region to improve the efficiency of translocation.

Preferably, the Tat-derived polypeptide lacks amino acids 73-86 of the Tat protein or amino acids 73-86 of the Tat protein. More preferably, the membrane translocation sequence comprises a Tat-derived protein which lacks both domains.

Drosophila Antennapedia homeodomain protein (Antp-HD) Agents may be conjugated or fused with all or part of the Drosophila Antennapedia homeodomain protein, preferably, the third helix of Antp-HD, which also has cell penetration properties (reviewed in Prochiantz (1999), Ann. N. E Acad. Sci. 886, 172-9). Cell internalization of the third helix of Antp-HD appears to be receptor-and endocytosis-independent. Derossi et al. (1996), J Biol. Chem. 271,18188-93 suggest that the translocation process involves direct interactions with membrane phospholipids.

The region responsible for translocation in Antp-HD has been localised to amino acids 43-58 (third helix), a 16 amino acid long peptide rich in basic amino acids having the sequence RQIKIWFQNRRMKWKK (Derossi, et al., (1994), J ; Biol. Chem. 269,10444- 50). This peptide is known as PenetratinQ'and has been used to direct biologically active substances to the cytoplasm and nucleus of cells in culture (Theodore, et al. (1995), J Neurosci. 15,7158-7167). Chimeric peptides less than 100 amino acids and oligonucleotides up to 55 nucleotides are capable of being internalised. Thoren et al.

(2000) FEBS Lett. 6,265-8 show that Penetratino traverses a lipid bilayer, further supporting the idea that cell internalization of the third helix of Antp-HD is receptor-and endocytosis-independent. We therefore provide for the use of Antp-HD or fragments of Antp-HD (including preferably fragments comprising, more preferably consisting of, RQIKIWFQNRRMKWKK, i. e., Penetratin) for intracellular delivery of agents.

Antp-HD and its fragments may be conjugated with proteins and nucleic acids by methods known in the art, for example as described in WO 99/11809. This document also describes sequences homologous to Antp-HD isolated from other organisms, including vertebrates, mammals and humans; homologues of Penetratin43 are also described in EP 485578. The use of these and other homologues and fragments of these for delivery of agents into cells is included. Truncated and modified forms of Antp-HD and Penetratin are described in WO 97/12912, UK 9825000.4 and UK 9902522.3. For example, truncated polypeptides of 15 and 7 amino acids such as RRMKWKK have been found to be active in translocation. Accordingly, such truncated and modified forms of Antp-HD and its homologues may be used in the methods and compositions described here.

To improve intracellular delivery, Antp-HD and/or its fragments may be conjugated to peptide nucleic acid (PNA), as described by Nielsen et al. (1991) Science 254,1497-1500. PNA is resistant to proteases and nucleases and is much more stable in cells than regular DNA. Pooga et al. (1998) Nat Biotechnol. 16,857-861 show that a 21- mer PNA complementary to human galanin receptor mRNA, coupled to Antp-HD, is efficiently taken up into Bowes melanoma cells, thus suppressing the expression of galanin receptors. The methods and compositions described here therefore include the use of conjugates and/or fusions of agents, membrane translocation proteins (and/or fragments) and peptide nucleic acid.

Herpes Simplex-1 virus VP22 protein The VP22 tegument protein of herpes simplex virus also exhibits membrane translocation activity. Thus, VP22 protein expressed in a subpopulation of cells spreads to other cells in the population (Elliot and O'Hare, 1997, Cell 88, 223-33). Fusion proteins consisting of GFP (Elliott and O'Hare, 1999, Gene Ther 6, 149-51), thymidine kinase protein (Dilber et al., 1999, Gene Ther 6, 12-21) or p53 (Phelan et al., 1998, Nat Biotechnol 16, 440-3) with VP22 have been targeted to cells in this manner.

HSV-VP22 has the amino acid sequence NAATATRGRSAASRPTERPRAPARSASRPRRPVE and agents may be conjugated or fused to this polypeptide (or fragments exhibiting translocation activity) for delivery into cells. As noted above, an important property of HSV-VP22 is that when applied to the surrounding medium, VP-22 is taken up by cells and accumulates in the nucleus. Thus, fusion proteins of HSV-VP22 conjugated to GFP (Elliott and O'Hare (1999), Gene Ther.

6,149-51), thymidine kinase protein (Dilber et al. (1999), Gene Ther. 6,12-21) and p53 (Phelan et al. (1998), Nat. Biotechnol. 16,440-3) have been targeted to cells in this manner. The mechanism of transport is thought to be via a Golgi-independent pathway.

Fusion proteins comprising HSV-VP22 (and sub-sequences) and a protein of interest, and the transport of such fusions into a cell are described in US 6017735.

Proteins capable of being transported by the methods described in US 6017735 include those involved in apoptosis, suicide proteins and therapeutic proteins. A feature of HSV-VP22 is that it binds to microtubules in cells as described in WO 98/42742.

Therefore, fusions, conjugates, etc of HSV-VP22 (including its fragments) with agents may be delivered into cells to stabilise microtubules and retard or enhance cell growth.

Variants of VP22 may be prepared in which the potency of this property is altered. Agents which enhance or inhibit microtubule polymerisation or de-polymerisation may be delivered to enhance or retard cell growth. Furthermore, HSV-VP22 fusions/conjugates may be employed where microtubule transport of an agent to a particular intracellular compartment or location is desired.

Signal-Sequence-Based Peptides Signal sequences of peptides are recognised by acceptor proteins that aid in addressing the pre-protein from the translation machinery to the membrane of appropriate intracellular organelles. The core hydrophobic region of a signal peptide sequence may be used as a carrier for cellular import of relevant segments or motifs of intracellular proteins (Lin et al, 1995, JBiol Chem 270,14255-14258; Liu et al., 1996, Proc Natl Acad Sci USA, 93,11819-11824). Synthetic membrane translocation domains and sequences containing such hydrophobic regions are able to translocate into cells.

The hydrophobic region, also known as the h region, consists of 7-16 non- conserved amino acids, and has been identified in 126 signal peptides ranging in length from 18-21 amino acids (Prabhakaran, 1990, Biochem J, 269,691-696). Any of these sequences may be employed. Signal sequence based translocators are thought to function by acting as a leader sequence ("leading edge") to carry peptides and proteins into cells (reviewed by Hawiger (1999), Curr. Opin. Cell. Biol. 3,89-94). Use of signal peptides for delivery of biologically active molecules is disclosed in US Patent No. l 5,807,746.

It is known that import of polypeptides comprising the signal sequence h-region does not require membrane caveolae (Torgerson et al. J Immunol. 161,6084-6092) or endosomal uptake (Lin et al. (1995), J Biol. Chem. 270,14255-14258; Hawiger (1997),

Curr. Opin. Immunol. 9, 189-194) but requires an intact plasma membrane (Lin et al.

(1995), J BioL Chem. 270,14255-14258). Furthermore, the uptake mechanism is concentration-and temperature-dependent, independent of cell type and receptor. Signal sequence based peptides can translocate into a number of cell types that include five human cell types (monocytic, endothelial, T lymphocyte, fibroblast and erythroleukemia) and three murine lines. Accordingly, we disclose the use of membrane translocation sequences, including signal sequence h-regions, conjugates, fusions, etc for intracellular delivery of agents.

Membrane translocation sequences comprising signal sequence based peptides coupled to nuclear localisation sequences (NLSs) may also be utilised. Thus, for example, the MPS peptide (Signal-sequence-based peptide I) is a chimera of the hydrophobic terminal domain of the viral gp41 protein and the NLS from the SV40 large antigen (GALFLGWLGAAGSTMGAWSQPKKKRKV) (Morris et al. (1997), Nucleic Acids Res.

25,2730-2736), and has been found to be active in membrane translocation. The peptide AAVALLPAVLLA1LLAP (Signal-sequence-based peptide II) is derived from the nuclear localisation signal of NF-KB p50 (Lin et al. (1996), Proc. Natl. Acad. Sci. USA 93,11819- 11824) and USF2 (Frenkel et al. (1998), J Immunol. 161,2881-2887). A peptide having the sequence AAVLLPVLLAAP is derived from from the Grb2 SH2 domain (Rojas et al.

(1998), Nat. Biotechnol. 16,370-375) and VTVLALGALAGVGVG from the Integrin 3 cytoplasmic domain (Liu et al. (1996), Proc. Natl. Acad. Sci. USA 93,11819-11824).

Peptides comprising membrane translocation sequence-nuclear localisation sequence have been shown to enter several cell types. Membrane translocation sequences derived from the hydrophobic regions of the signal sequences from Kaposi's sarcoma fibroblast growth factor 1 (K-FGF ; Lin et al. 1995, J. Biol. Chem. 271,5305-5308) and human integrin (Liu et al. 1996, Proc. Natl. Acad. Sci. USA 93,11819-11824), the fusion sequence of HIV-1 gp41 (Morris et al, 1997, Nucleic Acid Res, 25,2730-2736) and the signal sequence of the variable immunoglobulin light chain Ig (v) from Caiman crocodylus (Chaloin et al., 1997, Biochemistry 36,11179-11187) conjugated to NLS peptides originating from nuclear transcription factor kB (NF-KB ; Zhang et al., 1998, Proc Natl Acad Sci USA 95, 9184-9189), SV40 T-antigen (Chaloin et al., 1998, Biochem. Biophys. Res. Commun. 243,

601-608) or K-FGF (Lin et al., 1995, J BioL Chem. 270,14255-14258) may also be employed. Any of the peptides described above may be used alone or in combination, preferably in conjunction with nuclear localisation sequences, to deliver fused or conjugated agents into a cell.

Transportan Agents for delivery may be conjugated or fused or joined with transportan.

Transportan comprises a fusion between the neuropeptide galanin and the wasp venom peptide mastoparan. It is found to be localised in both the cytoplasm and nucleus (Pooga et al. (1998) FASEB J. 12,67-77). Transportan comprises the sequence GWTLNSAGYLLKINLKALAALAKKIL. Transportan may be used as a carrier vector for hydrophilic macromolecules. Cell-penetrating ability is not restricted by cell type and seems to be a general feature of this membrane translocation domain. Cellular uptake is not inhibited by unlabeled transportan or galanin and shows no toxicity at concentrations of 20 uM or less. However, concentrations of 50 uM decrease GTPase activity (Pooga et al. (1998), Ann. New YorkAcad. Sci. 863,45-453). The mechanism of cell penetration by transportan is not clear; however, it is known to be energy independent and that receptors and endocytosis are not involved. Deletion analogues of transportan have been prepared (Soomets et al. (2000), Biochim. Biophys. Acta. 1467,165-176) to identify those regions of the sequence responsible for translocation. Deletion of six amino acids from the N- terminus of transportan does not impair cell penetration. Deletions at the C-terminus or in the middle of the protein decrease or abolish translocation activity. Accordingly, we disclose the use of transportan, as well as deletions of transportan comprising translocation activity (preferably N-terminal deletions of 1,2,3,4,5 or 6 amino acids) in the delivery of agents into cells. We further disclose the use of novel short analogues disclosed by Lindgren et al., 2000, Bioconjug Chem 11 (5): 619-26 with similar translocation properties but with reduced undesired effects such as inhibition of GTPase activity.

Amphiphilic Model Peptide Agents may be conjugated with amphiphilic model peptide. Amphiphilic model peptide is a synthetic 18-mer (KLALKLALKALKAALKLA) first synthesised by Oehlke et al. (1998), Biochim. Biophys. Acta. 1414,127-139. Analogues that show less toxicity andhigheruptakehavebeensynthesisedbyScheller etal. (1999) J. Peptide Sci. 5,185- 194. The only essential structural requirement for amphiphilic model peptides is a length of four complete helical turns. The membrane translocation sequence crosses the plasma membranes of mast cells and endothelial cells by both energy-dependent and-independent mechanisms. The uptake behaviour shows analogy to several membrane translocation domain sequences including Antp-HD and Tat.

While it is clear from the above that any of the membrane translocation sequences (including domains and/or sequences and/or fragments of these exhibiting membrane translocation activity) may be used for the purpose of delivery of an agent into a cell, it should also be appreciated that other variations are also possible. For example, variations such as mutations, (including point mutations, deletions, insertions, etc) of any of the sequences disclosed here may be employed, provided that some membrane translocation activity is retained. Furthermore, it will be clear that any homologues of the membrane translocation proteins identified above, for example, from other organisms (as well as variations), may also be used.

Particular domains or sequences from proteins capable of translocation through the nuclear and/or plasma membranes may be identified by mutagenesis or deletion studies.

Alternatively, synthetic or expressed peptides having candidate sequences may be linked to reporters and translocation assayed. For example, synthetic peptides may be conjugated to fluoroscein and translocation monitored by fluorescence microscopy by methods described in Vives et al. (1997), JBiol Chem 272,16010-7. Alternatively, green fluorescent protein may be used as a reporter (Phelan et al., 1998, Nat Biotechnol 16, 440- 3).

The membrane translocation sequence may be linked to the agent to be delivered such that more than one agent can be delivered into a cell. The protein or fragment may contain components that facilitate the binding of multiple agents, for example drugs such as naturally occurring or synthetic amino acids. In this manner up to 32 different agents can be linked to the membrane translocation sequence and delivered. Such a method of using a membrane translocation sequence to facilitate the transfer of drugs is described in detail in WO 00/01417.

Agents may be fused to membrane translocation sequences, including proteins or fragments, using a variety of methods. Using peptide synthesis, the membrane translocation sequence can be chemically synthesised and linked with any peptide sequence or chemical compound (Lewin et al. (2000), Nat. Biotechnol. 18,410-414) using methods well known in the art. Peptides can also be chemically cross-linked to larger peptides and proteins (Fawell et al. (1994), Proc. Natl. Acad. Sci. USA 91, 664-668).

Furthermore, fusion proteins comprising the polypeptide agent fused to a membrane translocation sequence may be expressed in any suitable host, for example, a bacterial host (Nagahara et al. (1998), Nat. Med. 4,1449-1452). The cDNA of interest (including sequences encoding the membrane translocation protein or fragment as well as the polypeptide agent of interest) may be cloned in-frame downstream of an N-terminal leader, for example, comprising a 6-Histidine tag. This enables purification of the expressed recombinant fusion proteins using methods known in the art.

Advantageously, and as described above, polypeptides for delivery are expressed as fusion proteins with such sequences and/or fragments, the fusion proteins being expressed from a transgene in a transgenic animal. Thus, the transgene is capable of encoding a fusion protein comprising a membrane translocation sequence together with the polypeptide of iterest. Delivery of red blood cells containing the fusion protein, disruption and release in the vicinity of the target cell or tissue etc enables efficient intracellular delivery of agent into the target.

Thus the translocation domains described above may be conjugated or fused with the polypeptide (s) and second agents by any means known in the art. For example, the

transgene may be constructed by recombinant DNA technology to include a nucleotide sequence capable of expressing a translocation domain, such that the transgene is expressed as a fusion protein comprising the polypeptide sequence of interest fused to the translocation domain or sequence. This enables the packaging and loading of the fusion protein into the red blood cell by the animal. Subsequently, the cells are isolated, sensitised and introduced into the recipient animal. Lysis by ultrasound or other energy means enables release of the fusion protein, which is then able to enter the cells in the surrounding tissue.

The agent (s) may also be chemically coupled, either directly or indirectly, to the membrane translocation proteins, fragments, etc. The coupling may be permanent or transient, and may involve covalent or non-covalent interactions.

Direct linkage may be achieved by means of a functional group on the agent such as a hydroxyl, carboxy or amino group. Indirect linkage can occur through a linking moiety such as, but not limited to, one or more of bi-functional cross-linking agents, as known in the art. In this manner, a second agent comprising such fusion and/or conjugate, etc to be easily loaded into a transgenic red blood cell.

IMMUNOCOMPATIBILITY We provide a method for transplanting RBCs containing therapeutic payloads from transgenic donors, to an allogenic recipient animal.

According to this embodiment, red blood cells are preferably treated or modified to evade clearance from the body of an animal due to any naturally occurring clearance process. Such modified or treated red blood cells demonstrate immune evasion properties, e. g., reduced clearance mediated by the reticuloendothelial system (RES). Preferably, the red blood cells are treated or modified so that they exhibit reduced clearance by any one or more of the phagocytic system, macrophages, monocytes, or neutrophils.

The term"immune evasion"should be taken in this context to mean prevention of recognition by any or all of antibodies, complement, phagocytic cells to the cell, surface antigens, cell surface antigen or receptors. Red blood cells treated according to the methods and compositions described here demonstrate any or all of these properties.

To permit the survival of the heterologus donor cells, it is necessary to mask the cellular antigenic determinants of the donor RBCs. In order to do this, the RBC vehicles containing polypeptides may be coated with an agent which masks cell surface antigenic epitopes. For example, PEGylated RBCs evade the host immune response and thereby enjoy prolonged circulation. According to one such method, methoxy (polyethylene glycol), or mPEG, is covalently bound to RBCs (Scott et al., 1997, Proc. Natl. Acad. Sci.

U. S. A., 94: 7566-7571), and results from this study indicate that human RBC modified with mPEG in this manner resist agglutination by appropriate antisera, show decreased anti-blood group antibody binding, and are structurally normal, having normal osmotic fragility, and normal in vivo survival. Additionally, RBC modified in this manner demonstrate increased resistance to phagocytosis by peripheral blood monocytes upon xenogenic transplantation; the survival of mPEG treated sheep RBCs transfused into mice is increased 360-fold over that of untreated control cells (Scott et al., 1997, supra).

A second coating which may be useful is one which comprises distearoyl-phosphatidylethanolamine (DSPE)-conjugated PEG (Du et al., 1997, Biochim.

Biophys. Acta-Biomembranes, 1326: 236-248). When applied as a monolayer film to a glass plate, DSPE-PEG inhibits protein adsorption and cell adhesion to the glass plate (Du et al., 1997, supra). Furthermore, increasing percentages of grafted PEG in these supported lipid surfaces inhibit the adhesion of erythrocytes. The ability of PEG-grafted lipids to inhibit the binding of erythrocytes is directly related to the molecular weight of the grafted PEG, in that as the molecular weight of PEG increases, the erythrocyte adhesion decreases, making PEG-grafted lipids a potentially useful tool in the inhibition of antigen binding on xenogenic RBCs.

Furthermore, the transgenic donor animal may be genetically engineered to match the immunological specificity of the recipient. This reduces the chance of rejection of the

red blood cells. In a preferred embodiment, the transgenic animal expresses one or more blood group determinant antigens at a reduced level. Preferably, the donor animal is null for one or more blood group determinant antigens. The blood group determinant antigen may comprise an antigen involved in the ABO blood group system, or a Rhesus antigen, preferably a Rhesus D antigen.

POLYPEPTIDE PRODUCTION AND ISOLATION Production of large quantities of recombinant therapeutic molecules has long been a goal of modern biotechnology. Bacteria have been exploited for the production of useful recombinant therapeutic molecules, as have yeast. However, the production of therapeutically useful polypeptides in higher eukaryotic systems is often preferred, since the post-translational processing (e. g., glycosylation) of the recombinant products more closely resembles the processing that occurs in human cells. Many efforts to date have focused on the generation of transgenic mammals that secrete the transgene products in their milk. There have been difficulties in obtaining high level expression of recombinant polypeptides in the milk of transgenic animals, and the approach also has the disadvantages that only female animals are useful, and they must generally reach sexual maturity and bear offspring before the transgene product may be obtained in quantity. We provide a method, utilising the initial steps of RBC isolation, sensitization and ultrasound lysis, to isolate the therapeutic compounds described above, wherein said RBCs are isolated from a transgenic animal, in which the transgene encodes said therapeutic compound. In contrast to other transgenic production methods, animals expressing a transgene in RBCs will produce the transgene protein regardless of gender, age or reproductive history, providing a continuous source of the protein product.

The starting material for the isolation of the therapeutic compound is RBCs, including erythrocytes and reticulocytes. It is important to note that it is not necessary to isolate RBCs from among other blood cells in the present production method, although this step may be performed according to methods known in the art (e. g., by centrifugation and removal of the buffy coat), if desired. The RBCs are subjected to electrosensitization and ultrasound lysis under parameters described herein above

Briefly, the steps involved in the production and isolation of therapeutic transgene products are as follows: 1) isolating red blood cells (be sure to include reticulocytes in RBC definition) from an animal carrying and expressing a transgene encoding said pharmaceutical composition; 2) sensitizing the red blood cells; 3) exposing the red blood cells to energy sufficient to break the sensitized red blood cells generated in step (2); and 4) isolating the therapeutic transgene product.

In order to repel the proteolytic attack of enzymes present in the hemolysate, it is recommended to add a polyvalent proteinase inhibitor to the medium in which RBC disruption is performed. The particular components of the erythrocytes are separated from the so-called hemolysate. It is advisable to use high speed centrifugation for this purpose; the stroma-free supernatant obtained is then used for the preparation of the polypeptide.

The polypeptide can then be isolated by the non-limiting methods described below and, for example, in U. S. Patent No. 4,297,274. Methods of protein isolation and/or purification will necessarily vary with the nature of the individual protein product. However, methods as known in the art will include, for example, differential precipitation, separations based on molecular size or charge density, and immuno-or other affinity-based separation methods. Most preparative methods will entail a combination of two or more such methods.

Protein products can be precipitated, for example, with the aid of neutral salts.

With ammonium sulphate, which is usually employed for such precipitations, the protein is precipitated from its 1% strength aqueous solution at a concentration of between 1.5 and 2 moles/1 in a pH-range in proximity of the neutral point Furthermore, the protein can be precipitated with the aid of organic solvents, which are usually employed in protein chemistry, for example ethanol at a volume concentration of 10%, from an aqueous solution having a weakly acid pH-value, for example in a 0.04 molar acetate buffer having a pH of 5.5 at 0°C

Protein products may be precipitated with the water-soluble organic bases of the acridine and quinoline series. For example, 2-ethoxy-6,9-diaminoacridine lactate in a concentration of 0.01 mole/1 at a pH % 8.0 may be used to precipitate protein products from aqueous solution The protein can furthermore be precipitated with the organic acids usually employed in peptide chemistry for precipitations for example with trichloroacetic acid in a concentration of 0.2 mole/l and perchloric acid in a concentration of 0.6 mole/1 Upon reduction of the electrical conductivity of the protein solution as can be obtained through the removal of ions, some proteins precipitate in the weakly acid range.

Thus, precipitation of the protein is reached, for example, by dialysis against distilled water having a pH value of 5.0 If the electrical properties of the transgenic protein are known, the agent may be fractionated with the aid of an ion exchanger. Basic anion-exchangers, in particular those containing diethylaminoethyl, triethylaminoethyl or quaternary bases or their derivatives as functional groups, bind the protein from a buffer solution which has a relatively low concentration. Adsorption can be prevented by increasing the salt concentration. On the other hand, there exists the possibility of adsorbing the protein and of eluting it again with the use of salt solutions having a higher concentration, or of buffer solutions having an elevated pH value The behaviour of a given transgenic protein product under different conditions used to fractionate proteins may be exploited by one skilled in the art of protein fractionation to achieve purification of such protein from RBCs of transgenic animals The immunological affinity of the protein polypeptide for antibodies of said agent may be employed for enriching the protein with the aid of the so-called immuno-affinity processes. For this purpose, an immuno-adsorbent, i. e. a carrier-bound antibody against the protein which is capable of binding to the protein specifically, may be prepared in a

manner known in the art. The protein can then be eluted again by modification of the conditions of the medium (e. g., salt concentration). The techniques for this process, including the generation of antibodies specific for the polypeptide of interest, or an epitope thereof are readily available to those skilled in the art (See, for example Ausubel et al., Short Protocols in Molecular Biology, 3 d Edition, John Wiley & Sons, Inc. (1995)).

A method similar to the one disclosed in US Patent No 5,627,268, in which the desired peptide is linked via a cleavable peptide bond to a globin polypeptide, may also be used.

NUCLEIC ACIDS As explained elsewhere in this document, nucleic acids are useful in the production of transgene constructs and transgenic animals. A nucleic acid may comprise a viral or non-viral DNA or RNA vector, where non-viral vectors include, but are not limited to, plasmids, linear nucleic acid molecules, artificial chromosomes and episomal vectors.

Expression of heterologous genes has been observed after injection of plasmid DNA into muscle (Wolff J. A. et al., 1990, Science, 247: 1465-1468; Carson D. A. et al., US Patent No. 5,580,859), thyroid (Sykes et al., 1994, Human Gene Ther., 5: 837-844), melanoma (Vile et al., 1993, Cancer Res., 53: 962-967), skin (Hengge et al., 1995, Nature Genet., 10: 161-166), liver (Hickman et al., 1994, Human Gene Therapy, 5: 1477-1483) and after exposure of airway epithelium (Meyer et al., 1995, Gene Therapy, 2: 450-460).

As used herein, the term"nucleic acid"is defined to encompass DNA and RNA or both synthetic and natural origin which DNA or RNA may contain modified or unmodified deoxy-or dideoxy-nucleotides or ribonucleotides or analogues thereof. The nucleic acid may exist as single-or double-stranded DNA or RNA, an RNA/DNA heteroduplex or an RNA/DNA copolymer, wherein the term"copolymer"refers to a single nucleic acid strand that comprises both ribonucleotides and deoxyribonucleotides. The term"nucleic acid"is also intended to include oligonucleotides and modified oligonucleotides.

The term"synthetic", as used herein, is defined as that which is produced by in vitro chemical or enzymatic synthesis.

Therapeutic nucleic acid sequences useful according to the methods described here include those encoding receptors, enzymes, ligands, regulatory factors, and structural proteins. Therapeutic nucleic acid sequences also include sequences encoding nuclear proteins, cytoplasmic proteins, mitochondrial proteins, secreted proteins, plasmalemma- associated proteins, serum proteins, viral antigens, bacterial antigens, protozoal antigens and parasitic antigens. Therapeutic nucleic acid sequences also include sequences encoding proteins, lipoproteins, glycoproteins, phosphoproteins and nucleic acids (e. g., RNAs such as ribozymes or antisense nucleic acids). Ribozymes of the hammerhead class are the smallest known, and lend themselves both to in vitro synthesis and delivery to cells (summarised by Sullivan, 1994, J. Invest. Dermatol., 103: 85S-98S ; Usman et al., 1996, Curr. Opin. Struct. Biol., 6: 527-533). Proteins or polypeptides which can be expressed and delivered by nucleic acid molecules include hormones, growth factors, neurotransmitters, enzymes, clotting factors, apolipoproteins, receptors, drugs, oncogenes, tumour antigens, tumour suppressers, structural proteins, viral antigens, parasitic antigens and bacterial antigens. The compounds which can be incorporated are only limited by the availability of the nucleic acid sequence encoding a given protein or polypeptide. One skilled in the art will readily recognise that as more proteins and polypeptides become identified, their corresponding genes can be cloned into the gene expression vector (s) of choice and transgenic animals created which express these genes.

TREATMENT OF DISEASES The methods and compositions described here allow for the treatment of various diseases, by enabling the ready production and delivery of an agent, in particular a polypeptide, useful in the treatment of a disease to a vertebrate. Such agents including polypeptides are known in the art, or may be determined by methods known in the art.

Examples of diseases which may be treated include, but are not limited to, cancer, non-malignant tumors, localized infections, autoimmune disorders (e. g., rheumatoid arthritis, lupus erythematosus, Graves's disease), neurodegenerative disorders (e. g., Alzheimer's disease, amyotrophic lateral sclerosis and others), stroke, neuromuscular disorders (e. g., muscular dystrophy), heart disease (e. g., heart attack or arrhythmia), arterial or venous disease (e. g., blood clots or other arterial/venous blockage, such as from cholesterol or amyloid deposition), liver disease (e. g., cirrhosis), renal disease (e. g., kidney stones, bleeding or failure), diabetes or pulmonary disease (e. g., emphysema, asthma or bronchitis). The methods and compositions described here are additionally useful in the treatment of injury, such as surface wounds, puncture wounds, bone fractures, tearing of muscle, ligament or tendon, sprains/strains of a muscle or joint, burns (surface and internal, e. g., from extreme heat or cold, or from a chemical agent), inflammation, carpal tunnel syndrome or conditions resulting from injury, such as osteoarthritis.

TARGETING THE DELIVERY OF POLYPEPTIDES Any desired site in a vertebrate or mammal may be targeted using RBCs as described here and appropriate application of energy such as ultrasound or laser energy.

As used herein, the term"site"refers to a region of the body of a vertebrate, which region may comprise an anatomical area, a tissue, a system, an organ, a group of tissues, a cell, a group of cells or even substantially all of the cells of the vertebrate. The target is preferably a cell, tissue or vessel. As used herein, the term"tissue"refers to a population or physical aggregation of cells within an organism, wherein the cells are of the same cell type or are of cell different types resident within a single organ or other functional unit.

The efficacy of delivery of an agent to a site (such as a cell, tissue, organ, etc) may be assayed by any means. A particularly preferred means for assaying agent delivery is provided in the section below.

As used here, the term"tissue"refers to intact tissue or tissue fragments, such that the cells are sufficiently aggregated (associated) so as to form a cohesive mass.

Alternatively, the term"tissue"refers to a collection of individual cells, such as those which circulate (e. g., in blood or lymphatic fluid) within the vertebrate. A tissue may comprise an entire organ (e. g. the pancreas, the thyroid, a muscle, bone or others) or other system (e. g. the lymphatic system) or a subset of the cells thereof; therefore, a tissue may comprise 0. I-10%, 20-50% or 50-100% of the organ or system (e. g., as is true of islets of the pancreas).

As used herein the term"vessel"means any artery, vein or other"lumen"in an organism to which ultrasound can be applied and to which an agent may be delivered. A lumen is a channel within a tube or tubular organ. Examples of preferred vessels in the include but are not limited to the jugular vein, pulmonary vein, femoral vein, coronary artery, carotid artery, the femoral artery, the hepatic artery, the renal artery and the iliac artery.

In one embodiment, an ultrasound energy source may be focused at the target site (such as a vessel) as sensitized transgenic RBCs circulate through it. Furthermore, ultrasound may be focussed on red blood cells circulating through a tissue mass such as arterioles in the liver. Ultrasound may also be focussed on a target cell or tissue. For example, a diagnostic and/or therapeutic ultrasound energy source or a combination thereof may be applied to a target tissue. This is particularly applicable to target tissues located on the surface of the subject vertebrate, although deep targets may also be treated with an ultrasound energy source. Disruption using ultrasound is described in further detail below.

According to a method disclosed in U. S. Patent No. 4,669,481, limited targeting of RBCs to a small subset of vertebrate tissues may be achieved, if desired, as follows: Treating the red blood cell under mild heating conditions will damage the cells, resulting in rapid sequestration by the reticuloendothelial system. The cells can be specifically targeted for the spleen by heating for 10 minutes at 49°C. Greater temperature or length of heating produces increased cell damage, with resultant hepatic uptake. Thus, if desired, payload delivery to the spleen or liver can be preferentially enhanced; however, the degree to which the payload polypeptide is lost from damaged cells prior to administration is not

known. Sequestration of red blood cells may also be facilitated by cross-linking with compounds such as glutaraldehyde.

ASSAYS FOR AGENT DELIVERY In another aspect, we provide an assay for measuring the effectiveness of delivery of an agent to a cell, and in particular, intracellular delivery of an agent to a cell.

Preferably, such an assay employs transgenic animals, as described in further detail below.

The method for assaying delivery of an agent relies on detecting a reporter in a target cell. The reporter is encoded by and expressed from a first nucleotide sequence comprised in the cell. The delivery assay also makes use of a first polypeptide, which is capable of interacting with the first nucleotide sequence to modulate expression of the reporter. The interaction between the first polypeptide and the first nucleotide sequence may be direct (i. e., the first polypeptide binds to the first nucleotide sequence, or a subsequence of this), or indirect (i. e., the first polypeptide binds to the first nucleotide sequence via one or more other molecules, such as polypeptides, for example, transcription factors). What is important is that binding or other interaction between the first polypeptide and the first nucleotide sequence is capable of giving rise to, promoting or inhibiting (i. e., modulating) expression of the reporter sequence.

The delivery assay may also employ a molecule ("modulator"), which is capable of modulating (as detailed below) the interaction between the first polypeptide and the first nucleotide sequence, and hence expression of the reporter.

The delivery assay generally detects delivery of agent to target cells comprising a first nucleotide sequence encoding a reporter, and embodiments are described in which delivery of the first polypeptide to the cell are assayed, as well as those in which delivery of the"modulator"molecule are assayed. In each of these cases, the other entity (i. e., the entity whose delivery is not being assayed) is constitutively present, or at least present or provided when the assay is being undertaken or the reporter is being detected.

Thus, in a first embodiment, the assay determines successful delivery of the "modulator"molecule to the cell, where the cell comprises the first nucleotide sequence encoding the reporter, as well as the first polypeptide. In a second embodiment, the assay determines the successful delivery of the first polypeptide to the target cell (comprising the first nucleotide sequence encoding the reporter), in the presence of an agent. It will therefore be seen that the presence of all three components (first nucleotide sequence encoding reporter, first polypeptide, modulator molecule) will cause modulation of expression of the reporter, and detection of the reporter or such modulation indicates successful delivery of the relevant entity.

In highly preferred embodiments, the entity or agent whose delivery is being assayed is present in an extracellular environment. Preferably, the delivery assay comprises assaying delivery of the entity or agent into an intracellular environment, preferably a environment where gene expression (including transcription and/or translation) takes place. The delivery assay preferably assays delivery to the nucleus, mitochondria or cytoplasm.

In preferred embodiments, the entity or agent to be assayed is provided in the vicinity or area of a target cell. Preferably, the entity or agent is released from a delivery vehicle at or near a target cell. Preferably, the delivery vehicle is disrupted to effect such release. Preferably, the delivery vehicle is sensitised in order to effect such disruption.

Preferably, the delivery vehicle is exposed to a stimulus, preferably ultrasound, to cause it to be disrupted. Preferably, the entity or agent is loaded into the delivery vehicle. In highly preferred embodiments, the delivery vehicle comprises a red blood cell, preferably a sensitised, preferably electrosensitised, red blood cell.

In highly preferred embodiments of the delivery assay, the agent or entity is loaded into a red blood cell as described above, whether by hypotonic dialysis, osmotic shock, or by any other means such as in a ready loaded form derived from a suitable transgenic animal. Thus, in particular, the delivery assay is highly suited for assaying successful delivery of any agent which is loaded, sensitised and/or delivered as described in WO 01/58431, WO 01/07011 or WO 01/58432.

ASSAY FOR DELIVERY OF MODULATOR MOLECULE In the first embodiment of the delivery assay, the delivery of a molecule ("modulator") is assayed.

In this embodiment, the molecule is capable of modulating (as detailed below) the interaction between the first polypeptide and the first nucleotide sequence. The first nucleotide sequence and the first polypeptide are present constitutively in the cell, or are at least provided when the assay is being undertaken. The first polypeptide is present in the cell, preferably in the nucleus, and may be provided in the form of a protein which is capable of diffusing or otherwise entering the cell or nucleus. The protein may be provided in the medium or cellular environment of the cell, or it may be expressed in the cell.

The protein may be expressed from a suitable nucleic acid sequence (a second nucleotide sequence, for example, a nucleic acid sequence encoding the first polypeptide, cloned into an expression vector, which is introduced into the cell). In this case, the nucleotide sequence encoding the reporter and the nucleotide sequence encoding the first polypeptide may be present on the same nucleic acid construct, or in separate nucleic acid constructs. Thus, a single construct comprising the nucleotide sequence encoding the reporter and the nucleotide sequence encoding the first polypeptide may be transfected or otherwise introduced into a cell to provide a target cell. Alternatively, the nucleotide sequence encoding the reporter and the nucleotide sequence encoding the first polypeptide may be provided as separate constructs. Transfection may be permanent or transient, and the construct or constructs may be present episomally, for example as plasmids, or integrated into the genome of the target cell. Expression of the first polypeptide within the cell may be constitutive; production of constructs which are capable of constitutive expression of a nucleic acid sequence is known in the art.

In either case, where the modulator molecule is successfully delivered to or preferably into the target cell, modulation of the interaction between the first polypeptide and the first nucleotide sequence occurs, with the result that the expression of the reporter

from the first nucleotide sequence is changed. Thus, detection of the reporter, preferably, detection of modulation of reporter level or activity, provides a measure of whether delivery of the agent (in this case the"modulator"molecule) to the cell has been successful or not.

Examples of such an embodiment of the delivery assay are provided in Examples 10,11 and 12. In Example 10, delivery of the"modulator"molecule oestrogen is assayed ; Example 11 assays delivery of a"modulator"molecule in the form of tetracycline.

Example 12 details delivery of an antisense modulator molecule.

It will be appreciated that the assay is not only suitable for detection of delivery of the modulator molecule per se, but any molecules or agents which comprise the modulator molecule, conjugated in any way. Thus, for example, delivery of a polypeptide may be assayed by conjugating for example chemically coupling the modulator molecule to the polypeptide. Accordingly, the term"modulator"molecule should be understood as encompassing such joined or fused entities, so long as they comprise a portion corresponding to a"modulator"molecule which is capable of modulating the interaction between a first polypeptide and a first nucleotide sequence to modulate expression of a reporter.

ASSAY FOR DELIVERY OF A FIRST POLYPEPTIDE In a second embodiment, we provide a delivery assay to measure delivery of a first polypeptide.

In this embodiment, as in the first embodiment, the molecule is capable of modulating (as detailed below) the interaction between the first polypeptide and the first nucleotide sequence.

The first nucleotide sequence encoding the reporter is present in the cell, and the "modulator"molecule is provided constitutively in the cell, or is at least provided when

the assay is being undertaken. The"modulator"molecule may be provided in the cell medium or cell environment, and will any case be present at the point or locality of interaction of the first nucleotide sequence and the first polypeptide (e. g., the nucleus). In particular, and especially in the case of transgenic animal embodiments, the"modulator" molecule may be naturally expressed by the animal, for example, oestrogen.

The first polypeptide, whose delivery is being measured, may be produced by any means known in the art. In a preferred embodiment, the first polypeptide is loaded into a delivery vehicle (e. g., a red blood cell), which is disrupted at or near the target cell, thus releasing the first polypeptide.

The target cell is exposed to the first polypeptide in the presence of the "modulator"molecule. Preferably, the"modulator"molecule is present in the intracellular environment, i. e., intracellularly, of the cell.

Thus, where the first polypeptide is successfully delivered to or preferably into the target cell, interaction between the first polypeptide and the first nucleotide sequence occurs, with the result that the expression of the reporter from the first nucleotide sequence is changed. Thus, detection of the reporter, preferably, detection of modulation of reporter level or activity, provides a measure of whether delivery of the agent (in this case the first polypeptide) to the cell has been successful or not.

Examples of such an embodiment of the delivery assay are provided in Examples 8, 9 and 13. Example 8, delivery of the first polypeptide (Gal-ER-VP16) is assayed ; Example 9 assays delivery of a first polypeptide in the form of Gal-ER-VP16. Example 13 details delivery of a Tet-On first polypeptide.

It will be appreciated that the assay is not only suitable for detection of delivery of the first polypeptide per se, but any molecules or agents which comprise the first polypeptide, conjugated in any way. Thus, for example, delivery of a polypeptide may be assayed by conjugating for example chemically coupling the first polypeptide to the

polypeptide. Alternatively, the two polypeptides may be expressed as a fusion protein; other ways of performing this aspect may also be envisaged by a person skilled in the art.

Accordingly, the term"first polypeptide"should be understood as encompassing such joined or fused entities, so long as they comprise a portion corresponding to a first polypeptide which is capable of interacting with a first nucleotide sequence encoding a reporter to modulate expression of the reporter.

MODULATION OF REPORTER EXPRESSION The assay for delivery of an agent or entity to a cell may be performed in several ways, depending on whether the first polypeptide interacts with the first nucleotide sequence to promote or repress expression of the reporter.

In a first embodiment, the first polypeptide interacts with the first nucleotide sequence to promote expression of the reporter, and the"modulator"molecule is capable of modulating in a negative manner the interaction between the first polypeptide and the first nucleotide sequence. Thus, in the absence of the"modulator"molecule (or the first polypeptide), the cell expresses the reporter at a particular level. Where the"modulator" molecule (or first polypeptide) has been successfully delivered to the cell, expression of the reporter is repressed or reduced. Detection of reduction of reporter level and/or activity may therefore be conducted to detect delivery of the entity or agent to the cell.

In a second embodiment, the first polypeptide interacts with the first nucleotide sequence to inhibit or reduce expression of the reporter, and the"modulator"molecule is capable of modulating in a positive manner the interaction between the first polypeptide and the first nucleotide sequence. Thus, in the absence of the"modulator"molecule or first polypeptide, the cell expresses the reporter at a low level, or not at all. Where the "modulator"molecule (or first polypeptide) has been successfully delivered to the cell, expression of the reporter is induced or increased. Detection of a higher level and/or activity of the reporter may therefore be conducted to detect delivery of an entity or agent to the cell.

Preferably, the difference between the level and/or activity of the reporter in the absence and presence of delivery of the agent or entity to be assayed is maximised. Thus, preferably in assays where the first polypeptide and the first nucleotide sequence interact positively to promote reporter expression, delivery of agent or entity to the cell results in substantially complete inhibition of reporter. Similarly, where the first polypeptide interacts with the first nucleotide sequence to inhibit or reduce reporter expression, preferably the cell exhibits substantially complete inhibition of expression of reporter in the absence of agent.

Other embodiments of the delivery assay are also possible. For example, the first polypeptide may interact with the first nucleotide sequence to inhibit or reduce expression of the reporter, and the"modulator"molecule is capable of modulating in a negative manner the interaction between the first polypeptide and the first nucleotide sequence.

Furthermore, the first polypeptide may interact with the first nucleotide sequence to promote expression of the reporter, and the"modulator"molecule modulates the interaction between the two in a positive manner.

INDUCIBLE EXPRESSION SYSTEMS Preferably, the first polypeptide and the first nucleotide sequence form part of a conventional inducible expression system, or are derived from such a system. Examples of such inducible expression systems are known in the art, and include the Tet repressor system, an oestrogen inducible system and an ecdysone inducible system. These are described in further detail below.

Thus, in one embodiment, the first polypeptide comprises a Tet repressor (TetR), and the first nucleotide sequence comprises a Tet-responsive element (TRE). In a further embodiment, the first polypeptide comprises an oestrogen receptor, and the first nucleotide sequence comprises an oestrogen responsive element (ORE), while in a third embodiment, the first polypeptide comprises an ecdysone receptor, and the first nucleotide sequence comprises an ecdysone responsive element (EcRE).

The first polypeptide may optionally comprise a transcriptional activator domain.

Preferably, such a transcriptional activator domain is selected from a VP16, a VP64, a maize C1, and a PI domain. The first polypeptide may alternatively optionally comprise a transcriptional transcriptional repressor domain, preferably selected from a KRAB-A domain, an engrailed domain or a snag domain.

The."modulator"molecule may modulate the interaction between the first polypeptide and the first nucleotide sequence by any means, so long as expression of the reporter is modulated. As noted above, in some embodiments, the first polypeptide is expressed from a nucleic acid sequence comprised in the cell. Therefore, the"modulator" molecule may promote, inhibit, interfere or otherwise modulate any of the steps leading to expression of the first polypeptide from its encoding sequence. Accordingly, the "modulator"molecule may be capable of modulating the expression, preferably the transcription, of the nucleic acid sequence encoding the first polypeptide. The"modulator" molecule may therefore comprise a known repressor of transcription of the gene encoding the first polypeptide.

Furthermore, the"modulator"molecule may act by modulating the mRNA which is transcribed. Thus, the"modulator"molecule may modulate any of the post- transcriptional steps, for example, RNA transport, RNA processing, RNA splicing, RNA stability, RNA turnover or RNA degradation of a mRNA encoding the first polypeptide.

The"modulator"molecule may modulate the translation of the first polypeptide from the mRNA, by, for example, interfering in ribosome binding, tRNA synthesis, etc.

The"modulator"molecule may act directly on the first polypeptide itself, by regulating the transport, processing, post-translational modification, stability, turnover or degradation of the first polypeptide.

It will be appreciated that the"modulator"molecule may act physically to modulate the interaction between the first polypeptide and the first nucleotide sequence.

Thus, the"modulator"molecule may sterically hinder binding or interaction of the first polypeptide and the first nucleotide sequence.

Furthermore, the"modulator"molecule may, instead of modulating the interaction between the first polypeptide and the first nucleotide sequence, may modulate directly the expression of the reporter itself. This may be achieved by modulating the transcription of the reporter, the processing, etc of an RNA encoding the reporter, the processing, etc, of the reporter polypeptide, etc.

In a particular embodiment, the"modulator"molecule comprises an antisense nucleic acid. Binding of the antisense nucleic acid to a nucleotide sequence encoding the first polypeptide and inhibits its expression. Such a nucleic acid is preferably antisense to an mRNA sequence encoding the first polypeptide. However, the antisense nucleic acid may also be capable of binding to the DNA sequence encoding the first polypeptide and interfering with its expression.

The reporter may be capable of being expressed in a non-tissue specific manner.

Thus, in this embodiment, the control sequences regulating the expression of the reporter are capable of directing expression in any cell type. Such an arrangement is useful where it is desired to determine the locations within the body of an animal where the agent or entity (such as a"modulator"molecule or first polypeptide) has been delivered. For example, an agent to be assayed may be loaded into a red blood cell, the red blood cell sensitised and administered to the body of an animal. Disruption of the sensitised red blood cell with ultrasound enables release, whether systemically or locally depending on the exposure of the animal to the ultrasound source, of the agent. The reporter is then monitored throughout the entire body of the animal, and locations where modulation of expression of the reporter occurs determined. This enables a determination of whether the agent has been successfully delivered into a particular cell type, tissue, subsystem or organ within the body.

However, in a preferred embodiment, expression of the reporter is potentially restricted to a subset of cell or tissue types. Such tissue specific expression may be achieved by use of particular control regions, e. g., tissue specific enhancers and/or promoters to drive expression of the reporter. Tissue specific expression is preferably achieved by the use of Locus Control Regions (LCR). LCRs are capable of directing high- level integration site independent expression of transgenes integrated into host cell chromatin, which is of importance especially where the gene is to be expressed in the context of a permanently-transfected or transgenic cell in which chromosomal integration of the vector has occurred.

For example, expression of the reporter may be restricted only to vascular endothelial cells. Such an arrangement may be useful if it is desired to determine the extent of penetration of an agent ("modulator"molecule or first polypeptide) into surrounding tissues of a blood vessel, in the locality or vicinity of the position at which agent is released. The kinetics of diffusion or penetration of an agent into the locality may also be examined using this arrangement.

In embodiments where delivery of a"modulator"molecule is assayed, the cell may express the first polypeptide and/or the reporter in a transient manner. Such a cell may be transfected with relevant plasmid constructs encoding the first polypeptide and the first nucleotide sequence, whether as part of the same construct or as a separate construct.

Alternatively and preferably, the cell is capable of persistent expression of the first polypeptide and/or the reporter; preferably, the cell comprises sequences encoding the first polypeptide and the first nucleotide sequence integrated within its genome.

In a highly preferred embodiment, the cell is provided from a transgenic animal.

The transgenic animal carries and expresses a transgene encoding at least the the first nucleotide sequence in this embodiment. In certain embodiments, the transgenic animal may carry and express a transgene encoding the first polypeptide as well, particularly in assays for delivery of a"modulator"molecule. In this case, the sequence encoding the first polypeptide and the sequence comprising the first nucleotide sequence may be adjacent or separated within the genome of the transgenic animal. The transgenic animal may be

selected from the group consisting of : mouse, rat, rabbit, sheep, goat, cow, and pig.

Transgenic animals comprising cells expressing first nucleotide sequences and/or first polypeptides as described above, and methods for their production, are described in detail in separate sections of this document.

The cell and the animal of which it forms a part may be genetically identical, i. e., the cell may be derived from the animal. However, the cell may also be a heterologous or foreign cell, and accordingly, the cell may form part of a tissue mass grafted onto a host animal. For example, the cell may be a human cell, whether derived from a primary or established cell line, which has been engineered to express the system described here. The human cell is transplanted into a host animal, for example, a mouse or a pig.

The reporter is preferably detected in situ in an animal comprising the cell, by any means known in the art depending on the nature of the reporter.

Expression of the reporter is such that it is capable of being modulated by interaction of a first polypeptide and a first nucleotide sequence The methods disclosed here may be used for assaying any means of delivery, for example, delivery of agents by use of liposomes, protein vesicles, etc. In particular, we envisage that the methods described here will be especially useful to assay delivery of agents loaded into carriers such as red blood cells, in particular red blood cells which have been sensitised as described in our International Patent Application Number PCT/GB00/02848 (published as WO/01/07011). Such sensitised red blood cells may be disrupted to effect release of agent, as described in WO/01/07011.

Particular applications include optimisation of drug release systems in animals prior to trials in man (for example, by use of focused ultrasound), as well as gathering of pharmacogenomic and toxicology data on distribution and sites of action of any suitable drug delivered by any suitable means.

TETRACYCLINE BASED SYSTEMS The methods of assaying agent delivery may make use of a tetracycline inducible system. The first polypeptide comprises a Tet-repressor, while the first nucleotide sequence comprises a Tet operator ; the"modulator"molecule comprises a tetracycline, doxycycline or any other suitable derivative or molecule.

Such a system is described in detail in l. Gossen, M. & Bujard, H. (1992) Proc.

Natl. Acad. Sci. USA 89: 5547-5551 and also in US Patent Numbers 5,464,758 and 5,814,618. Such systems are commercially available, for example, the Tet-Off and Tet- OnTM systems, from Clonetech Laboratories, Inc. The following description is based on the use of the Tet-Off and Tet-On systems, but should not be regarded as limiting.

Briefly, Tet repressor systems are based on two regulatory elements derived from the tetracycline-resistance operon of the E. coli TnIO transposon: the tet repressor protein (TetR) and the Tet operator DNA sequence (tetO) to which TetR binds. The gene to be expressed (i. e., a reporter) is cloned into a suitable"response"plasmid which contains a suitable promoter comprising the operator sequence, in such a way that the promoter controls the expression of the reporter. For example, in the Tet-OffTM system, the reporter may be cloned into the pTRE"response"plasmid, which contains a PhCMV*-I promoter upstream of a multiple cloning site (MCS). PhCMV*-l is a compound promoter consisting of the tet-responsive element (TRE), which contains seven copies of tetO, and the minimal immediate early promoter of cytomegalovirus (PminCMV).

The second key component of the Tet inducible system is a"regulator"plasmid which expresses a tet-controlled transcriptional activator. (tTA) Such an activator may comprise a hybrid protein. In the Tet-Off TM system, tTA is encoded by plasmid pTet-Off and is a fusion of the wild-type Tet repressor (TetR) to the VP 16 activation domain (AD) of herpes simplex virus. tTA binds the Tet operator sequence (tetO)-and thereby activates transcription-in the absence of tetracycline (Tc). Thus, as Tc is added to the culture medium, transcription is turned off in a dose-dependent manner. Both the response

plasmid (pTRE-GeneX) and the regulator plasmid (pTet-Off) must be integrated into the host cell line to create a"double-stable"cell line in the Tet-Off TM system.

The tetracycline inducible system may be configured in such a way that addition of tetracycline induces expression of the reporter, for example in the Tet-On system of Clonetech. Thus, the Tet-On System is based on the"reverse"TetR (rTetR), which differs from the wild-type TetR by four amino acid changes (Gossen, M., et al. (1995) Science 268: 1766-1769). When fused to the VP16 AD, rTetR creates a"reverse"tTA (rtTA) that activates transcription in the presence of Dox. The rtTA regulatory protein in Tet-On Systems is 100 times more sensitive to Dox than it is to Tc. Consequently, Dox may be used for optimal expression in Tet-On Systems. In contrast, Tet-Off Systems respond well to either Tc or Dox.

In general, a Tet-Off or Tet-On gene expression system may be set up using two stable transfections. The mammalian cell line of choice is initially transfected with either pTet-Off or pTet-On. Because the integration site can significantly affect expression levels of tTA or rtTA, multiple clonal, G418-resistant lines may be isolated and tested to identify a clone that gives a wide range of tet-dependent induction of luciferase when transiently transfected with pTRE-Luc. Such a cell line may be referred to as a Tet-Off or a Tet-On cell line.

In the second transfection, the gene of interest (i. e., the reporter gene, cloned into pTRE) is cotransfected with the pTK-Hyg selection plasmid into the Tet-Off or Tet-On cell line described above. Again, it is necessary to screen multiple clonal cell lines to identify a cell line that gives high expression of the reporter in the induced state, and low background expression of the reporter in the noninduced state. Many clones will exhibit high background expression of the reporter. Such background is presumably due to integration of pTRE-Reporter near an endogenous enhancer in the host genome.

A cell line may be established as described above. Furthermore, transgenic animals expressing components of a tetracycline inducible system may be made, as described in further detail below.

The agent whose delivery is to be assayed may be chosen to disrupt the interaction between the Tet Response Element and the Tet repressor (in the case of a system based on Tet-OffrM), in the absence of tetracycline. An decrease in reporter level or activity is detected as an indication of successful delivery. The system may also be used in the presence of Tet, but with an agent which promotes the interaction between the Tet Response Element and the Tet repressor. Here, detection of an increase of reporter level or activity indicates successful delivery of an agent to the cell.

A Tet-On system may also be used as a basis for the assay. Thus, the agent may be chosen to promote the interaction between the modified Tet repressor (reverse TetR) and the Tet Response Element, and the cell assayed in the absence of tetracycline or doxy dine. Detection of an increase in reporter level or activity indicates successful delivery of the agent to the cell. Furthermore, in the presence of tetracycline or doxycline, delivery of an agent which is capable of disrupting the interaction between the modified Tet repressor (reverse TetR) and the Tet Response Element may be detected by detecting a decrease in level or activity of a reporter.

ECDYSONE BASED SYSTEMS As noted above, the method for assaying agent delivery may be based on an ecdysone inducible system. In this embodiment, the first polypeptide comprises an ecdysone receptor, and the first nucleotide sequence comprises an ecdysone responsive element (EcRE).

Ecdysone is an insect steroid hormone that is involved in insect molting. Ecdysone binds to a heterodimeric receptor composed of the ecdysone receptor (VgEcR) and USP (ultraspiracle). Each of these proteins has a DNA binding domain that is unique. ecdysone

binding causes the dimeric receptor to bind its target DNA sites and activate transcription of genes involved in molting.

This system has been modified to make it bind to sequences on a vector that have been engineered to be unique in mammalian cells. The VgEcR receptor has had the VP16 transactivation domain (used in two hybrid systems) added to it to make it a better activator of transcription. In addition, the ecdysone receptor's DNA binding domain has been altered to recognize a hybrid response element. One half of the response element is the normal ecdysone response element. The other half is the glucocorticoid response element. This hybrid response element is called (E/GRE). Instead of using USP that is specific for insects, the mammalian homolog of USP, the retinoid X receptor (RXR), is used for the second subunit of the heterodimer. With all this modification, the only site the modified ecdysone heterodimer can recognize is a synthetic sequence that is only found on a vector upstream of the gene to be expressed. In fact the vector has 5 of these modified E/GRE elements in front of the minimal heat shock promoter.

To make this system work in mammalian cells, two vectors are needed to be transfected into the cells. One contains both subunits of the receptor, and the other contains the gene of interest with the E/GRE elements upstream. An analog of ecdysone called muristerone A is used. Each vector has a different resistance gene, one is for neomycin resistance and the other is for Zeocin resistance. (Zeocin is a drug similar to bleomycin that intercalates into DNA and cleaves it). With both vectors in a cell, addition of muristerone A causes a greater than 200 fold induction of expression of the target gene over basal levels.

Accordingly, a reporter may be cloned into the vector such that it is operatively linked to the response element. The agent whose delivery is to be assayed will in this system be capable of inhibiting or interfering with the interaction between the response element (s) and the VgEcR-RXR heterodimeric receptor, by, for example, inhibiting the expression of the receptor.

REPORTERS The reporter may comprise any entity which may be detected, either directly or indirectly. For example, the detection may be non-destructive, e. g., detection of fluorescence, or it may be a destructive detection, for example, detection of reporter by staining and sectioning of tissues (i. e., histopathology).

In a preferred embodiment, the reporter comprises a fluorescent polypeptide.

Examples of fluorescent polypeptides and proteins include Green Fluorescent Protein (GFP) from Aequorea victoria and Red Fluorescent Protein (RFP) from Discosoma spp.

Derivatives and variants of these proteins, such as Cyan Fluorescent Protein, Blue Fluorescent Protein, Enhanced Green Fluorescent Protein (EGFP; GFPmutl ; Yang, T. T., et al. (1996) Nucleic Acids Res. 24 (22): 4592-4593;. Cormack, B. P., et al. (1996) Gene 173: 33-38.), Enhanced Blue Fluorescent Protein (EBFP), Enhanced Yellow Fluorescent Protein (EYFP; Ormo, et al. (1996) Science 273: 1392-1395), Destablised Enhanced Green Fluorescent Protein (d2EGFP ; Living Colors Destabilized EGFP Vectors (April 1998) CLONTECHniques XIII (2): 16-17), Enhanced Cyan Fluorescent Protein (ECFP), and GFPuv (Haas, J., et al. (1996) Curr. Biol. 6: 315-324). may also be used. These fluorescent proteins are available from CLONTECH Laboratories, Inc. (Palo Alto, California, USA).

Methods of detecting, quantitating and generally assaying fluorescence are known in the art.

In a preferred embodiment, the reporter is detected by means of Fluorescence Resonance Energy Transfer (FRET). FRET is detectable when two fluorescent labels which fluoresce at different frequencies are sufficiently close to each other that energy is able to be transferred from one label to the other. FRET is widely known in the art (for a review, see Matyus, 1992, J ; Photochen. Photobiol. B : Biol., 12: 323-337, which is herein incorporated by reference). FRET is a radiationless process in which energy is transferred from an excited donor molecule to an acceptor molecule; the efficiency of this transfer is dependent upon the distance between the donor an acceptor molecules, as described

below. Since the rate of energy transfer is inversely proportional to the sixth power of the distance between the donor and acceptor, the energy transfer efficiency is extremely sensitive to distance changes. Energy transfer is said to occur with detectable efficiency in the 1-10 nm distance range, but is typically 4-6 nm for favourable pairs of donor and acceptor.

Accordingly, this embodiment of the delivery assay may be practised by choosing suitable pairs of donor and acceptor molecules, in which the reporter comprises a fluorescent polypeptide which is either a donor or an acceptor. The cell comprises the other of the donor and acceptor, within the intracellular environment (for example, by comprising a nucleic acid sequence encoding a donor or acceptor fluorescent protein).

When the reporters is expressed, the donor molecule and the acceptor molecule are brought together so that energy transfer occurs.

Fluorescent techniques are particularly preferred, as the signal may be detected from outside the cell and allows cell sorting by FACS or other optical sorting techniques.

The reporter may also be detected by detecting the generation of an enzymatic activity, for example, transcriptional activity. In this case, the reporter may be capable of transcriptional activation. The transcriptional activity may be detected by assaying the expression of a reporter gene such as CD4, by fluorescent antibodies and FACs for example.

Alternatively, the reporter may manifest its effects by way of influencing cell growth, cell division or differentiation. The reporter may stably interact with another entity to produce a transcriptional activity, which may activate a program of differentiation or cell growth. For example, VEGF (vascular endothelial growth factor) and/or other angiogenic growth factors may also be expressed to induce angiogenesis. It has been shown that the Lmo2 LIM-only protein is specifically needed for angiogenesis (Yamada et al., 2000, Proc Natl Acad Sci USA 97, 320-324), and accordingly the transcriptional

activity may be Lmo2 activity. Cell growth and detection may be detected by microscopy, detecting appropriate cell surface markers, etc as known in the art.

TRANSGENIC ANIMALS FOR USE IN DELIVERY ASSAYS In a highly preferred embodiment of the delivery assay, the cell for use in a delivery assay is comprised in, or otherwise derived from a transgenic animal. The transgenic animal may comprise one or more such cells; preferably, all of the cells of the transgenic animal comprise a first nucleotide sequence encoding a reporter, and optionally a sequence encoding a first polypeptide. Thus, a transgenic animal for use in a delivery assay is such that it comprises at least one cell comprising a first nucleotide sequence, which interacts with a first polypeptide, to modulate expression of a reporter.

Transgenic animals generally, and their construction, are described in a separate section of this document. These techniques may equally be employed for construction of a transgenic animal expressing a first polypeptide, which interacts with the first nucleotide sequence to modulate expression of the reporter.

Constructs useful for creating transgenic animals useful according to the invention comprise genes encoding reporters, preferably under the control of nucleic acid sequences directing their expression in a tissue specific manner, as well as genes encoding polypeptides capable of interacting with response elements to modulate expression of reporters. Alternatively, the constructs may be under the control of their native promoters, or inducibly regulated.

A transgenic animal expressing one transgene can be crossed to a second transgenic animal expressing second transgene such that their offspring will carry both transgenes. Thus, a transgenic animal carrying a transgene expressing a first polypeptide may be crossed with a transgenic animal carrying a nucleotide sequence encoding a reporter, to produce an animal which carries both transgenes, and in whose cells the

polypeptide may interact with the nucleotide sequence to modulate expression of the reporter.

SENSITISATION The agent whose delivery is being assayed may be delivered to the vicinity of the cell by any means whatsoever. In a particularly preferred embodiment, the agent is loaded into a red blood cell. Accordingly, the method of assaying agent delivery as disclosed here preferably makes use of sensitised red blood cells, as described elsewhere in this document. In a highly preferred embodiment of the delivery assay, sensitisation of red blood cells is achieved by administration of an electric field ; this is known as "electrosensitisation", as described elsewhere in this document.

Furthermore, and as noted above, ultrasound may be used as an energy source to disrupt sensitised red blood cells to effect release of the agent in the vicinity of the cell for assaying delivery.

AGENT The delivery assay as disclosed here is suitable for assaying whether an agent has been delivered to a cell. Such an"agent"may be anything that is capable of modulating the interaction between the first polypeptide and the first nucleotide sequence encoding a reporter to modulate expression of the reporter. The agent may be one which is capable of being loaded in any kind of vehicle whatsoever, for delivery to the cell (for example, and preferably, a red blood cell vehicle).

As noted above, the assays depend on delivery of a"modulator"molecule or a first polypeptide, and the agents whose delivery are to be assayed may be linked, conjugated, fused with or otherwise joined to the"modulator"molecule (or first polypeptide), by means known in the art. Thus, the term"agent"or"entity"in the context of delivery preferably comprises such entities.

For example, the agent may comprise an atom or molecule, wherein a molecule may be inorganic or organic, a biological effector molecule and/or a nucleic acid encoding an agent such as a biological effector molecule, a protein, a polypeptide, a peptide, a nucleic acid, a peptide nucleic acid (PNA), a virus, a virus-like particle, a nucleotide, a ribonucleotide, a synthetic analogue of a nucleotide, a synthetic analogue of a ribonucleotide, a modified nucleotide, a modified ribonucleotide, an amino acid, an amino acid analogue, a modified amino acid, a modified amino acid analogue, a steroid, a proteoglycan, a lipid, a fatty acid and a carbohydrate. An agent may be in solution or in suspension (e. g., in crystalline, colloidal or other particulate form). The agent may be in the form of a monomer, dimer, oligomer, etc, or otherwise in a complex. The agent may be coated with one or more molecules, preferably macromoleucles, most preferably polymers such as PEG (polyethylene glycol). Use of a PEGylated agent increases the circulating lifetime of the agent once released.

The agent may comprise an imaging agent; such an agent may be loaded into a red blood cell to allow the progress and efficiency of introduction of the polypeptide to be monitored. Furthermore, the imaging agent may also be detected within the target cell (s) to determine whether the agent has been delivered. The term"imaging agent"is intended to include an agent which may be detected, whether in vitro in the context of a tissue, organ or organism in which the agent is located. The imaging agent may emit a detectable signal, such as light or other electromagnetic radiation. The imaging agent may be a radio- isotope as known in the art, for example 32P or 35S or 99Tc, or a molecule such as a nucleic acid, polypeptide, or other molecule as explained below conjugated with such a radio- isotope. The imaging agent may be opaque to radiation, such as X-ray radiation, by (for example) having high electron density. The imaging agent may be detected by any means such as magnetic resonance imaging. The imaging agent may also comprise a targeting means by which it is directed to a particular cell, tissue, organ or other compartment within the body of an animal. For example, the agent may comprise a radiolabelled antibody specific for defined molecules, tissues or cells in an organism.

The imaging agent may be combined with, conjugated to, mixed with or combined with, any of the agents or polypeptides disclosed here.

It will be appreciated that it is not necessary for a single agent to be used, and that it is possible to assay the delivery of two or more agents ino a cell. Accordingly, the term "agent"also includes mixtures, fusions, combinations and conjugates, of atoms, molecules etc as disclosed herein. For example, an agent may include but is not limited to: a nucleic acid combined with a polypeptide; two or more polypeptides conjugated to each other; a protein conjugated to a biologically active molecule (which may be a small molecule such as a prodrug); or a combination of a biologically active molecule with an imaging agent.

As used herein, the term"biological effector molecule"or"biologically active molecule"refers to an agent that has activity in a biological system, including, but not limited to, a protein, polypeptide or peptide including, but not limited to, a structural protein, an enzyme, a cytokine (such as an interferon and/or an interleukin) an antibiotic, a polyclonal or monoclonal antibody, or an effective part thereof, such as an Fv fragment, which antibody or part thereof may be natural, synthetic or humanised, a peptide hormone, a receptor, a signalling molecule or other protein; a nucleic acid, as defined below, including, but not limited to, an oligonucleotide or modified oligonucleotide, an antisense oligonucleotide or modified antisense oligonucleotide, cDNA, genomic DNA, an artificial or natural chromosome (e. g. a yeast artificial chromosome) or a part thereof, RNA, including mRNA, tRNA, rRNA or a ribozyme, or a peptide nucleic acid (PNA); a virus or virus-like particles; a nucleotide or ribonucleotide or synthetic analogue thereof, which may be modified or unmodified ; an amino acid or analogue thereof, which may be modified or unmodified; a non-peptide (e. g., steroid) hormone; a proteoglycan; a lipid; or a carbohydrate. Small molecules, including inorganic and organic chemicals, are also of use. In a particularly preferred embodiment, the biologically active molecule is a pharmaceutically active agent, for example, an isotope.

A preferred embodiment of the delivery assay comprises the use of a ribozyme or an oligonucleotide such as an antisense oligonucleotide, which may optionally comprise a membrane translocation sequence (as described elsewhere in this document). Such molecules have the potential to disrupt the expression of the first polypeptide, as described elsewhere.

Particularly useful classes of biological effector molecules include, but are not limited to, antibiotics, anti-inflammatory drugs, angiogenic or vasoactive agents, growth factors and cytotoxic agents (e. g., tumour suppressers). Cytotoxic agents of use include, but are not limited to, diptheria toxin, Pseudomonas exotoxin, cholera toxin, pertussis toxin, and the prodrugs peptidyl-p-phenylenediamine-mustard, benzoic acid mustard glutamates, ganciclovir, 6-methoxypurine arabinonucleoside (araM), 5-fluorocytosine, glucose, hypoxanthine, methotrexate-alanine, N- [4- (a-D-galactopyranosyl) benyloxycarbonyl]-daunorubicin, amygdalin, azobenzene mustards, glutamyl p- phenylenediamine mustard, phenolmustard-glucuronide, epirubicin-glucuronide, vinca- cephalosporin, phenylenediamine mustard-cephalosporin, nitrogen-mustard-cephalosporin, phenolmustard phosphate, doxorubicin phosphate, mitomycin phosphate, etoposide phosphate, palytoxin-4-hydroxyphenyl-acetamide, doxorubicin-phenoxyacetamide, melphalan-phenoxyacetamide, cyclophosphamide, ifosfamide or analogues thereof. If a prodrug is loaded in inactive form, a second biological effector molecule may be loaded into the red blood cell. Such a second biological effector molecule is usefully an activating polypeptide which converts the inactive prodrug to active drug form, and which activating polypeptide is selected from the group that includes, but is not limited to, viral thymidine kinase (encoded by Genbank Accession No. J02224), carboxypeptidase A (encoded by Genbank Accession No. M27717), a-galactosidase (encoded by Genbank Accession No.

M13571), B-glucuronidase (encoded by Genbank Accession No. M15182), alkaline phosphatase (encoded by Genbank Accession No. J03252 J03512), or cytochrome P-450 (encoded by Genbank Accession No. D00003 N00003), plasmin, carboxypeptidase G2, cytosine deaminase, glucose oxidase, xanthine oxidase, B-glucosidase, azoreductase, t- gutamyl transferase, B-lactamase, or penicillin amidase.

Preferably the biological effector molecule is selected from the group consisting of a protein, a polypeptide, a peptide, a nucleic acid, a virus, a virus-like particle, a nucleotide, a ribonucleotide, a synthetic analogue of a nucleotide, a synthetic analogue of a ribonucleotide, a modified nucleotide, a modified ribonucleotide, an amino acid, an amino acid analogue, a modified amino acid, a modified amino acid analogue, a steroid, a proteoglycan, a lipid and a carbohydrate or a combination thereof (e. g., chromosomal

material comprising both protein and DNA components or a pair or set of effectors, wherein one or more convert another to active form, for example catalytically).

The first polypeptide and/or the reporter may be encoded by a nucleic acid, and described in detail elsewhere in this document.

Further Aspects Paragraph 1. A method of assaying delivery of an agent to a cell, the method comprising the steps of : (a) providing a cell comprising a first nucleotide sequence encoding a reporter, in which the cell expresses a first polypeptide capable of interacting directly or indirectly with the first nucleotide sequence to modulate expression of the reporter; (b) contacting the cell with an agent capable of modulating the interaction between the first polypeptide and the first nucleotide sequence; and (c) detecting the reporter.

Paragraph 2. A method according to Paragraph 1, in which the first polypeptide interacts with the first nucleotide sequence to promote expression of the reporter.

Paragraph 3. A method according to Paragraph 1, in which the first polypeptide interacts with the first nucleotide sequence to inhibit expression of the reporter.

Paragraph 4. A method according to any preceding Paragraph, in which the first polypeptide interacts with an operator sequence in the first nucleotide sequence.

Paragraph 5. A method according to any preceding Paragraph, in which the agent is capable of inhibiting the interaction between the first polypeptide and the first nucleotide sequence.

Paragraph 6. A method according to any preceding Paragraph, in which the agent modulates the interaction between the first polypeptide and the first nucleotide sequence

by: (a) modulating the transcription of an mRNA encoding the first polypeptide; (b) modulating the transport, processing, splicing, stability, turnover or degradation of a mRNA encoding the first polypeptide; (c) modulating the translation of an mRNA encoding the first polypeptide; (d) modulating the transport, processing, post-translational modification, stability, turnover or degradation of the first polypeptide; or (e) sterically hindering the interaction between the first polypeptide and the first nucleotide sequence.

Paragraph 7. A method according to any preceding Paragraph, in which the agent comprises an antisense nucleic acid which binds to a nucleotide sequence encoding the first polypeptide and thereby inhibits its expression.

Paragraph 8. A method according to any preceding Paragraph, in which the antisense nucleic acid comprises an antisense RNA capable of binding to a messenger RNA encoding the first polypeptide.

Paragraph 9. A method according to any preceding Paragraph, in which the first nucleotide sequence comprises a sequence capable of directing tissue specific expression of the reporter.

Paragraph 10. A method according to any preceding Paragraph, in which: (a) the first polypeptide comprises a Tet repressor (TetR), and the first nucleotide sequence comprises a Tet-responsive element (TRE); (b) the first polypeptide comprises an oestrogen receptor, and the first nucleotide sequence comprises an oestrogen responsive element (ORE); or (c) the first polypeptide comprises an ecdysone receptor, and the first nucleotide sequence comprises an ecdysone responsive element (EcRE).

Paragraph 11. A method according to Paragraph 10, in which the first polypeptide comprises a transcriptional activator domain, preferably selected from a VP16, a VP64, a maize Cl, and a PI domain, or a transcriptional repressor domain, preferably selected from a KRAB-A domain, an engrailed domain or a snag domain.

Paragraph 12. A method according to any preceding Paragraph, in which the first nucleotide sequence encodes a reporter selected from the group consisting of : a fluorescent protein, luciferase, P-galactosidase, or chloramphenicol acetyl transferase (CAT).

Paragraph 13. A method according to any preceding Paragraph, in which the reporter comprises a fluorescent protein selected from the group consisting of : a Green Fluorescent Protein, a Cyan Fluorescent Protein, a Yellow Fluorescent Protein, a Blue Fluorescent Protein and a Red Fluorescent Protein.

Paragraph 14. A method according to any preceding Paragraph, in which the reporter is detected by detecting fluorescent resonance energy transfer (FRET).

Paragraph 15. A method according to any preceding Paragraph, in which the agent is provided as an agent-MTS (membrane translocation sequence) conjugate, or in which the agent is provided as a virus or a virus-like particle comprising the agent.

Paragraph 16. A method according to any preceding Paragraph, in which the agent is loaded into a red blood cell for delivery to the cell.

Paragraph 17. A method according to Paragraph 16, in which the red blood cell is sensitised.

Paragraph 18. A method according to Paragraph 16 or 17, in which the agent is released from the red blood cell by application of ultrasound energy.

Paragraph 19. A method according to any preceding Paragraph, in which the cell is provided from a transgenic animal carrying and expressing a transgene encoding the first polypeptide and a transgene comprising the first nucleotide sequence.

Paragraph 20. A method according to Paragraph 19, in which the transgenic animal is selected from the group consisting of : mouse, rat, rabbit, sheep, goat, cow, and pig.

Paragraph 21. A method according to any preceding Paragraph, in which the cell forms part of an animal and the reporter is detected in situ in the animal.

Paragraph 22. A method according to any preceding Paragraph, in which the cell forms part of a tissue mass grafted onto a host animal.

Paragraph 23. A method according to any preceding Paragraph, in which the reporter is expressed under the control of a tissue specific promoter or enhancer, preferably a Locus Control Region (LCR).

Paragraph 24. A method according to any preceding Paragraph, in which expression of the reporter is substantially restricted to vascular endothelial cells.

EXAMPLES Example 1. Production of Transgenic Animals In one embodiment, the RBCs which carry and express the polypeptide are derived from transgenic animals which carry and express the transgene encoding the polypeptide.

Transgenic technology is standard in rodents, and is also well known in higher mammals.

Clearly, for the large scale production of polypeptides either for isolation in pure form or for in vivo delivery using transgenic RBCs, larger animals are preferred. Among the larger mammals, pigs are particularly preferred due to cardiovascular and vascular similarities with humans. The following example is intended to provide one skilled in the art with an understanding of the general procedures employed to produce a transgenic pig.

Estrus is synchronized in sexually mature gilts (>7 months of age) by feeding an orally active progestogen (allyl trenbolone, AT: 15 mg/gilt/day) for 12 to 14 days. On the last day of AT feeding all gilts are given an intramuscular injection (IM) of prostaglandin F2a (Lutalyse: 10 mg/injection) at 0800 and 1600. Twenty-four hours after the last day of AT consumption all donor gilts are given a single IM injection of pregnant mare serum gonadotropin (PMSG: 1500 IU). Human chorionic gonadotropin (HCG: 750 IU) is administered to all donors at 80 hours after PMSG.

Following AT withdrawal, donor and recipient gilts are checked twice daily for signs of estrus using a mature boar. Donors which exhibit estrus within 36 hours following HCG administration are bred at 12 and 24 hours after the onset of estrus using artificial and natural (respectively) insemination.

Between 59 and 66 hours after the administration of HCG, one-and two-cell ova are surgically recovered from bred donors using the following procedure. General anesthesia is induced by administering 0.5 mg of acepromazin/kg of bodyweight and 1. 3 mg ketamine/kg of bodyweight via a peripheral ear vein. Following anesthetization, the reproductive tract is exteriorized following a midventral laparotomy. A drawn glass cannula (O. D. 5 mm, length 8 cm) is inserted into the ostium of the oviduct and anchored to the infundibulum using a single silk (2-0) suture. Ova are flushed in retrograde fashion by inserting a 20 g needle into the lumen of the oviduct 2 cm anterior to the uterotubal junction. Sterile Dulbecco's phosphate buffered saline (PBS) supplemented with 0.4% bovine serum albumin (BSA) is infused into the oviduct and flushed toward the glass canula. The medium is collected into sterile 17 X 100 mm polystyrene tubes. Flushings are transferred to 10 X 60 mm petri dishes and searched at lower power (50X). All one- and two-cell ova are washed twice in Brinster's Modified Ova Culture-3 medium (BMOC- 3) supplemented with 1.5% BSA and transferred to 50 ml drops of BMOC-3 medium under oil. Ova are stored at 38°C. under a 90% N2,5% Oz, 5% COa atmosphere until microinjection is performed.

One-and two-cell ova are placed in an Eppendorf tube (15 ova per tube) containing 1 ml HEPES Medium supplemented with 1.5% BSA and centrifuged for 6

minutes at 14000 X g in order to visualize pronuclei in one-cell and nuclei in two-cell ova.

Ova are then transferred to a 5-10 ml drop of HEPES medium under oil on a depression slide. Microinjection is performed using a Laborlux microscope with Nomarski optics and two Leitz micromanipulators. 10-1700 copies of a DNA construct which includes the gene which encodes the polypeptide of interest operably linked to a promoter as described above (1 ng/ml in Tris-EDTA buffer) are injected into one pronucleus in one-cell ova or both pronuclei in two-cell ova.

Microinjected ova are returned to microdrops of BMOC-3 medium under oil and maintained at 38°C. under a 90% N2, 5% CO2, 5% 02 atmosphere prior to their transfer to suitable recipients. Ova are transferred within 10 hours of recovery.

Only recipients which exhibit estrus on the same day or 24 hours later than the donors are utilized for embryo transfer. Recipients are anesthetized as described above.

Following exteriorization of one oviduct, at least 30 injected one and/or two-cell ova and 4-6 control ova are transferred in the following manner. The tubing from a 21 g X 3/4 butterfly infusion set is connected to a 1 cc syringe. The ova and one to two mls of BMOC-3 medium are aspirated into the tubing. The tubing is then fed through the ostium of the oviduct until the tip reached the lower third or isthmus of the oviduct. The ova are subsequently expelled as the tubing is slowly withdrawn.

The exposed portion of the reproductive tract is bathed in a sterile 10% glycerol/0.9% saline solution and returned to the body cavity. The connective tissue encompassing the linea alba, the fat and the skin are sutured as three separate layers. An uninterrupted Halstead stitch is used to close the linea alba. The fat and skin are closed using a simple continuous and mattress stitch, respectively. A topical antibacterial agent (Furazolidone) is then administered to the incision area.

Recipients are penned in groups of four and fed 1.8 kg of a standard 16% crude protein corn-soybean ration. Beginning on day 18 (day 0 = onset of estrus), all recipients are checked daily for signs of estrus using a mature boar. On day 35, pregnancy detection

is performed using ultrasound. On day 107 of gestation recipients are transferred to the farrowing suite. In order to ensure attendance at farrowing time, farrowing is induced by the administration of prostaglandin F2a, (10 mg/injection) at 0800 and 1400 hours on day 112 of gestation. Recipients should farrow within about 34 hours of PGF2a administration.

Tissue and/or blood samples from the potentially transgenic offspring can then be examined by a number of methods known to those skilled in the art for the expression of the transgene.

Example 2. RBC Preparation and Delivery The following example describes procedures employed to produce, harvest, electrosensitize, and deliver RBCs expressing a polypeptide.

Briefly, 10 ml of peripheral venous blood is collected from the transgenic animal donor by venipuncture, into lithium heparin anticoagulant containing tubes, and mixed gently. The whole blood is then poured into a polypropylene tube and centrifuged at 300g for 15min at room temperature. The plasma and white blood cells (buffy coat) are removed.

1X phosphate buffered saline (PBS, made from Oxoid tablets, code BR14a, pH7.3) is added and the cells are centrifuged at 700g for 5min. The supernatant is removed and the pellet of remaining cells resuspended in ice cold 1X PBS. The spin/wash procedure is then repeated once, and cells are suspended in ice-cold PBS at 6x108 cells/ml. Steps may be taken at this stage, if desired or necessary, to mask antigenic epitopes on the surface of the RBCs prior to administration. This includes, for example, treatment with mPEG as known in the art (Scott et al., 1997, Proc. Natl. Acad. Sci. U. S. A., 94: 7566-7571).

Cells are then electrosensitized by dispensing 800T1 of the RBC into sterile electroporation cuvettes, and placed on ice. To electrosensitize the cells, they are exposed to an electric field at 3.625kV/cm, 1TF (2 pulses). The RBCs are then removed, and pooled in polypropylene tubes.

Cells are centrifuged once at 700g for 5min at room temperature (RT). The cells may be diluted in PBS. Cells are then resuspended in PBS, and centrifuged at 700g for 5min, twice. Finally, cells are resuspended in PBS, at approximately 7xl08cells/ml, and rested for 30min at room temperature.

Stock potassium phosphate buffer: 5mM K2HP043H20 (FW 228.2g) => 141g/L ; 5mM KH2PO4 (MW136. 1g) =>0. 68g/L. Stored at 4°C. Mix as follows: For a pH7. 4 K2H/KH2 phosphate buffer =>approx. 6.1: 3.9 parts. Mix the 2 stock solutions as and when required Isoosmotic PBS: pH7.4 K2H/KH2 phosphate buffer. 150mM NaCl => 8.76g/L.

Check and adjust pH (1M NaOH) Following electrosensitization, sensitized RBCs are infused into either the venous or arterial circulation of a mammal at a concentration of 7x10 cells/ml, where the volume of RBC cell suspension infused will vary with the size of the recipient mammal.

Approximately one hour after sensitized RBC infusion, the mammal will be treated with ultrasound at an energy of 1.25W/cm2 using a 3MHz probe for 30s. Tissue samples from treated mammals can then be assayed for the presence of the polypeptide by methods routinely used in the art.

Example 3. Light-Mediated Release of a Transgene Product from Photosensitised Erythroyctes Derived from a Transgenic Mouse Line In these experiments the objective is to determine whether or not it is possible to photosensitise erythrocytes containing a transgene product and to facilitate light activated release of that product.

A transgenic mouse line containing the E. coli ! p-galactosidase gene inserted downstream from the globin LCR/ß-globin promoter is obtained from the Dept. of Cell Biology and Genetics, Erasmus University, Rotterdam, Holland. The construction of this

trangenic mouse line is described in Collis & Grosveld [1990], EMBO J. 9,233-240; and Grosveld et al., 1987, Cell, 51,975-985. Erythrocytes harvested from these animals have been shown to contain functional p-galactosidase enzyme activity.

In order to facilitate light-activated release of enzyme from the cells, erythrocytes are harvested by cardiac puncture and treated as follows: Cells are washed by centrifugation in phosphate buffered saline (PBS) and diluted to yield a concentration of 6.6x108 cells/ml. 0.4ml aliquots of cells are mixed together with 0.4ml aliquots of hematoporphyrin derivative (HPD) (Dougherty et al., 1984 In"Porphyrin Localisation and Treatment of Tumours", Eds: Doirin, D. R. & Gower, C. J., Published By Alan R. Liss, N. Y. p302) at a concentration of lmg/ml. Samples are protected from light for a period of 1 hour during which photosensitisation is accomplished.

Samples are subsequently washed by centrifugation and suspended in PBS to yield a cell concentration of 7.2x108 cells/ml. In order to achieve activation and payload release, samples are aliquoted into the wells of a 96-well plate (O : lml per well) and individually exposed to radiation emitted by a HeNe laser (1 OmW) for the periods of time indicated in Figure 1. The HeNe laser is placed at a distance of 17cm during exposures and samples are retained for 2 hours following irradiation. Cell concentrations are then determined by direct counting and P-galactosidase activity is determined by assaying at 37°C using the colorimetric substrate p-nitrophenyl-p-D-galactopyranoside. The absorbance at 405nm is determined using a spectrophotometer. Enzyme activities in these experiments are expressed as a percentage of the overall quantity of enzyme recovered from control populations of cells following lysis by immersion in distilled water and subsequent freeze thaw cycles (x3). Control samples consist of sensitised cells in the absence of light and are represented by the zero irradiation point in Figure 1. In addition, samples of non-sensitised cells are exposed to laser irradiation up to 10 minutes The results obtained are shown in Figure 1 and they demonstrate that light- activated cell lysis increased with increasing exposure to the HeNe laser, reaching a maximum at approx. 5 minutes exposure. When cell lysates are assayed for the presence

of the transgene product (i. e. p-galactosidase activity), enzyme activity released from the cells increased with increasing exposure to laser radiation. Release of activity increased to a maximum at exposures of approx. 2.5minutes. The maximum amount of activity recovered in these studies following exposure to light is about 75% of the maximum detected in cells which are lysed using distilled water and freeze/thaw cycles. This probably results from entrapment of enzyme in cell fragments. In addition, the zero radiation point on the curve indicates a limited degree of lysis and enzyme release and this is the result of light leakage into the cell preparations during experimentation. Exposure of non-sensitised cells to laser radiation for up to 10 minutes fails to result in either cell lysis or enzyme release.

The results clearly demonstrate activated cell lysis and transgene product release from erythrocytes derived from a transgenic animal, following treatment with laser irradiation conditions which have no effect on normal cells.

Example 4. Ultrasound-Mediated Release of a Transgene Product from Sensitised Erythrocytes Derived from a Transgenic Mouse Line In order to demonstrate that the transgene product can also be released from the erythrocytes using ultrasound, we electrosensitised the cells and examined enzyme release following direct exposure of those sensitised cells to increasing ultrasound power densities at 3MHz.

Cells are harvested as described for Example 3 above and suspended at a concentraton of 6.5x108 cells/ml prior to electrosensitisation. 0.8ml aliquots of cells are dispensed into electroporation cuvettes (0.4cm electrode gap) and retained on ice for 1 Omin. Samples are electrosensitised by exposure to double pulses at 1.46kV at a capacitance of 1 : F and generated by a Biorad GenePulsar I apparatus. Cells are then recovered by centrifugation, suspended in PBS containing 4mM MgCla and retained at room temperature for 30 minutes. Cells are then washed by centrifugation, suspended in PBS containing 4mM MgCl2/10mM glucose and retained at room temperature for 1 hour.

Control cell populations are treated as described above except that the electrosensitisation step is omitted. Cell preparations are counted directly and the cell recoveries following electrosensitisation are determined by comparing with the untreated population of cells. In addition, samples of supernatant are retained following electrosensitisation in order to determine whether or not the transgene product galactosidase) leaks from the cells during that procedure. Following counting, all cell suspensions are adjusted to between 6.7 and 6.9 X108 cells/ml for ultrasound treatment and the latter is performed by placing 100. 1 aliquots of the suspensions directly in contact with a 3MHz ultrasound head. Cells are exposed to increasing ultrasound power densities for 30 sec., as are samples of non-sensitised control cell populations. Following treatment, cell lysis is determined by direct counting and activated release of transgene product (P- galactosidase) is determined by assaying lysates for the enzyme as described for Example 3. Enzyme activity is expressed as a percentage of the total enzyme released from control cells following lysis by immersion of cells in distilled water and freeze/thaw cycling (x3).

Since exposure of cells to electric fields may result in transient permeabilisation of the cell membrane it is of interest to determine whether or not significant quantities of transgene product leak from the cells during the electrosensitisation procedure. When cells are electrosensitised the amount of enzyme released into the medium during the process is determined as is the total content of enzyme before and after electrosensitisation. Cell recoveries following electrosensitisation are in the region of 70% and it is found that the medium harvested following electrosensitisation contains approximately 5% of the overall enzyme contained within the cells prior to treatment. This demonstrates that electosensitisation does not result in significant leakage of the transgene product from the erythroyctes.

The results obtained following ultrasound treatment of electrosensitised and normal erythrocytes derived from the transgenic animal are shown in Figure 2 and they demonstrate that cell lysis of the electrosensitised cells increases with increasing ultrasound power density, reaching a maximum of 90% lysis at 1.5W/cm2. Treatment of control non-sensitised cells (derived from the transgenic animals) with similar ultrasound

conditions fails to elicit any cell lysis (Figure 2). In addition when lysates obtained following treatment of the electrosensitised cells with increasing ultrasound power densities are assayed for the transgene product (p-galactosidase activity) enzyme release appears to mimic cell lysis in terms of increased enzyme release with increasing ultrasound power density (Figure 2). It is interesting to note that greater than 100% of the enzyme is recovered from the erythrocytes following treatment with ultrasound at power densities of 1.25 and 1.5W/cm2 and we believe that this may be due to more efficient cell lysis achieved using ultrasound. When normal non-sensitised cells (derived from the transgenic animals) are treated with similar ultrasound conditions, no enzyme is detected in supernatants harvested following treatment.

These results clearly demonstrate that ultrasound-mediated release of the transgene product is achieved using ultrasound conditions which have no effect on normal non- sensitised erythrocytes derived from the transgenic animals.

Example 5. Ultrasound-Mediated-Release of Transgene Product from Cells Derived from a Transgenic Animal During Treatment in a Tissue Mimicking Medium Since Example 4 demonstrates that transgene product can be actively released from erythrocytes derived from a transgenic animal following direct contact with the ultrasound head, we determined whether or not this might also be achievable at a distance from the ultrasound head. In addition, we performed the latter using a system which incorporated a tissue mimicking material between the sample and the emitting surface of the ultrasound head.

Cells are therefore harvested from the transgenic animals and electrosensitised as described above in Example 4. In these experiments the target cell populations are placed at a distance of 1.3cm from the emitting surface of the ultrasound head and the intervening space is filled with a tissue mimicking material (TMM) which attenuates ultrasound in the same manner as a soft tissue. The TMM chosen for this work is described in Madsen et al.

(1998, Ultrasound Med. & Biol., 24,535-542) and following preparation, care is taken to

ensure that the material has a density of 1.03g/ml. Cells are treated with ultrasound (lMHz) for 35 sec. at increasing power densities and both cell lysis and transgene product release is determined as described for Example 4 above.

The results obtained from these experiments are shown in Figure 3 and they demonstrate that cell lysis in the tissue mimicking system increases with increasing ultrasound power density as does release of the transgene product. Again the amount of enzyme recovered at treatments of 3W/cm2 is slightly greater than 100% and we believe that this may be due to more efficient cell lysis using ultrasound. No cell lysis or enzyme release is detected when non-sensitised cells are treated with ultrasound on the system at 3W/cm2.

The results clearly demonstrate release of the transgene product even when the activating ultrasound is transmitted through 1. 3cm of a tissue mimicking material. In addition the release of transgene product is facilitated using ultrasound conditions which had no effect on non-sensitised erythrocytes derived from the transgenic animals.

Example 6. Electrosensitisation of Pig Erythroyctes to Ultrasound The objective of the concept outlined above is to exploit'ready loaded' erythrocytes from transgenic animals in humans. The possibility of exploiting transgenic porcine organs for xenotransplantation in humans has become more realistic with recent advances such as producing transgenic pigs expressing human complement-regulatory proteins (Logan, 2000, Curr. Opin. Immunol., 12,563-568; Cozzi et al., 2000, Transplantation, 15,12-21). It is therefore possible to produce the'ready loaded' erythrocytes in transgenic pigs and subsequently employ those as vehicles for delivery of therapeutic agents in humans.

In this example cells are harvested from pigs, sensitised and subsequently treated with ultrasound to determine whether or not they exhibit hypersensitivity to ultrasound.

The protocol employed is as briefly set out here. The cells are washed and subjected to

electrosensitising pulses, hypotonic dialysis and rested overnight. Cell are again subjected to electrosensitising pulses and suspended at a concentration of 7x108 cells/ml for ultrasound treatment. Control cells are untreated but exposed to all buffers (except the hypotonic buffer) and washes employed in the protocol. Samples are then stored at 4°C for 4 days. Ultrasound treatment is performed as described above for Example 5 and this involves the use of the tissue mimicking system. Following ultrasound treatment cell lysis is determined by direct counting.

The results obtained following treatment of sensitised porcine erythrocytes with increasing ultrasound power density are shown in Figure 4. The results demonstrate that ultrasound-mediated lysis of the sensitised cells increases with increasing ultrasound power density. In addition, these conditions of ultrasound have no effect on normal porcine erythrocytes. These results demonstrate that it is possible to render'ready-loaded' erythrocytes from transgenic pigs sensitive using the technology described here. This offers potential for the production of'humanised'transgenic porcine erythrocyte-based delivery vehicles containing transgene encoded therapeutic agents.

Example 7. Sensitization of Transgenic Murine Erythrocytes Over a Range of Voltages and Examination of Enzyme Release During Electroporation This example describes the sensitisation of murine erythrocytes from a transgenic mouse (as described above in Example 3). These erythrocytes are examined to determine whether they can be effectively sensitised using electricity, and whether such electrosensitised transgenic red blood cells are capable of being lysed effectively to allow release of the contents of the RBCs.

A 10 ml LiHep tube is filled with PBS and mixed. 5ml of this is used to collect the transgenic murine erythrocytes. Blood is taken by posthumous cardiac puncture. The cells are centrifuged for 15 mins at 1300 RPM and the supernatant transferred to a fresh tube for assay. The cells are washed twice in PBS and re-suspended to approximately 5ml, cell count = 7.1 x 108 cells/ml. The cells are electrosensitized over a range of voltages by administration of two pulses at the appropriate voltage at ljj. F, 4°C. No. Voltage Cell counts Resuspended Counts for US prior to ES 0 Non-ES control 7.1 NA 7.05 x 108 cells/ml 1 1KV 7.1 5001 7. 0x10 cells/ml 2 1.25KV 7.1 5501 6.8 x 108 cells/ml 3 1.45KV 7.1 400tl 7. 15 x 108 cells/ml 4 1.8KV 7.1 250p1 6.9 108 cells/ml 5 2KV 7.1 2001 6. 8x10 cells/ml

The cells are then centrifuged and the supernatants transferred to fresh tubes for assay (labelled 0-5 as above). The cells are washed twice in PBS/MgClz and rested for 30mins in PBS/MgCl2. After this time the cells are again centrifuged and the supernatant again discarded. The cells are washed twice in mBax buffer and rested for one hour in mBax. After this the cells are resuspended to the above volumes to approx 7 x 108 cells/ml.

100u. l of each sample is taken and lysed by freeze thaw in TWEEN containing buffer.

A 100111 aliquot of the cells is taken for ultrasound treatment in the TMM (tissue mimicking medium), 3 W/cm-, I MHz ultrasound for 35 seconds. Count safter treatment are taken and percentage lysis calculated. The sample is centrifuged and the supernatant transferred to fresh tubes. No. Sample Counts before US Counts after US Percentage (electroporation lysis voltage) 0 NES 7. 1 x 108 cells/ml 6.85 x 108 cells/ml 3.5% 1 1KV 7. 2 x 108 cells/ml 4 x 108 cells/ml 43.7% 2 1.25KV 7. 05 x 108 cells/ml 0 x 108 cells/ml 100% 3 1.45KV 6.9 x 108 cells/ml 0 x 108 cells/ml 100% 4 1*8KV 7 x 108 cells/ml 0 x 108 cells/ml 100% 5 2KV 7.05 x 108 cells/ml 0 x 108 cells/ml 100% No. Samples mABS/Percentage of % Calc. from min Highest mABS (i. e., Lysed 1.24 kV lysed by by F/T for US) each sample 0 NES : Supernatant after ES 0.175 2. 3% 5.99% 1 lKV : Supernatant after ES-0.092 0% 0% 2 1. 25KV: Supernatant after ES 0. 099 1. 3% 2.2% 3 1. 45KV: Supernatant after ES 0. 008 1% 0. 1% 4 1. 8KV: Supernatant after ES 0. 568 7. 5% 8.8% 5 2KV : Supernatant after ES-0.620 0% 0% 6 NES Control Lysed by freeze/thaw 2.923 38.7% N/A 7 lav : Lysed by freeze/thaw 4. 832 64% N/A 8 1. 25KV: Lysed by freeze/thaw 4. 564 60. 4% N/A 1. 45KV : Lysed by freeze/thaw 7. 556 100% N/S 10 1. 8KV: Lysed by freeze/thaw 6. 409 84. 8% N/A 11 2KV : Lysed by freeze/thaw 33844. 2% N/A 12 NES Control : Supernatant after US 0.738 9.8% 25% 13 lKV : Supernatant after US 2. 642 35% 54.7% 14 1. 25KV: Supernatant after US 6. 872 90. 9% 150.5% 15 1. 45KV: Supernatant after US 3. 282 43. 4% 43.4% 16 1. 8KV : Supernatant after US 2. 372 31. 4% 37% 17 2KV : Supernatant after US 1. 029 13. 6% 30.8% Figure 5 shows the results of the experiment. This Figure, as well as the above results, show that very little enzyme is released during the electrosensitisation process.

Furthermore, it is clear that the transgenic red blood cells can successfully be made sensitive to ultrasound over a range of voltages, with effective cell lysis and enzyme release.

Example 8. A Gal-ER-VP16 in vitro Assay System for Release/Delivery of an Entity to a Target Cell Line In this example the Gal-ER-VP 16 activator protein is loaded into the ultrasound- sensitive vehicle, a red blood cell. Gal-ER-VP16 chemically coupled to HIV TAT is also loaded into a red blood cell. A gene product resulting from expression of a gene fusion encoding GAL-ER-VP16 and HIV TAT (Braselmann et al., 1993, Proc. Natl. Acad. Sci.

U. S. A. 90,1657-1661) is also loaded into the ultrasound-sensitive vehicle.

The target for assaying release or delivery of the loaded entity consists of NIH 3T3 cells transfected with constructs consisting of a gene sequence encoding GFP downstream from the transactivator target promoter (Braselmann et al., 1993, Proc. Natl. Acad. Sci.

U. S. A. 90,1657-1661). A positive fluorescent signal from the cells following exposure to ultrasound-mediated lyses in the presence of oestrogen is indicative of both release of the GAL-ER-VP16 payload and delivery of this entity into the target cell.

To the above ends, recombinant Gal-ER-VP16 protein is produced and purified preparations of this protein (or the described derivatives thereof) are loaded into ultrasound-sensitive human erythrocytes as described previously (WO0158431). A conjugate comprising Gal-ER-VP16 chemically coupled to HIV TAT or a fusion product (Gal-ER-VP16-HIV TAT) is also loaded as described in WO0158431.

0.2ml preparations of the loaded and sensitised erythrocytes are exposed to ultrasound at 2.5W/cm2 using a tissue mimicking system as described previously (WOO 107011). These lysates are then stored for future use as described in that document.

As a target system, NIH3T3 cells are transfected with constructs containing a reporter GFP-encoding sequence downstream from a synthetic Gal4-responsive promoter.

The latter consists of four Gal4-binding sites, an inverted CCAAT element, TATA box and the adenovirus major late initiation region fused to the strong activating domain of the

herpes virus protein VP16 as described previously. (Braselmann et al., 1993, Proc. Natl.

Acad. Sci. U. S. A. 90,1657-1661).

Transfected cells are grown to confluence and exposed to 0. lml aliquots of the lysates containing the activator proteins in the presence of oestrogen (lllM 17 P-estradiol).

Cells are analysed for oestrogen-dependant expression of the GFP using fluorescent microscopy.

Results 8 In cell samples which are exposed to erythrocyte lysates containing either Gal-ER- VP16 or this entity fused to HIV TAT no significant fluorescence is observed. However, in cell preparations that are exposed to erythrocyte lysates containing either entity in the presence of oestrogen, strong fluorescence is observed. Of the latter, cells exposed to the transactivator fused to or chemically linked to HIV TAT exhibit both the highest degree of fluorescence and the greatest number of cells which are fluorescent.

The results demonstrate that Gal-ER-VP16 is functionally released from the erythrocytes and this has a positive effect on expression of the target gene in the presence of oestrogen. The results also demonstrate that when the membrane translocating peptide, HIV TAT is fused to the Gal-ER-VP16, again a positive effect on expression is observed in the presence of oestrogen, although in this case a greater number of cells in the target population appear fluorescent. The latter indicates that the addition of the membrane translocation peptide enhances uptake of the hormone responsive element and further indicates that such a system can be employed to assay delivery of either the hormone or hormone responsive entity in vitro.

Example 9. A Gal-ER-VP16 ira vivo Assay System for Release/Delivery of an Agent to a Target in a Transgenic Animal In this example a similar approach to that described above is employed except that the target is transgenic with respect to both the promoter and reporter sequences.

In this case the activator is loaded into the vehicle and this is injected into a target transgenic mouse. Release/delivery of the activator (Gal-ER-VP 16, that protein chemically coupled to the HIV TAT peptide or a gene fusion product in which the partners consist of Gal-ER-VP 16 and HIV-TAT peptide) from the vehicle following treatment with ultrasound will is indicated by a fluorescent signal at the treatment site as a result of the presence of endogenous oestrogen.

Transgenic mice are generated as described in the literature (Wang et al., 1999, Proc. Natl. Acad. Sci. U. S. A., 96,8483-8488) using a construct containing a GFP reporter gene downstream from the Gal4-responsive reporter as described above for Example 8.

Ultrasound-sensitive and loaded mouse erythrocytes are prepared and injected into the transgenic target.

Animals are anaesthetised using 2% isofluorane in an oxygen carrier at a flow rate of 2L/min. Animals are treated over the kidney region with ultrasound for 5 min. using a frequency of lMHz and a power density of 6W/cm2. The ultrasound is delivered in pulsed mode at 35% continuous wave. Animals are allowed to recover for a period of 12h after treatment and both the treated and untreated kidneys are subsequently harvested from animals. Organs are fixed, sectioned and subsequently examined using fluorescence microscopy.

Results 9 Examination of the untreated kidney using fluorescent microscopy fails to show a fluorescent signal whereas the ultrasound treated kidney yields a strong fluorescent signal.

In the cases where the activator either chemically coupled to HIV TAT or the activator- HIV TAT fusion produced are employed as the payload, the fluorescent signal is much more intense. These results indicate that such a system can be employed as a convenient means of assaying delivery payload to a target site in vivo.

Example 10. A Gal-ER-VP16 in vitro Assay System for Release/Delivery of an Entity (Oestrogen) to a Target Cell Line In the following example a similar approach is taken to assay delivery of an agent to a target cell except that in this case the inducer of gene expression, rather than the activator of gene expression is employed as the payload in the vehicle.

In this case oestrogen is loaded into the erythrocyte and release of payload is again monitored in the target. However, in this case the target cell line contains one or more gene constructs encoding both the activator (Gal-ER-VP16) and the promoter-reporter sequences. In this case introduction of the inducer (oestrogen) to the target cell results in an interaction between Gal-ER-VP 16 and oestrogen and this results in an interaction between the activator-oestrogen complex and the promoter sequence. This subsequently results in enhanced transcription of the reporter and appearance of a fluorescent signal.

In this case, the target cell line is produced by transfecting NIH 3T3 cells with a construct encoding the Gal-ER-VP16 activator and the promoter sequence upstream from the GFP encoding sequence. Human erythrocytes (from a male donor) are loaded with 17 p-estradiol as described previously (WO0158431) and the sensitised vehicle is exposed to ultrasound as described for Example 8. Lysates are then placed in contact with the transfected cells for 24h and cell populations are then analysed for expression of GFP using fluorescent microscopy.

Results 10 When cells are exposed to lysates prepared from control unloaded human erythrocytes and subsequently examined using fluorescence microscopy, no significant fluorescent signal is detected. However, when target cells are exposed to lysates from oestrogen-loaded cells a fluorescent signal is detected. These results demonstrate that this method provides a convention means of measuring delivery of an entity to a target cell line.

A corresponding experiment, in which the NIH-3T3 cells are transfected with two separate constructs, one encoding the Gal-ER-VP16 activator and the other construct comprising promoter sequence upstream from the GFP encoding sequence, is conducted.

Similar results to those described above are observed.

Example 11. Tet-On Based Assay of Delivery of an Agent to a Target Site ill vivo In this example the Tet-On system as described earlier in this document is employed and ultrasound mediated delivery of tetracyclin is assayed using a Lac Z reporter gene at the target site.

In this system transgenic mice are generated in which the Tet-On transactivator is under the control of the human cytomegalovirus promoter and the responsive element is used to drive expression of the lacZ reporter gene (Puttini et al., 2001, Am. J. Physiol.

Renal Physiol., 281, F1164-1172). Such a system has been shown to up-regulate expression of the reporter in the presence of tetracyclin, particularly in the renal collecting duct of transgenic mice.

Ultrasound-sensitive and tetracyclin-loaded mouse erythrocytes are prepared as described previously (WO0158431). Recipient animals are anaesthetised using 2% isofluorane in an oxygen carrier delivered at 2L/min. The sensitised and loaded cells are injected into the animals through the tail vein and animals are treated over the kidney region using 1MHz ultrasound at 6W/cm2 for 5 min. Ultrasound is delivered using pulsed mode at 35% continuous wave. Following treatment animals are allowed to recover from anaesthesia and 12h after treatment animals are sacrificed and both the treated and untreated kidneys are harvested. Both kidneys are frozen and cryosections are prepared.

These are then histologically stained for the presence of (3-galactosidase using the chromogenic substrate bromochloroindolyl- (3-galactoside and examined using light microscopy. In addition, animals are injected with free tetracycline as a positive control and kidney tissue is examined in a similar manner.

Results 11 When sections from the control, untreated kidney are examined using light microscopy no staining for lacZ product is evident. However, when sections from the ultrasound-treated kidney are examined, staining for p-galactosidase activity is evident.

This indicates that tetracycline is deposited to the ultrasound treated kidney and this results in transactivator-mediated up-regulation of the reporter gene.

Kidney tissues from control animals injected with tetracycline are positive for p- galactosidase activity. The results demonstrate that this system may be employed to assay delivery of a gene modulator from the erythrocyte-based vehicle.

Result 12. Tet-On Based Delivery of Antisense to a Target Site in vivo.

In this experiment it was decided to determine whether or not the approach described in Example 11 can be employed to assay delivery of antisense molecules.

To this end the Tet-On transgenic mouse strain, similar to that employed in Example 11, is used as the target. Antisense to the tet transactivator is loaded into the ultrasound-sensitive vehicle and this preparation is injected into the Tet-On transgenic mouse strain. Animals are then anaesthetised and treated with ultrasound over the kidney area as described for Example 11.

Following treatment, animals are then injected with tetracycline and allowed to recover from anaesthesia. After 8h, animals are sacrificed and both treated and untreated kidneys are removed. Cryosections re stained for the presence of (3-galactosidase activity and examined using light microscopy as described for Example 11.

Results 12 When sections from the untreated kidney are examined using light microscopy, staining for ß-galactosidase activity is evident. However, sections from the treated kidney

fail to exhibit staining for activity. This result indicates that functionality of the transactivator is inhibited by the presence of the antisense payload. The results further indicate that this system may be employed to assay delivery of antisense to a target site in vivo.

Example 13. Tet-OnAssay for Delivery of a Transgene Product to a Target Site in vivo In this experiment the objective is to demonstrate that delivery of a transgenic product (the transactivator) in donor erythrocytes may be assayed using the approach.

A transgenic mouse line containing the Tet-On transactivator (or a transactivator fused to a HIV TAT encoding sequence) inserted downstream from the globin LCR/P- globin promoter is generated in a manner similar to that described by Collis & Grosveld (1990), EMBO J. 9,233-240 and Grosveld etal., (1987), Cell, 51,975-985. Erythrocytes harvested from these animals contain the transaetivator (or the above-described modification thereof) already loaded. Erythrocytes containing the transgene product are harvested and sensitised as described previously (WOO 107011) and these serve as the loaded and sensitised vehicle.

As a target, a transgenic line was generated which is similar to that employed in Examples 11 and 12, except that the sequence encoding the transactivator is deleted. The animals were anaesthetised using 2% isofluorane in an oxygen carrier delivered at 2L/min.

Animals are then injected with the loaded and sensitised vehicle containing the transgene product. Animals are also injected with tetracycline.

Animals re then treated with pulsed ultrasound (35% continuous wave) using a lMHz ultrasound probe at a power density of 6W/cm2 for 5min. Animals re then allowed to recover for 12h and kidneys were subsequently harvested. Cryosections from both the ultrasound-treated and untreated kidneys are stained for P-galactosidase activity using the

chromogenic substrate bromochloroindolyl-p-D-galactoside and examined using light microscopy.

Results 13 In sections stained from the untreated kidney, no significant staining for galactosidase is detected. In sections from the ultrasound-treated kidney evidence of galactosidase activity is present and these results indicate that the transgene product payload (the transactivator) is delivered during treatment. Again, these results demonstrate that this approach may be employed as an assay for determining delivery of an agent to a target site.

Each of the applications and patents mentioned in this document, and each document cited or referenced in each of the above applications and patents, including during the prosecution of each of the applications and patents ("application cited documents") and any manufacturer's instructions or catalogues for any products cited or mentioned in each of the applications and patents and in any of the application cited documents, are hereby incorporated herein by reference. Furthermore, all documents cited in this text, and all documents cited or referenced in documents cited in this text, and any manufacturer's instructions or catalogues for any products cited or mentioned in this text, are hereby incorporated herein by reference.

Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims.