WO2002060416A1

WO2002060416A1 - Polypeptide delivery system and method for their preparation

Info

Publication number: WO2002060416A1
Application number: PCT/GB2002/000437
Authority: WO
Inventors: Anthony Patrick Mchale; Roger Craig
Original assignee: Gendel Limited
Priority date: 2001-02-01
Filing date: 2002-02-01
Publication date: 2002-08-08

Abstract

We describe a method of preparing a red blood cell vehicle suitable for delivering an agent to a target site in a vertebrate, the method comprising the steps of: (a) providing a red blood cell; and (b) loading the red blood cell with a virus or a virus-like particle comprising the agent.

Description

POLYPEPTIDE DELIVERY SYSTEM AND METHOD FOR THEIR PREPARATION

FIELD

This invention relates to the delivery of an agent to a target site, including drug delivery. In particular, the present invention relates to methods for delivering an agent using a red blood cell loaded with the agent, which cell may be sensitised to assist in agent release.

BACKGROUND

The delivery of therapeutic agents to specific tissues is desirable typically to ensure that a sufficiently high dose of a given agent is delivered to a selected tissue. Examples of therapeutic agents which are currently sought to be delivered include antibodies, enzymes, transcription factors, nucleic acids (DNA, RNA, etc), ribozymes, oligonucleotides, peptides and aptamers, among others. The target location where it is intended for the agent to act is, however, typically within the cell (for example, within the nucleus if the agent is to affect gene transcription). However, therapeutic agents of interest, for example, those listed above, typically cross cell membranes at low efficiency. A particular problem therefore arises in ensuring the agent is delivered into the intracellular environment of a cell.

The failure of agents to penetrate cell membranes may be due to various factors, such as their intrinsic size, charge, polarity and chemical composition.

A number of different methods have been developed for the delivery of agents into cells. For example, direct micro-injection of the agent into cells of interest may be used. Furthermore, modified viruses have also been proposed as delivery vehicles or vectors. For example, viruses such as adeno associated virus (AAV), adenovirus, baculovirus, modified Semliki Forest Virus (SFV), retroviruses, lentiviruses (such as Human Imnunodeficiency Virus HIV), herpesviruses (such as Herpes Simplex Virus HSV) have been proposed as vectors for intracellular delivery of agents. Furthermore, non- viral particles have been used comprising condensed nucleic acids and combinations of lipids, condensing peptides and peptides or proteins of viral origin to facilitate cellular uptake and intracellular delivery. Thus, viral agents and virus-like particles been used to deliver agents in gene therapy.

Although methods of delivery of agents into cells have been proposed, no one to our knowledge has disclosed particular methods of targeting the delivery vehicles to appropriate tissues or organs. Thus, the delivery methods proposed previously rely on general systemic administration of the delivery vector, or on injection to a particular part of a patient's body. Each of these techniques has its disadvantages, including wastage, imprecise targeting, and takeup at inappropriate sites, leading to unwanted side effects. In the case of injection, there is restriction of targeting to accessible sites of the patient, and surgical intervention may be needed to target internal sites.

SUMMARY

The present invention seeks to overcome the problems associated with the prior art methods of delivery. The invention is based on the demonstration that it is possible to load viruses and virus-like particles comprising agents of interest into red blood cells. We also demonstrate that it is possible to subsequently release the loaded virus and virus-like particles in a biologically active form.

According to a first aspect of the present invention, we provide a method of preparing a red blood cell vehicle suitable for delivering an agent to a target site in a vertebrate, the method comprising the steps of: (a) providing a red blood cell; and (b) loading the red blood cell with a virus or a virus-like particle comprising the agent.

Preferably, the method further comprises the step of sensitising the red blood cell, whether before or after the loading step, to render the red blood cell more susceptible to disruption by exposure to a stimulus than an unsensitised red blood cell. There is provided, according to a second aspect of the present invention, a method of preparing a red blood cell vehicle suitable for delivering an agent to a target site in a vertebrate, the method comprising the steps of: (a) providing a red blood cell loaded with a virus or a virus-like particle comprising an agent; and (b) sensitising the red blood cell.

We provide, according to a third aspect of the present invention, a method of preparing a red blood cell vehicle suitable for delivering an agent to a target site in a vertebrate, the method comprising the steps of: (a) providing a sensitised red blood cell; and (b) loading the red blood cell with a virus or a virus-like particle comprising an agent.

As a fourth aspect of the present invention, there is provided a method for delivering an agent to a target site in a vertebrate, the method comprising the steps of: (a) providing a red blood cell; (b) loading the red blood cell with a virus or a virus-like particle comprising an agent; (c) sensitising the red blood cell to render it more susceptible to disruption than an unsensitised red blood cell; (d) introducing the red blood cell into a vertebrate; and (e) causing the virus or a virus-like particle comprising the agent to be released from the sensitised red blood cell by applying energy to the sensitised red blood cell; in which steps (b) and (c) may be performed in any order.

We provide, according to a fifth aspect of the present invention, a red blood cell vehicle suitable for delivery of an agent to a vertebrate, the red blood cell comprising a virus or a virus-like particle comprising an agent. Preferably, the red blood cell is sensitised so that it is rendered more susceptible to disruption by exposure to a stimulus than an unsensitised red blood cell. More preferably, the red blood cell is sensitised by applying an electric field to the red blood cell. Most preferably, the electric field has a field strength of from about O.lkVolts/cm to about 10 kVolts/cm under in vitro conditions. The red blood cell may be sensitised by application of an electric pulse for between lμs and 100 milliseconds.

In a preferred embodiment, the sensitised red blood cell is capable of being disrupted by exposure to ultrasound. The ultrasound may be selected from the group consisting of diagnostic ultrasound, therapeutic ultrasound and a combination of diagnostic and therapeutic ultrasound. Preferably, the applied ultrasound energy source is at a power level of from about 0.05W/cm to about lOOW/cm .

In a preferred embodiment, the red blood cell vehicle is pre-sensitised so that it is capable of being loaded with a larger amount of agent than a red blood cell which has not been pre-sensitised. Preferably, the pre-sensitisation comprises exposing the red blood cell to an electric field and/or ultrasound.

Preferably, the virus or a virus-like particle is capable of penetrating a membrane of a target cell to deliver the agent into an intracellular compartment. The virus may be selected from the group consisting of: adeno associated virus (AAV), adenovirus, baculovirus, modified Semliki Forest Virus (SFV), retroviruses, lentiviruses (such as Human Imnunodeficiency Virus HIV), herpesviruses (such as Herpes Simplex Virus HSV), a eukaryotic virus, a prokaryotic virus, a bacteriophage, and bacteriophage lambda.

Furthermore, the agent may be selected from a group consisting of a biologically active molecule, a protein, a polypeptide, a peptide, a nucleic acid, a virus, a virus-like particle, a nucleotide, a ribonucleotide, a deoxyribonucleotide, a modified deoxyribonucleotide, a heteroduplex, a nanoparticle, 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 carbohydrate, and mixtures, fusions, combinations or conjugates of the above.

The agent may comprise a nucleotide sequence comprised in the viral genome, or which is transcribed, reverse-transcribed, translated, or otherwise expressed from, a nucleotide sequence comprised in the viral genome. Preferably, the agent comprises a viral protein, or a fusion protein comprising a viral protein. Preferably, the agent is conjugated to, fused to, mixed with or combined with an imaging agent, or in which the virus or the virus-like particle comprises an imaging agent. The present invention, in a sixth aspect, provides a red blood cell prepared according to a method as described. Preferably, the invention provides for a red blood cell prepared according to a method as described, or a red blood cell as described, for use in the delivery of a therapeutic agent to a target site in a vertebrate.

In a seventh aspect of the present invention, there is provided use of a red blood cell prepared according a method as described, or a red blood cell as described, in the preparation of a medicament for delivery of a therapeutic agent to a target site in a vertebrate.

According to an eighth aspect of the present invention, we provide a kit comprising a red blood cell prepared by a method as described, or a red blood cell as described, a virus or a virus-like particle comprising an agent to be delivered to a target site and suitable for loading into said red blood cell and packaging materials therefor.

We provide, according to a ninth aspect of the invention, a pharmaceutical composition comprising a red blood cell prepared by a method as described, or a red blood cell as described, together with a physiologically compatible buffer.

There is provided, in accordance with a tenth aspect of the present invention, a method of loading a red blood cell with an agent, the method comprising the steps of: (a) providing a red blood cell; and (b) exposing the red blood cell to a virus or a virus-like particle comprising an agent. The method may further comprise the additional step of allowing the virus or virus-like particle to infect the red blood cell to load the red blood cell with the agent.

As an eleventh aspect of the invention, we provide use of a virus or a virus-like particle in a method of delivery of an agent to a vertebrate, in which the method comprises the steps of: (a) providing an agent to be delivered; (b) modifying a virus or a virus-like particle to produce a virus or a virus-like particle comprising the agent; and (c) loading the virus or a virus-like particle into a red blood cell vehicle. The method may comprise the additional steps of: (d) sensitising the red blood cell to render it more susceptible to disruption than an unsensitised red blood cell; (e) introducing the red blood cell into a vertebrate; and (f) causing the virus or virus-like particle comprising the agent to be released from the sensitised red blood cell by applying energy to the sensitised red blood cell; in which the loading step and the sensitisation step may be performed in any order.

According to a twelfth aspect of the invention, there is provided use of a red blood cell prepared by a method as described, or a red blood cell as described, for the delivery of one or more agents to a vertebrate.

According to a thirteenth aspect of the present invention, we provide a method of treatment or prevention of a disease in a patient, the method comprising administering a red blood cell loaded with a virus or a virus-like particle comprising a therapeutic agent to the patient.

There is provided, according to a fourteenth aspect of the present invention, use of a red blood cell prepared according to a method as described, or a red blood cell as described, in the preparation of a medicament for delivery of an agent to or at a target site.

We provide, according to a fifteenth aspect of the present invention, a method comprising the steps of (a) providing a red blood cell; (b) loading the red blood cell with a virus or a virus-like particle comprising the agent; (c) releasing the virus or virus-like particle comprising an agent; and (d) determining a biological activity or viral function of the released virus or virus-like particle comprising an agent.

Preferably, the biological activity or viral function is compared to a virus or viruslike particle comprising an agent which has not been loaded into a red blood cell. More preferably, the viral function is selected from the group consisting of: viral titre, viral infectivity, viral replication, viral packaging, and viral transcription. Most preferably, viral infectivity is determined by exposing a released virus or virus-like particle comprising an agent to a suitable host cell. According to a sixteenth aspect of the present invention, we provide an electrosensitised red blood cell loaded with a virus or virus-like particle comprising an agent to be delivered.

There is provided, according to a seventeenth aspect of the present invention, a kit comprising: (a) an agent to be delivered, a virus or virus-like particle and a red blood cell, preferably a sensitised red blood cell; or (b) a virus or virus-like particle comprising an agent and a red blood cell, preferably a sensitised red blood cell; together with packaging materials and optionally instructions for loading or use.

DETAILED DESCRIPTION

The methods and compositions described here generally provide for the delivery of an agent to a target site, by employing virus or virus-like particles. According to the methods and compositions disclosed here, a virus or virus-like particle comprising the agent is loaded into the red blood cell, such that the virus or virus-like particle is encapsulated in the red blood cell and the red blood cell acts as a carrier for the agent.

The agent of interest may be coupled, fused, mixed, combined, or otherwise joined to a virus or a virus-like particle, by permanent or transient means, as described in further detail below. The red blood cells may be optionally sensitised by, for example, application of an electric field, to increase their sensitivity to external stimulus, such as an energy source. The loaded red blood cell may be used as a carrier for the agent, and delivered to an organism. Disruption of the red blood cell at a target site in the organism results in release of virus and agent. In a preferred embodiment, the red blood cell is electrosensitised, before, during or after loading, and disrupted by means of ultrasound.

It will be understood that viruses and virus-like particles are capable of fusing with cellular membranes and delivering their payload, and that once conveyed to their target site, they are capable of delivering agents into the cells of the tissues, etc at or in the vicinity of the target site. Infection of cells at or in the vicinity of the target site by the virus or a virus-like particle thus enables entry of the agent of interest into a cell. The agent is then able to act within the intracellular environment. Accordingly, the methods and compositions described here enable the local release and delivery, as well as intracellular delivery, of agents to be accomplished.

Virus and virus-like particle carriers may be chosen which are capable of infecting red blood cells; use of such agents enables the red blood cell to be automatically loaded with virus or virus-like particle comprising agent (referred to here as "passive" loading). Alternatively, or in addition, "active" loading (by means of, for example, hypotonic dialysis) may be used, as described below.

According to a general principle, agents to be delivered to a vertebrate comprise, and/or are coupled, fused, mixed, combmed, or otherwise joined to a virus or a virus-like particle, and loaded into a red blood cell delivery vehicle for delivery. Modified viruses or virus-like particles comprising agent(s) are capable of infecting cells, and therefore have the property of being capable of crossing the plasma or other membrane of a cell. As described below, this is accomplished in the case of viruses or virus-like particles by fusion of the virus with the plasma membrane or other cellular membrane such as an endosomal membrane during the infection process. The virus or virus-like particle thus acts as a "secondary" carrier to enable the agent to be delivered into the intracellular environment once released at the target site.

The coupling, etc between the agent and the virus or virus-like particle may be permanent or transient, and coupling or association between the virus or virus-like particle and the agent to be delivered is described in further detail below.

Viruses, and their use as secondary carriers of agents to be delivered, are described in further detail below. Virus-like particles are known in the art, and are described in further detail below. The viruses or virus-like particles may be themselves agents of interest, but preferably they are capable of carrying and delivering other agents of interest, such as drugs, polypeptides, nucleic acids, or other small molecules. In one embodiment, the agent to be delivered comprises a nucleic acid sequence. In this embodiment, the viral genome may comprise the nucleic acid sequence. Furthermore, where a virus-like particle is employed, the nucleic acid may be packaged or otherwise encompassed in the virus-like particle, and may form part of the nucleic acid of the virus- like particle. Thus, the nucleic acid sequence may encode a viral RNA or protein, or may comprise a heterologous sequence which has been engineered into the viral genome. The agent may, instead of comprising a nucleic acid sequence, comprise a molecule derived from the nucleic acid sequence. For example, the agent may comprise a RNA transcribed from a nucleic acid sequence comprised in the virus or virus-like particle. The agent of interest may therefore comprise a ribozyme, which is engineered into the viral genome or packaged in the virus-like particle such that it is capable of being expressed once delivered into the cell. Furthermore, the agent may comprise a DNA sequence which has been reverse transcribed from a viral RNA genome, in the case of retroviral carriers, or reverse transcribed from RNA packaged in a virus-like particle.

Furthermore, the agent of interest may include a protein or polypeptide (the two terms being used synonymously). The polypeptide may comprise a viral protein, which may form part of the virus or virus-like particle. The protein or polypeptide may form part of a viral structure, such as a viral coat. The protein or polypeptide may comprise a fusion protein comprising a heterologous polypeptide of interest fused with a viral polypeptide. The virus or virus-like particle may comprise a polypeptide of interest as a coat protein or as a fusion with a coat protein, together with a wild type viral genome or other nucleic acid. Alternatively, and preferably, the protein or polypeptide to be delivered is provided in the form of a nucleic acid, for example, a viral nucleic acid, encoding the protein or polypeptide.

Thus, the virus may be engineered so that the coding sequence of the protein or polypeptide is engineered into the viral genome. Furthermore, where a virus-like particle is employed, the coding sequence of the protein or polypeptide may be engineered into the nucleic acid which is packaged in the virus-like particle. The virus or virus-like particle is capable therefore of expressing the agent of interest as a viral coat protein, or as a fusion with a viral coat protein. Use of such viruses or virus-like particles comprising agents is advantageous as a reduced titre is needed, leading to a more limited immune response against the virus or particle.

The expression of the polypeptide or nucleic acid may be controlled by a tissue specific promoter, or a cell cycle specific promoter. Use of such specific promoters enables the expression of the polypeptide or nucleic acid to be restricted to sub- populations of the cells within the release area, and enables finer control of expression.

Viral genomes comprising agents of interest, or sequences encoding agents of interest, may be integrated into the host genome by the use of suitable integrating viruses such as adeno associated virus. This allows for long term expression and maintenance of agent in the target cell.

Furthermore, the use of other means of attachment of an agent of interest to a viral structure such as a viral coat protein or a viral nucleic acid, is envisaged, as are other means for attaching to a structure or component of a virus-like particle. Agents of interest may, for example, be conjugated or coupled by chemical means to a viral capsid. These and other means of joining the virus or virus-like particle to the agent are disclosed in further detail below.

The viruses or virus-like particles comprising agents may be loaded into red blood cells by any suitable means, as described in further detail below. It will be appreciated that the nature of some viruses or virus-like particles means that they are capable of crossing the red blood cell membrane and therefore can "self-load" into the red blood cell vehicle with little or no further assistance. Such viruses include Semliki Forest Virus and adeno associated virus AAV. Thus, we disclose a method of loading a red blood cell with an agent, the method comprising exposing a red blood cell to a virus or a virus-like particle comprising an agent. The virus or virus-like particle is preferably capable of infecting a red blood cell. While it is appreciated that energy may be required for viral infection, for simplicity, such auto-loading of viruses or virus-like particles in RBCs is referred to here as "passive loading". It will be appreciated however, that "active" loading means may also be employed, in place of, or in conjunction with passive loading. Loading procedures are described in detail below, including a preferred means of active loading using hypotonic dialysis.

The RBC vehicles described here, whether loaded or unloaded, may be subjected to a "pre-sensitising" step to increase the efficiency of loading of agent. A preferred pre- sensitising step involves applying an electric field to the red blood cells, as described in our International Patent Application Number PCT/GB00/03056 (published as WO 01/58431), and also in detail below. The RBC may be further loaded with a second agent, which itself may be an agent comprised in a virus or a virus-like particle. Such loading may be active or passive.

As described in further detail below, in a highly preferred embodiment the red blood cells are sensitised to render them more susceptible to disruption by a stimulus than unsensitised red blood cells. We therefore envisage the use of sensitising agents and/or processes to increase the susceptibility of RBC vehicles to disruption using energy such as ultrasound or light energy. The RBC vehicles disclosed here are therefore preferably capable of being selectively disrupted at a target site by exposure to a stimulus, for example laser light or ultrasound. Accordingly, "sensitised" is intended to indicate that the cells have been treated in order to render them more susceptible to a stimulus. Preferred sensitisation procedures such as electrosensitisation are set forth in our International Patent Application Number PCT/GB00/02848, and are described in detail below.

Such sensitisation may take place during, before or after loading. The loaded red blood cells (optionally sensitised) are subsequently introduced into a recipient animal, including a vertebrate or mammal such as a human, as described in detail elsewhere in this document. Lysis by ultrasound or other energy means enables release of the agent, which is then able to enter the cells in the surrounding tissue, either passively or as part of the process of viral infection. Disruption may be focused in a single tissue, or may be generalised throughout the body. Equally, the energy levels used may be intended to release the contents of substantially all of the RBC vehicles, or only part of these. In the second case, repeated applications of the required energy may be used to provide further doses of the relevant agent. This is referred to for convenience as "pulsatile delivery". The methods and compositions described here are therefore useful for the delivery of therapeutic or diagnostic agents to specific sites in vertebrate organisms, without the problems associated with agents being unable to cross the cell membrane. The ability to selectively disrupt RBC vehicles permits the person skilled in the art to achieve release of the contents of the RBC at any desired site to which the stimulus required may be directed.

The RBC vehicles loaded with virus or virus-like particle comprising agent(s) may be used for a variety of purposes. Advantageously, the RBC vehicles described here are useful for the delivery of agents (comprised in viruses or virus-like particles) to the body of a vertebrate. Such agents may comprise therapeutic agents for the treatment or prevention of any number of diseases in the vertebrate.

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; Current 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 ofEnzymology: 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. In this document, where reference is made to the loading, release, etc of an agent, this should be taken to include reference (where the context permits) to loading, release, etc of a virus or virus-like particle comprising an or the agent.

RED BLOOD CELLS

As used in this document, the term "red blood cell" (RBC) refers to a living, enucleate red blood cell (i.e., a mature erythrocyte) of a vertebrate.

Preferably the red blood cell is a mammalian red blood cell, advantageously a human red blood cell. As used in this document, the term "mammal" refers to a member of the class Mammalia including, but not limited to, a rodent, lagomorph, pig or primate. More preferably, the animal is selected from the group consisting of: mouse, rat, rabbit, sheep, goat, horse, cow, and pig. Most preferably, the mammal is a human.

As used in this document the term "introducing" includes but is not limited to the administration of a red blood cell and/or an agent (for example as a virus or virus-like particle comprising an agent) into a vertebrate. As used in this document in reference to administration of an agent to a vertebrate, the term "introducing" includes but is not limited to causing the agent (for example as a virus or virus-like particle comprising an agent) to enter the circulatory system of the vertebrate by transfusion or to infusing an agent to a target site. It is contemplated that a hollow needle, such as a hypodermic needle or cannula, is inserted through the wall of a blood vessel (e.g., a vein or artery) and the red blood cell is either injected using applied pressure or allowed to diffuse or otherwise migrate into the blood vessel. It is understood that the diameter of the needle is sufficiently large and the pressure sufficiently light to avoid damage of the cell by shear forces. Preferably, introduction of a red blood cell into a vertebrate in a method as described here is intra-arterial or intravenous. Methods of blood cell transfusion are well known in the art.

As used in this document, the term "red blood cell delivery vector" means a red blood cell that has been loaded with one or more virus(es) or one or more virus-like particle(s) comprising agent(s) and which can be used to deliver the agent to a vertebrate. The term also refers to red blood cells which are capable of being so loaded. The red blood cell delivery vector is typically made to release the agent at a site of interest in the vertebrate using ultrasound as described above. The terms "vehicle" and "delivery vehicle", where the context permits, are also intended to be synonymous with "red blood cell delivery vector".

SENSITISATION AND PRE-SENSITISATION

The agents (in the form of viruses or virus-like particles comprising agents) are loaded into a red blood cell, which may be sensitised. Such a sensitised red blood cell preferably is rendered more susceptible to disruption by exposure to a stimulus than an unsensitised red blood cell. The stimulus may include any energy source, for example, ultrasound. One or more sensitisation steps may therefore be employed to increase the sensitivity of the cells to ultrasound.

Furthermore, in order to enhance the loading efficiency of an agent into the red blood cell, the red blood cell may be subject to a "pre-sensitisation" step. Although the purpose of the pre-sensitisation step is to enhance the loading of the agent, an increase in sensitivity to lysis (for example, ultrasound mediated lysis) may also be achieved. Where more than one sensitisation step is involved, additional sensitisation steps may be performed at any stage in the process after the pre-sensitisation step. Thus, a second sensitisation step may be carried out either after the pre-sensitisation step but prior to loading, or after loading. Further sensitisation steps may be performed as required.

Generally, where present, 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. However, where passive loading is used, the sensitisation step may be effectively carried out at the same time as the agent is being loaded.

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 below. Alternatively, or in addition, the pre-sensitisation may be effected by for example the use of ultrasound, 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.

Preferably, the sensitisation step comprises an electrosensitisation procedure as described below. We have found that the efficiency of sensitisation for given electrical parameters varies depending on the cell density and it may therefore be necessary to perform a titration of cell density and or electrical parameters to establish the optimum concentration. By way of guidance, we have found that human red blood cells sensitised at a density of about 6-8x10⁸ cells/ml have good sensitivity to ultrasound.

It will be appreciated that pre-sensitisation of a red blood cell may enhance the efficiency of loading of virus or virus-like particle comprising an agent, even where passive loading is used.

PRE-SENSITISATION USING ULTRASOUND

Where a pre-sensitisation step is present, this typically involves electrosensitisation (described in detail below); however, as noted above, ultrasound may also be used to pre- sensitise red blood cells. Such use of ultrasound is also referred to in this document as "sonoporation". Exposure of red blood cells to ultrasound is believed to result in nondestructive and transient membrane poration (Miller et al, 1998, Ultrasonics 36, 947-952).

As used in this document, 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/cm (FDA recommendation), although energy densities of up to 750m W/cm² have been used. In physiotherapy, ultrasound is typically used as an energy source in a range up to about 3 to 4 W/cm (WHO recommendation). In other therapeutic applications, higher intensities of ultrasound may be employed, for example, HIFU at 100 W/cm up to IkW/cm (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 of Magnetic Resonance Imaging 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. inAcustica, 1997, Vol.83, No.6, pp.1103-1106.

Preferably, the red blood cells are pre-sensitised by exposure to 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 1MHz 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.

LOADING

As used in this document, the term "loading" refers to introducing into a red blood at least one agent and the term "loaded" is to be construed accordingly. Although the term "loading" is to be construed generally, in specific terms, it refers to the introduction of a virus or virus-like particle comprising an agent into a red blood cell.

The agent (including a virus or virus-like particle comprising an agent) may be loaded by becoming internalised by, affixed to the surface of, or anchored into the plasma membrane of a red blood cell. Where the agent, etc is affixed or anchored to the plasma membrane, loading may be achieved by cross-linking the agent, etc to any cell surface molecule. Alternatively, the agent, etc may be conjugated to or fused with an antibody specific for a cell surface molecule. Preferably, however, the agent, etc is encapsulated within the red blood cell.

Viruses or virus-like particles comprising agents may be loaded into red blood cells by any suitable means. Passive loading means, where the viruses or virus-like particles infect red blood cells and "self-load" into the red blood cell vehicle, are included, as well as "active" loading, such as hypotonic dialysis.

In passive loading, a source of red blood cells is provided. The red blood cells are then exposed to a virus or a virus-like particle comprising an agent under conditions which allow the translocation of the virus/virus-like particle/agent into the red blood cell. The red blood cells are exposed for a sufficient amount of time to allow a suitable loading level to be achieved. Progress of loading may be monitored by any suitable means. Passive loading may be aided by the concurrent, prior or post- application of an active loading method, as described in detail below. As mentioned above, in addition to loading a virus or virus-like particle comprising an agent into a red blood cell, second or further agents may be loaded, concurrently, before, or after the virus or virus-like particle comprising an agent.

Loading of a red blood cell with more than one agent may be performed such that the agents are loaded individually (in sequence) or together (simultaneously or concurrently). Such co-loading may involve any combination of passive and active loading. The second, third, et seq agents may be in the form of viruse(s) or virus-like particle(s) comprising agent(s), or they may be loaded on their own. Loading is generally performed in a separate procedure to the "sensitising" procedure, where this is performed. The agents may be first admixed at the time of contact with the red blood cells or prior to that time.

Where a pre-sensitisation step is undertaken, the red blood cells may be loaded either after the pre-sensitisation procedure or after one or more sensitisation procedures, preferably after the cells have rested. In this embodiment, the loading may be performed by any desired technique. Thus, a pre-sensitised and loaded cell may be sensitised. Furthermore, a pre-sensitised and subsequently sensitised cell may be loaded.

The loading may be performed by a procedure selected from the group consisting of electroporation, iontophoresis, sonoporation, microinjection, calcium precipitation, membrane intercalation, microparticle bombardment, lipid-mediated transfection, viral infection, osmosis, osmotic pulsing, osmotic shock, diffusion, endocytosis, mechanical perforation/restoration of the plasma membrane by shearing, single-cell injection or a combination thereof. These are referred to here as "active" loading means.

Sonoporation as a method for loading an agent into a cell is disclosed in, for example, Miller et al (1998), Ultrasonics 36, 947-952.

Iontophoresis uses electrical current to activate and to modulate the diffusion of a charged molecule across a biological membrane, such as the skin, in a manner similar to passive diffusion under a concentration gradient, but at a facilitated rate. In general, iontophoresis technology uses an electrical potential or current across a semipermeable barrier. By way of example, delivery of heparin molecules to patients has been shown using iontophoresis, a technique which uses low current (d.c.) to drive charged species into the arterial wall. The iontophoresis technology and references relating thereto is disclosed in WO 97/49450.

In a highly preferred embodiment, the red blood cell is pre-sensitised by electrosensitisation, and loaded using osmotic shock. If more than one agent is employed, the same or a different technique may be used to load the second agent into the red blood cell. Preferably the red blood cells disclosed here are pre-sensitised, sensitised and loaded in vitro or ex-vivo. Preferably loading is carried out by an osmotic shock procedure. The term "osmotic shock" is intended to be synonymous with the term "hypotonic dialysis" or "hypoosmotic dialysis".

A preferred osmotic shock/hypotonic dialysis method is based on the method described in Eichler et al., 1986, Res. Exp. Med. 186: 407-412. This preferred method is as follows. Washed red blood cells are suspended in 1 ml of PBS (150 mM NaCl, 5 mM K₂HPO₄/KH PO₄; pH 7.4) to obtain a hematocrit of approximately 60%. The suspension is placed in dialysis tubing (molecular weight cut-off 12-14,000; Spectra-Por; prepared as outlined below) and swelling of cells obtained by dialysis against 100 ml of 5 mM K₂HPO₄/KH₂PO , pH 7.4 for 90 minutes at 4°C. Resealing is achieved by subsequent dialysis for 15 minutes at 37°C against 100ml of PBS containing 10 mM glucose. Cells are then washed in ice cold PBS containing 10 mM glucose using centrifugation.

Other osmotic shock procedures include the method described in U.S. Pat. No. 4,478,824. That method involves incubating a packed red blood cell fraction in a solution containing a compound (such as dimethyl sulphoxide (DMSO) or glycerol) which readily diffuses into and out of cells, rapidly creating a transmembrane osmotic gradient by diluting the suspension of red blood cell in the solution with a near-isotonic aqueous medium. This medium contains an anionic agent to be introduced (such as inosine monophosphate or a phosphorylated inositol, for example inositol hexaphosphate) which may be an allosteric effector of haemoglobin, thereby causing diffusion of water into the cells with consequent swelling thereof and increase in permeability of the outer membranes of the cells. This increase in permeability is maintained for a period of time sufficient only to permit transport of the anionic agent into the cells and diffusion of the readily-diffusing compound out of the cells. This method is of limited effectiveness where the desired agent to be loaded into cells is not anionic, or is anionic or polyanionic but is not present in the near-isotonic aqueous medium in sufficient concentration to cause the needed increase in cell permeability without cell destruction.

U.S. Patent No. 4,931,276 and WO 91/16080 also disclose methods of loading red blood cells with selected agents using an osmotic shock technique. Therefore, these techniques can be used to enable loading of red blood cells in the methods and compositions described here.

Effective agents which may advantageously be loaded into red blood cells using the modified method provided in U.S. Patent No. 4,931 ,276 include peptides, purine analogues, pyrimidine analogues, chemotherapeutic agents and antibiotic agents. These agents frequently present drug delivery problems. Specific compounds include but are not limited to tryptophan, phenylalanine and other water-soluble amino acid compounds. Several derivatives of the unnatural analogues of the nucleic acid bases adenine, guanine, cytosine and thymine are well known as useful therapeutic agents, e.g. 6-mercaptopurine (6MP) and azathioprine, which are commonly used as immunosuppressants and inhibitors of malignant cell growth, and azidothymidine (AZT) and analogues thereof which are useful as anti- viral agents, particularly in the treatment of AIDS. It has been shown that the action of these unnatural base derivatives is dependent on intra-cellular conversion thereof to phosphorylated forms (Chan et al., 1987, Pharmacotherapy, 7: 165;14 177; also Mitsuya et al., 1986, Proc. Natl. Acad. Sci. U.S.A., 83: 1911-1915).

An alternative osmotic shock procedure is described in U.S. Patent No. 4,931,276 which is incorporated herein by reference. Alternatively, loading may be carried out by a microparticle bombardment procedure. Microparticle bombardment entails coating gold particles with the agent to be loaded, dusting the particles onto a 22 calibre bullet, and firing the bullet into a restraining shield made of a bullet-proof material and having a hole smaller than the diameter of the bullet, such that the gold particles continue in motion toward cells in vitro and, upon contacting these cells, perforate them and deliver the payload to the cell cytoplasm.

It will be appreciated by one skilled in the art that combinations of methods may be used to facilitate the loading of a red blood cell with agents of interest. Likewise, it will be appreciated that a first and second agent, may be loaded concurrently or sequentially, in either order, into a red blood cell in any method disclosed here.

As is apparent to one of skill in the art, any one or more of the above techniques can be used to load red blood cells for use in the methods and compositions described here, either prior to, simultaneously with, separate from or in sequence to the sensitisation procedure. For example, U.S. Patent No. 4,224,313 discloses a process for preparing a mass of loaded cells suspended in a solution by increasing the permeability of the cell membranes by osmotic pressure or an electric field, or both, loading agents by passage from a solution through the membranes of increased permeability, restoring the original permeability by sealing the membranes by regeneration effect, and separating the cells from the solution in which they are suspended. In that procedure, the agents in solution which are to be loaded include i) a pharmaceutical substance which reacts chemically or physically with substances in the extracellular milieu and which, when loaded into the cell, would prematurely destroy the cell membranes, and ii) at least one blood-compatible sugar and protein capable of providing hydrogen bridge bonding- or of entering into covalent bonds with the pharmaceutical substance, thereby inhibiting the reaction of the pharmaceutical substance with the cell membranes.

It will be appreciated by one skilled in the art that combinations of methods may be used to facilitate the loading of a red blood cell with agents of interest. Likewise, it will be appreciated that a first and second agent, may be loaded concurrently or sequentially, in either order, into a red blood cell in any method as disclosed in this document. The concentration of agent used in the loading procedure may need to be optimised. Preferably loading takes place over a period of at least 30 mins, more preferably about 90 mins.

ELECTROSENSITISATION

The red blood cell vehicles disclosed here may be sensitised to ultrasound or other sources of energy by the use of an electric field ("electrosensitisation"). Electrosensitisation may also be used as a means of pre-sensitising red blood cells.

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 lkV/cm, of about 100 μs 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.

Preferably, 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 WO97/49450). 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 μF, followed by a pulse of greater than 2.5 kV/cm and a capacitance of less than 2 μF, followed by another pulse of less than 1.5 kV/cm and a capacitance of greater than 5 μF. A particular example is 0.75 kV/cm, 10 μF; 3.625 kV/cm, 1 μF and 0.75 kV/cm, 10 μF. 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 in this document, 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 μF) using a BioRad Gene Pulser apparatus. Cells are immediately washed with PBS containing MgCl₂ (4mM) (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.

SELECTIVE RELEASE USING ULTRASOUND

The agents which are loaded into a red blood cell, for example, as comprised in viruses or virus-like particles may be released from the red blood cells and into their surroundings. Release may be effected at or into the target site, tissue or cell, by the application of ultrasound directed at a target site, tissue and/or cell. Furthermore, the agent may be delivered to the target site by application of ultrasound to vessels, for example, blood vessels, feeding the target site. A general discussion on ultrasound, including different types of ultrasound (for example, diagnostic, therapeutic and focussed ultrasound), is presented above.

Preferably, ultrasound within the diagnostic range or the therapeutic range is employed o effect selective release. Acombination of diagnostic ultrasound and a therapeutic ultrasound may also be used. This combination is not intended to be limiting, however, and the skilled reader will appreciate that any variety of combinations of ultrasound may be used. Additionally, the energy density, frequency of ultrasound, and period of exposure may be varied. What is important is that the application of ultrasound is able to selectively disrupt the sensitised red blood cells to effect release of agent, without substantially disrupting or damaging endogenous red blood cells or surrounding cells or tissues.

Preferably the ultrasound is applied to a target cell or target tissue with sufficient strength to disrupt loaded and sensitised red blood cells but without damaging the target tissue or surrounding tissues. In this context, the term "damage or damaging" does not include a transient permeabilisation of the target site by the ultrasound energy source. Such a permeabilisation may facilitate uptake of the released payload at the target site.

Preferably the exposure to an ultrasound energy source is at a power density of from about 0.05 to about 100 Wcm^"2. Even more preferably, the exposure to an ultrasound energy source is at a power density of from about 1 to about 15 Wcm^"2.

Preferably the exposure to an ultrasound energy source is at a frequency of from about 0.015 to about 10.0 MHz. More preferably the exposure to an ultrasound energy source is at a frequency of from about 0.02 to about 6.0 MHz.

Preferably the exposure is for periods of from about 10 milliseconds to about 60 minutes. More preferably the exposure is for periods of from about 1 second to about 5 minutes. Depending on the amount of agent which it is desired to release, however, the exposure may be for a longer duration, for example, for 15 minutes. Particularly preferably the patient is exposed to an ultrasound energy source at an acoustic power density of from about 0.05 Wcm^"2 to about 10 Wcm^"2 with a frequency ranging from about 0.015 to about 10 MHz (see WO 98/52609). However, alternatives are also possible, for example, exposure to an ultrasound energy source at an acoustic power density of above 100 Wcm^"2, but for reduced periods of time, for example, lOOOWcm^"2 for periods in the millisecond range or less.

Use of ultrasound is advantageous as, like light, it can be focused accurately on a target. Moreover, ultrasound is advantageous as it can be focussed more deeply into tissues unlike light. It is therefore better suited to whole-tissue penetration (such as but not limited to a lobe of the liver) or whole organ (such as but not limited to the entire liver or an entire muscle, such as the heart) delivery of agents. In addition, ultrasound may induce a transient permeabilisation of the target site so that uptake of a released payload is facilitated at the target site. Another important advantage is that ultrasound is a noninvasive stimulus which is used in a wide variety of diagnostic and therapeutic applications. By way of example, ultrasound is well known in medical imaging techniques and, additionally, in orthopaedic therapy. Furthermore, instruments suitable for the application of ultrasound to a subject vertebrate are widely available and their use is well known in the art.

In the methods described here, release of the agent is effected by exposure of red blood cells either in vitro or ex-vivo to an effective amount of a diagnostic ultrasound energy source or a therapeutic ultrasound energy source as described in US Patent No. 5558092 and WO94/28873. The agent, which is released from a red blood cell for use in the methods and compositions described here may be referred to as the "payload" of that cell.

Preferably the agent is released from the red blood cell by treatment of a target site, tissue or cell with ultrasound. The selective release of the agent at the target site can be determined by observing a) the amount which has been released at the target site, tissue or cell and b) its effect on the target site, tissue or cell, the latter determining whether its delivery should increase, decrease or be discontinued. The viability of viruses which have been loaded and released using the methods and compositions described here may be assayed by plaque assays, as known in the art, and as described in the Examples below.

AGENTSANDDELIVERYOFAGENTS

The delivery methods described here are useful for the delivery of agents to a selected site in a vertebrate body, whether an organ, part of an organ or otherwise, in the presence or absence of specific targeting means.

This may be achieved, as set out above, by the selective disruption by ultrasound at the selected target site of preferably electrosensitised red blood cells loaded with the agent of choice. The agents to be delivered according to the methods and compositions described here are virus(es) or virus-like particle(s) comprising agent(s). Such agents are able to cross the cell membrane and enter the intracellular environment of a target cell.

Examples of agents useful in the methods and compositions described here are set out below. Preferred agents include those useful for imaging of tissues in vivo or ex vivo. For example, imaging agents, such as 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 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 endothelial cells in lower body vasculature, for example, lower limbs, by releasing the antibody selectively in the lower body by applying ultrasound thereto. As used in this document, the term "agent" includes but is not limited to 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 an imaging agent, by which term is meant 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 P or S or Tc, 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. 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 disclosed in this document.

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 "agent" also includes mixtures, fusions, combinations and conjugates, of atoms, molecules etc as disclosed in this document. 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 in this document, 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, and a signalling 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/GBOO/00876, hereby incorporated by reference. Techniques for producing intracellular antibodies, such as anti-β-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.

An agent may include 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 in the methods and compositions described 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 virus or virus-like particle comprising an agent such as a ribozyme or an oligonucleotide (for example, an antisense oligonucleotide) into a red blood cell, which is optionally 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) benyloxycarbonyij-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 of. 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), -galactosidase (encoded by Genbank Accession No. M13571), β-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, β-glucosidase, azoreductase, t- gutamyl transferase, β-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 trans genically 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).

In a highly preferred embodiment, the methods and compositions described here enable the intracellular delivery of an agent. The intracellular environment may comprise the cytoplasm, a subcellular organelle such as the nucleus, mitochondria, Golgi, endoplasmic reticulum, vacuole, etc. Our methods and compositions preferably enable the delivery of an agent to any or all of the above target locations. TARGETING

As used herein, the term "target" is used in reference to the spatial coordinates (anatomical location) of the cell, tissue or site (such as a vessel) to which the agent is delivered.

The red blood cells as described here may be targeted to any desired site in a vertebrate, or mammal. 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 group of tissues, a cell, a group of cells or even substantially all of the cells of the vertebrate.

Preferably the target is a cell. As used in this document, the term "cell" refers to a viable, naturally-occurring or genetically engineered, single unit of an organism.

Preferably the target is a tissue. As used in this document, the term "tissue" refers to a population or physical aggregation of cells within an organism, in which the cells are of the same cell type or are of cell different types resident within a single organ or other functional unit. As used in this document, 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 0.1-10%), 20-50% or 50-100% of the organ or system (e.g., as is true of islets of the pancreas).

Preferably the target is a vessel. As used in this document the term "vessel" means any artery, vein or other "lumen" in an organism to which ultrasound can be applied and to and an agent may be delivered. A lumen is a channel within a tube or tubular organ. Examples of preferred vessels include but are not limited to the coronary artery, carotid artery, the femoral artery, and the iliac artery. Where ultrasound disruption is employed, an ultrasound energy source may be focused at the target cell, tissue or site (such as a vessel) as loaded red blood cells circulate through it. 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.

VIRUS

A virus of use in the methods and compositions described here may be an RNA virus or a DNA virus. Preferably, the virus is an integrating virus. More preferably, the virus is a vertebrate virus, a mammalian virus, a primate virus, or a human virus. The virus may be selected from a lentivirus and a herpesvirus, a Semliki Forest Virus, an adenovirus, an adeno associated virus, a baculovirus and a retro virus. The virus may comprise an HIV virus such as HIV-1 and HIV-2, or a herpesvirus, such as a HSV virus, for example HSV- 1, HSV-2, HSV-7 and HSV-8.

Examples of viruses which may be used to provide virus comprising agents using the methods and compositions described here are given in the tables below.

DNA VIRUSES

Genus or

Family Example Diseases [Subfamily]

[Alphaherpes- Herpes simplex virus type 1

Herpesviridae HHV-1) Encephalitis, cold sores, gingivostomatitis virinae] (aka Herpes simplex virus type 2

Genital herpes, encephalitis

(aka HHV-2)

Varicella zoster virus (aka

HHV-3) Chickenpox, shingles

[Gammaherpesviri Epstein Barr virus (aka HHV- Mononucleoisis, hepatitis, tumors (BL, NPC) nae] 4)

Kaposi's sarcoma associated ?Probably: tumors, inc. Kaposi's sarcoma herpesvirus, K.SHV (aka (KS) and some B cell lymphomas

Human herpesvirus 8)

Human cytomegalovirus (aka Mononucleosis, hepatitis, pneumonitis,

[Betaherpesvirinae]

HHV-5) congenital

Human herpesvirus 6 Roseola (aka E. subitum), pneumonitis

Adenoviridae Human herpesvirus 7 Some cases of roseola?

Papovaviridae Mastadenovirus Human adenoviruses 50 serotypes (species); respiratory infections

Papillomavirus Human papillomaviruses 80 species; warts and tumors

Hepadnaviridae Polyomavirus JC, BK viruses Mild usually; JC causes PML in AIDS

Poxviridae Orthohepadnavirus Hepatitis B virus (HBV) Hepatitis (chronic), cirrhosis, liver tumors Hepatitis C virus (HCV) Hepatitis (chronic), cirrhosis, liver tumors

Orthopoxvirus Vaccinia virus Smallpox vaccine virus Smallpox-like disease; a rare zoonosis (recent

Monkeypox virus outbreak in Congo; 92 cases from 2/96 - 2/97)

Parvoviridae Parapoxvirus Orf virus Skin lesions ("pocks")

E. infectiousum (aka Fifth disease), aplastic Erythrovirus B19 parvovirus crisis, fetal loss

Useful for gene therapy; integrates into

Circoviridae Dependovirus Adeno-associated virus chromosome Circovirus TT virus (TTV) Linked to hepatitis of unknown etiology

RNA VIRUSES

Genus or

Family Example Diseases [Subfamily]

3 types; Aseptic meningitis, paralytic Picornaviridae Enterovirus Polioviruses poliomyelitis

Echoviruses 30 types; Aseptic meningitis, rashes

Coxsackieviruses 30 types; Aseptic meningitis, myopericarditis

Hepatovirus Hepatitis A virus Acute hepatitis (fecal-oral spread) Rhinovirus Human rhinoviruses 115 types; Common cold

Caliciviridae Calicivirus Norwalk virus Gastrointestinal illness

4 types; Common cold, bronchiolitis, Paramyxoviridae Paramyxovirus Parainfluenza viruses pneumonia

Mumps: parotitis, aseptic meningitis (rare:

Rubulavirus Mumps virus orchitis, encephalitis)

Measles: fever, rash (rare: encephalitis,

Morbillivirus Measles virus

SSPE) Common cold (adults), bronchiolitis,

Pneumovirus Respiratory syncytial virus pneumonia (infants)

Orthomyxo- Flu: fever, myalgia, malaise, cough,

Influenzavirus A Influenza virus A viridae pneumonia

Flu: fever, myalgia, malaise, cough,

Influenzavirus B Influenza virus B pneumonia

Rabies: long incubation, then CNS disease,

Rhabdoviridae Lyssavirus Rabies virus death

Filoviridae Filovims Ebola and Marburg viruses Hemorrhagic fever, death

Uncertain; linked to schizophrenia-like

Bornaviridae Bornavirus Borna disease virus disease in some animals

Human T-lymphotropic virus Adult T-cell leukemia (ATL), tropical spastic

Retroviridae Deltaretrovirus type-1 paraparesis (TSP)

Spumavirus Human foamy viruses No disease known

Human immunodeficiency

Lentivirus AIDS, CNS disease virus type-1 and -2

Togaviridae Rubivirus Rubella virus Mild exanthem; congenital fetal defects

Equine encephalitis viruses

Alphavirus Mosquito-born, encephalitis

(WEE, EEE, VEE)

Mosquito-born; fever, hepatitis (yellow

Flaviviridae Flavivirus Yellow fever virus fever!)

Dengue virus Mosquito-born; hemorrhagic fever

St. Louis Encephalitis virus Mosquito-born; encephalitis

Hepacivirus Hepatitis C virus Hepatitis (often chronic), liver cancer

Hepatitis G virus Hepatitis???

Reoviridae Rotavirus Human rotaviruses Numerous serotypes; Diarrhea

Coltivirus Colorado Tick Fever virus Tick-bora; fever

Orthoreovirus Human reoviruses Minimal disease

Pulmonary Syndrome Rodent spread; pulmonary illness (can be

Bunyaviridae Hantavirus

Hantavirus lethal, "Four Corners" outbreak)

Rodent spread; hemorrhagic fever with renal

Hantaan virus syndrome

Phlebovirus Rift Valley Fever virus Mosquito-born; hemorrhagic fever

Crimean-Congo Hemorrhagic Nairovirus Mosquito-born; hemorrhagic fever

Fever virus

Lymphocytic

Arenaviridae Arenavirus Rodent-born; fever, aseptic meningitis

Choriomeningitis virus Rodent-born; severe hemorrhagic fever (BL4

Lassa virus agents; also: Machupo, Junin)

Deltavirus Hepatitis Delta virus Requires HBV to grow; hepatitis, liver cancer

Coronaviridae Coronavirus Human coronaviruses Mild common cold-like illness

Astroviridae Astrovirus Human astroviruses Gastroenteritis

"Hepatitis E-like

Unclassified Hepatitis E virus Hepatitis (acute); fecal-oral spread viruses"

The term "virus" should also be taken to include reference to those viruses whose hosts are any micro-organism, including prokaryotic viruses, i.e., viruses whose hosts are prokaryotic. Included are bacterial viruses such as bacteriophages, for example, bacteriophage lambda (also known as phage lambda).

Preferred bacterial viruses include those in the families, genus and species listed in the Table below.

Genus (Known Species or Type

Family also as) (Example)

Cystoviridae Cystovirus Pseudomonas Phage phi6

Fuselloviridae Fusellovirus Sulfobolus virus 1

Inoviridae Inovirus Coliphage fd

Inoviridae Plectrovirus Acholeplasma phage L51

Enterobacteria phage

Leviviridae Levi virus

MS2

Ungrouped Species of the

Family Leviviridae

Leviviridae Allolevirus

Enterobacteria phage

Qbeta

Lipothrixviridae Lipothrixvirus Thermoproteus virus 1 Microviridae Microvirus Coliphage phiXl 74 Microviridae Spiromicrovirus Spiroplasma phage 4

Bdellovibrio phage MAC

Microviridae Bdellomicrovirus

1

Chlamydiamicrovir

Microviridae Chlamydia phage 1 us

Myoviridae "T4-like phages" coliphage T4

Plasmaviridae Plasmavirus Acholeplasma phage L2

Podoviridae Podovirus Coliphage T7

"lambda-like

Siphoviridae coliphage lambda phages" enterobacteria phage Tectiviridae Tectivirus PRD1

It will be appreciated that recombinant, genetically modified or altered versions of any suitable virus, including the ones listed above, may be used. Preferably, viruses are modified to reduce their infectivity, or ability to replicate or propagate. Modification may be accomplished through chemical means, or by recombinant DNA technology.

Examples of recombinantly modified adeno viral vectors which have been employed for gene therapy, and which are suitable for loading and delivery as described here, are set out in in Tallone et al 2001 , PNAS, 98(14) p7910; Davis et al 2001 Mol

Biotechnol 18(1) p63; Rubinchik et al, 2000 Gene Ther 7(10) p875 or Tashiro et al, 1999, Hum Gen Ther 10(11) pi 845.

VIRUS-LIKE PARTICLES

As used in this document, the terms "virus-like particle(s)" or "VLPs" refer to a virus-like particle(s), fragment(s), capsomer(s) or portion(s) thereof produced from the capsid protein coding sequence of a virus and comprising one or more characteristic(s) similar to those of infectious virus particles.

Particular characteristics include infectivity, ability of the virus-like particle(s) to cross-react with wild-type particles (native infectious virus particles) as determined by antisera generated in animals and/or humans by immunization with either VLPs or infectious virus; the ability to recognize or detect antibodies in human sera from persons known to be infected with homologous virus, etc. Preferably, the virus-like particle retains at least one, some or all of these characteristics. Preferably, the virus-like particle retains the ability to infect a host cell. The term "virus-like particle" is understood to include reference to pseudo viruses and pseudovirions.

HIV-like viral particles are known in the art, and are disclosed in, for example, PCT applications WO 93/20220 published Oct. 14, 1993 and WO 91/05860 published May 2, 1990 (Whitehead Institute for Biomedical Research). These and other documents teach constructs comprising HIV genomes having an alteration in a nucleotide sequence which is critical for genomic RNA packaging, and the production of non-infectious immunogenic HIV particles produced by expression of these constructs in mammalian cells. PCT application WO 91/07425 published May 30, 1991 (Oncogen Limited Partnership) teaches non-replicating retroviral particles produced by coexpression of mature retroviral core and envelope structural proteins such that the expressed retroviral proteins assemble into budding retroviral particles. A particular non-replicating HIV-1 like particle is made by coinfecting mammalian host cells with a recombinant vaccinia virus carrying the HIV-1 gag and protease genes and a recombinant vaccinia virus carrying the HIV-1 env gene.

In published PCT application WO 91/05864, there is described particular non- infectious non-replicating retrovirus-like particles containing at least gag, pol and env proteins in their natural conformation and encoded by a modified retroviral genome deficient in long terminal repeats and containing gag, pol and env genes in their natural genomic arrangement. In WO 96/06177, there are described further mutations to the HIV genome of the constructs of U.S. Pat. Nos. 5,439,809 and 5,571,712 to reduce gag- dependent RNA packaging of the HIV-1 genome, to eliminate reverse transcriptase activity of the pol gene product, to eliminate integrase activity of the pol gene product and to eliminate RNAse activity of the pol gene product, through genetic manipulation of the gag and pol genes. In the preferred vectors described in the aforementioned U.S. Pat. Nos. 5,439,809 and 5,571,712 and U.S. Pat. No. 08/292,967, a metallothionein promoter is employed, which requires the addition of an inducer for expression to be effected.

US Patent No. 6,121,021 discloses constitutive expression of non-infectious HIV- like particles.

Virus-like particles derived from papillomavirus are disclosed in, for example, Kirnbauer et al., "Papillomavirus LI Major Capsid Protein Self- Assembles Into Virus-like Particles that are Highly Immunogenic," Proc. Natl. Acad. Sci. USA, 89:12180-12184 (1992). Rose et al., "Serological Differentiation of Human Papillomavirus Types 11, 16, and 18 Using Recombinant Virus-Like Particles," J. of Gen Virology, 75:2445-2449 (1994). Rose et al, "Human Papillomavirus Type 11 (HPV-11) Virus-Like Particles (VLPs) Induce the Formation of Neutralizing Antibodies," 12.sup.th International Papillomavirus Conference, Sep.-Oct. 1993 (abstract). Kirnbauer et al., "Efficient Self- Assembly of Human Papillomavirus Type 16 LI and L1-L2 into Virus-Like Particles," J. Virol. 67(12):6929-6936 (1993). O'Neal et al., "Rotavirus Virus-Like Particles Administered Mucosally Induce Protective Immunity," Journal of Virology, 71(11):8707- 8717 (1997). Ball et al., "Oral Immunization with Recombinant Norwalk Virus-Like Particles Induces a Systemic and Mucosal Immune Response in Mice," Journal of Virology, 72(2):1345-1353 (1998). The manufacture, characteristics and use of papillomavirus virus-like particles for roal immunization is disclosed in US Patent No. 6,153,201

US Patent No. 6,077,662 discloses virus-like particles, methods and immunogenic compositions. A particular method of making a retrovirus-like particle disclosed in that document, comprises the steps of: (a) operably linking a coding sequence for a truncated envelope protein of a retrovirus to a baculovirus early promoter, late promoter, or hybrid late/very late promoter, and inserting into baculovirus vector to form an envelope protein expression construct; (b) operably linking a protease-encoding sequence to regulatory sequences in a vector for expression in insect cells to form a protease expression construct; (c) simultaneously introducing into an insect cell the envelope protein expression construct of step (a) and the protease expression construct of step (b) and allowing for expression of the truncated retrovirus envelope protein and the protease. The retrovirus-like particles are formed by the insect cells of step (c), and a furin is disclosed as a particular protease which may be used. The method is suitable for making a variety of retrovirus-like particles including Simian Immunodeficiency Virus-like particles, Human Immunodeficiency Virus-like particles, bovine immunodeficiency virus-like particles, bovine leukemia viruslike particles, feline leukemia virus-like particles, feline immunodeficiency virus-like particles, equine infectious anemia virus-like particles and human T cell leukemia virus type I virus-like particles.

PCT/GB98/01626 discloses a composition comprising at least one baculoviral component and at least one retroviral component, wherein the retroviral component is capable of being packaged into a retroviral particle. A particular method of producing HPV virus-like particles follows in brief. A DNA fragment containing the entire HPV-11 LI open reading frame (ORF), is purified by agarose gel electrophoresis as described by Rose et al., "Expression of the full-length products of the HPV-6b and HPV-11 L2 open reading frames by recombinant baculovirus, and antigenic comparisons with HPV-11 whole virus particles," J. Gen. Virol. 71 :2725- 2729 (1990), and cloned into the corresponding sites of a baculovirus transfer vector, pVL-1392 (M. D. Summers, Texas A&M University, College Station, Tex.). The resulting construct, pVLl 1L1, is used to co-transfect Sf-9 cells with Autographa californica nuclear polyhedrosis virus (AcNPV) genomic DNA according to the methods of Summers et al., A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, 1987, Texas A&M University, College Station, Tex. Recombinant baculoviruses are recovered by visual examination and selection of occlusion-negative (occ-) plaques, and are subjected to two further rounds of plaque-purification according to the methods of Summers et al., A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, 1987, Texas A&M University, College Station, Tex. Protein expression from isolated virus stocks is determined by Western blot.

Recombinant VLPs are purified directly from the cell-free culture supernatant of Acl 1 LI -infected Sf-9 cell suspension cultures by a series of low and high speed centrifugation steps. Infected Sf-9 cells are pelleted from a 200 ml suspension culture a low speed (l,000.times.g) and the cell-free supernatant is centrifuged again at high speed (lOOjOOO.times.g) for 90 minutes at 4 degrees C. The high-speed pellet is resuspended in buffer A (50 mM Tris, pH 8.0; 1 M NaCl; 10 mM MgCl₂ ; 10 mM CaCl₂ ; 2 mM phenylmethylsulfonyl fluoride (PMSF); 10 μg/ml Leupeptin), 5.2 g solid CsCl are added, and the final volume is adjusted to a total of 13 ml with fresh buffer A (0.4 g/ml final concentration). After centrifugation (100,000 x g, 22 hours, 10 degrees C), the single band obtained is removed and diluted with 12 ml of fresh buffer A (without CsCl) and centrifuged again (100,000xg, 90 minutes, 4 degrees C.) to pellet purified VLPs. VLPs purified by sucrose density gradient centrifugation are identified by electron microscopy after staining with 2% neutral buffered phosphotungstic acid. Any virus-like particle, including those described above, may be loaded and/or delivered according to the methods and compositions described here.

VIRUSES AND VIRUS-LIKE PARTICLES COMPRISING AGENTS

In one embodiment, the agents are delivered in the form of one or more viruses or virus-like particles comprising the agent(s), which are capable of being internalised into a cell.

Preferred viruses include adeno associated virus (AAV), adenovirus, baculovirus, modified Semliki Forest Virus (SFV), retroviruses, lentiviruses (such as Human Imnunodeficiency Virus HIV), herpesviruses (such as Herpes Simplex Virus HSV). Other preferred viruses include bacterial viruses, in other words, viruses whose hosts are bacteria or other microorganisms. Included are bacteriophages, for example, phage lambda, and T viruses, including T4, etc. Such viruses are discussed in further detail above. Use of bacterial viruses is advantageous in that they may be employed to target bacterial cells present in the body, for example, as a result of an infection.

In a particular embodiment, we disclose the loading and delivery of a bacteriophage, which is capable of infecting host cells such as Salmonella typhimurium or Multiply Resistant Staphylococcus Aureus (MRSA).

Preferred virus-like particles are discussed elsewhere. However, any suitable viral vector, optionally expressing an agent of interest, may be delivered according to the methods and compositions described in this document.

The agent to be delivered is associated with a virus or virus-like particle (i.e., a "virus comprising an agent" or a "virus-like particle comprising an agent" as the case may be), in a permanent or transient manner in such a way that when the virus or virus-like particle infects a cell, the agent is directed or conveyed into an intracellular compartment of a cell. Thus, viruses and virus-like particles useful in the methods and compositions described here are those which are capable of delivering the agent associated with the virus or virus-like particle into the cell. Preferred viruses and virus-like particles are those which "self-load" into a red blood cell, i.e., are capable of "infecting" a red blood cell. Examples of such viruses are Semliki Forest Virus and Adeno Associated Virus.

Thus, a virus or virus-like particle may be modified to deliver an agent according to our methods. Such modifications are described in detail below, and include genetically engineering the genome of the virus to carry or express the agent, loading a virus-like particle with a nucleic acid comprising or capable of expressing an agent, as well as modification of other viral structures, such as the capsid, to enable association between the agent and the virus and the virus-like particle.

The agent of interest may be coupled, fused, mixed, combined, linked to, associated with or otherwise joined to a virus or virus-like particle. The association between the agent and the virus or virus-like particle may be permanent, semi-permanent or transient, and involve covalent or non-covalent interactions (including ionic interactions, hydrophobic forces, Van der Waals interactions, etc). The association may be reversible or non-reversible (i.e., permanent). In some cases, the virus or virus-like particle itself may be regarded as the agent to be delivered. Accordingly, the methods and compositions described here include the loading and delivery of a native (i.e., unmodified) virus to a target site.

The exact mode of coupling is not important, so long as it is effective in maintaining an association with the virus/virus-like particle and the agent to be delivered.

Preferably, the association between the agent and the virus or virus-like particle persists through one or more, preferably, all of the steps of: coupling, loading, release and delivery, preferably intracellular delivery. Where the red blood cell is pre-sensitised or sensitised, for example, electrosensitised, the coupling preferably also persists through either or both these steps. In a highly preferred embodiment, the agent to be delivered is associated with the virus or virus-like particle through all the above steps, including optional steps where these are performed. Preferably, the coupling persists at least through release and more preferably also during intracellular delivery. Thus, in this embodiment, the virus or virus-like particle comprising agent is effective in crossing the cell membrane of a target cell to deliver the agent into the intracellular environment. Accordingly, where reference is made to "comprising", "conjugation", "coupling", etc, these references should preferably be taken to include any form of interaction between the agent to be delivered and the virus or the virus-like particle, preferably in such a manner as to allow intracellular delivery of the agent.

Accordingly, the term "virus or virus-like particle comprising an agent" should be taken to include reference to an agent is coupled, fused, mixed, combined, or otherwise joined, permanently or transiently, etc to a virus or a virus-like particle, for example, as set out above.

Preferred means of association include the provision of recombinant viral genomes or genes comprising nucleotide sequences capable of expressing the agent of interest.

Particularly useful agents for viral delivery include nucleic acids, such as DNA and

RNA. In particular, viruses and virus-like particles are useful for delivery of RNAs having enzymatic activity, such as ribozymes. Nucleic acid agents may be delivered virally by engineering the viral genome to express the nucleotide of interest, or by packaging suitable nucleic acids comprising nucleotide sequences in virus-like particles. For example, the nucleic acid may be cloned in such a way that its expression is driven by one or more viral promoters. On infection, activation of such promoters as part of the viral replicative cycle enables expression of the nucleic acid agent of interest within the cell. Retroviruses may be used where expression of DNA species (by reverse-transcription of nucleic acid in a RNA genome) is desired.

The agent may further be a polypeptide. Such a polypeptide may be delivered as above, by cloning into a virus genome for expression from a viral promoter. Furthermore, where virus-like particles are employed, they may be packaged with nucleic acid expressing the polypeptide. The agent may also be provided as a fusion protein with another polypeptide, such as a virally encoded polypeptide. The virally encoded polypeptide may comprise a coat protein.

Viral expression vectors and techniques for manipulating these for expression of nucleic acids and/or polypeptides are known in the art. Methods of producing and manipulating virus-like particles are also known in the art, and are discussed elsewhere in this document. Accordingly, viruses and virus-like particles comprising agents may be constructed by standard recombinant DNA technology as known in the art. The recombinant viral vector may be transfected or transformed into a suitable host for large scale production of engineered virus or virus-like particle, by means known in the art. Purification of the virus or virus-like particle may also be carried out by known means.

For example, recombinant poxvirus (e.g., vaccinia, avipox virus) and exogenous DNA for expression in viral vector systems is described in U.S. Pat. Nos. 5,174,993 and 5,505,941 (e.g., recombinant avipox virus, vaccinia virus; rabies glycoprotein (G), gene, turkey influenza hemagglutinin gene, gp51,30 envelope gene of bovine leukemia virus, Newcastle Disease Virus (NDV) antigen, FelV envelope gene, RAV-1 env gene, NP (nudeoprotein gene of Chicken/Pennsylvania/1/83 influenza virus), matrix and preplomer gene of infectious bronchitis virus; HSV gD; entomopox promoter, inter alia), U.S. Pat. No. 5,338,683, e.g., recombinant vaccinia virus, avipox virus; DNA encoding Herpesvirus glycoproteins, inter alia; U.S. Pat. No. 5,494,807 (e.g., recombinant vaccinia, avipox; exogenous DNA encoding antigens from rabies, Hepatitis B, JEV, YF, Dengue, measles, pseudorabies, Epstein-Barr, HSV, HIV, SIN, EHV, BHV, HCMV, canine parvovirus, equine influenza, FeLV, FHV, Hantaan, C. tetani, avian influenza, mumps, ΝDV, inter alia); U.S. Pat. No. 5,503,834 (e.g., recombinant vaccinia, avipox, Morbillivirus [e.g., measles F, hemagglutinin, inter alia]); U.S. Pat. No. 4,722,848 (e.g., recombinant vaccinia virus; HSV tk, glycoproteins [e.g., gB, gD], influenza HA, Hepatitis B [e.g., HBsAg], inter alia); U.K. Patent GB 2269 820 B and U.S. Patent No. 5,514,375 (recombinant poxvirus; flavivirus structural proteins); WO 92/22641 (e.g., recombinant poxvirus; immunodeficiency virus, inter alia); WO 93/03145 (e.g., recombinant poxvirus; IBDV, inter alia); WO 94/16716 and U.S. application Ser. No. 08/184,009, filed Jan. 19, 1994 (e.g., recombinant poxvirus; cytokine and/or tumor associated antigens, inter alia); and PCT/US94/06652 (Plasmodium antigens such as from each stage of the Plasmodium life cycle).

Baculovirus expression systems, exogenous DNA for expression therein, and purification of recombinant proteins therefrom can be found in Richardson, C. D. (Editor), Methods in Molecular Biology 39, "Baculovirus Expression Protocols" (1995 Humana Press Inc.) (see, e.g., Ch.18 for influenza HA expression, Ch.19 for recombinant protein purification techniques), Smith et al., "Production of Huma Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector," Molecular and Cellular Biology, Dec, 1983, Vol. 3, No. 12, p. 2156-2165; Pennock et al., "Strong and Regulated Expression of Escherichia coli B-Galactosidase in Infect Cells with a Baculovirus vector," Molecular and Cellular Biology Mar. 1984, Vol. 4, No. 3, p. 399-406; EPA 0 370 573 (Skin test and test kit for AIDS, discussing baculovirus expression systems containing portion of HIV-1 env gene, and citing U.S. application Ser. No. 920,197, filed Oct. 16, 1986 and EP Patent publication No. 265785). U.S. Pat. No. 4,769,331 relates to herpesvirus as a vector.

There are also poliovirus and adenovirus vector systems known in the art (see. e.g., Kitson et al., J. Virol. 65, 3068-3075, 1991; Grunhaus et al., 1992, "Adenovirus as cloning vectors," Seminars in Virology (Vol. 3) p. 237-52, 1993; Ballay et al. EMBO Journal, vol. 4, p. 3861-65; Graham, Tibtech 8, 85-87, April, 1990; Prevec et al., J. Gen Virol. 70, 429- 434). Any of these virus vector systems may be loaded into a red blood cell for delivery according to the methods and compositions described in this document. As noted above, other means of association between the agent to be delivered and the virus are possible, for example, by chemical coupling, etc.

Retroviruses have been proposed as a delivery system (other wise expressed as a delivery vehicle or delivery vector) for ter alia the transfer of a nucleotide of interest to one or more sites of interest. Such retroviruses include lentiviruses such as HIV. The transfer can occur in vitro, ex vivo, in vivo, or combinations thereof. When used in this fashion, the retroviruses are typically called retroviral vectors or recombinant retroviral vectors. Retroviral vectors have even been exploited to study various aspects of the retrovirus life cycle, including receptor usage, reverse transcription and RNA packaging (reviewed by Miller, 1992 Curr Top Microbiol Immunol 158:1-24).

In a typical recombinant retroviral vector, at least part of one or more of the gag, pol and env protein coding regions may be removed from the virus. This makes the retroviral vector replication-defective. The removed portions may be replaced by a nucleotide sequence of interest in order to generate a virus capable of integrating its genome into a host genome but wherein the modified viral genome is unable to propagate itself due to a lack of structural proteins. When integrated in the host genome, expression of the nucleotide sequence of interest occurs - resulting in, for example, a therapeutic effect. Thus, the transfer of a nucleotide sequence of interest into a site of interest is typically achieved by: integrating the nucleotide sequence of interest into the recombinant viral vector; packaging the modified viral vector into a virion coat; and allowing transduction of a site of interest - such as a targeted cell or a targeted cell population.

It is possible to propagate and isolate quantities of retroviral vectors (e.g. to prepare suitable titres of the retroviral vector) for subsequent transduction of, for example, a site of interest by using a combination of a packaging or helper cell line and a recombinant vector.

In some instances, propagation and isolation may entail isolation of the retroviral gag, pol and env genes and their separate introduction into a host cell to produce a "packaging cell line". The packaging cell line produces the proteins required for packaging retroviral DNA but it cannot bring about encapsidation due to the lack of a. psi region. However, when a recombinant vector carrying a nucleotide sequence of interest and apsi region is introduced into the packaging cell line, the helper proteins can package the s't-positrve recombinant vector to produce the recombinant virus stock. This can be used to infect cells to introduce the nucleotide sequence of interest into the genome of the cells. The recombinant virus whose genome lacks all genes required to make viral proteins can infect only once and cannot propagate. Hence, the nucleotide sequence of interest is introduced into the host cell genome without the generation of potentially harmful retrovirus. A summary of the available packaging lines is presented in "Retroviruses" (1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 449). However, this technique can be problematic in the sense that the titre levels are not always at a satisfactory level. Nevertheless, the design of retroviral packaging cell lines has evolved to address the problem of ter alia the spontaneous production of helper virus that was frequently encountered with early designs. As recombination is greatly facilitated by homology, reducing or eliminating homology between the genomes of the vector and the helper has reduced the problem of helper virus production.

More recently, packaging cells have been developed in which the gag, pol and env viral coding regions are carried on separate expression plasmids that are independently transfected into a packaging cell line so that three recombinant events are required for wild type viral production. This strategy is sometimes referred to as the three plasmid transfection method (Soneoka et αl 1995 Nucl. Acids Res. 23: 628-633).

Transient transfection can also be used to measure vector production when vectors are being developed. In this regard, transient transfection avoids the longer time required to generate stable vector-producing cell lines and is used if the vector or retroviral packaging components are toxic to cells. Components typically used to generate retroviral vectors include a plasmid encoding the Gag/Pol proteins, a plasmid encoding the Env protein and a plasmid containing a nucleotide sequence of interest. Vector production involves transient transfection of one or more of these components into cells containing the other required components. If the vector encodes toxic genes or genes that interfere with the replication of the host cell, such as inhibitors of the cell cycle or genes that induce apotosis, it may be difficult to generate stable vector-producing cell lines, but transient transfection can be used to produce the vector before the cells die. Also, cell lines have been developed using transient infection that produce vector titre levels that are comparable to the levels obtained from stable vector-producing cell lines (Pear et αl 1993, PNAS 90:8392-8396).

In view of the toxicity of some HIV proteins - which can make it difficult to generate stable HIV-based packaging cells - HIV vectors are usually made by transient transfection of vector and helper virus. Some workers have even replaced the HIV Env protein with that of vesicular stomatis virus (VSV). Insertion of the Env protein of VSV facilitates vector concentration as HIV/VSV-G vectors with titres of 5 x 10⁵ (10⁸ after concentration) were generated by transient transfection (Naldini et al 1996 Science 272: 263-267). Thus, transient transfection of HIV vectors may provide a useful strategy for the generation of high titre vectors (Yee et al 1994 PNAS. 91 : 9564-9568). A drawback, however, with this approach is that the VSV-G protein is quite toxic to cells.

Thus, and as indicated, retroviral vectors are used extensively in biomedical research and for gene therapy. Current methods for the production of retroviral vectors make use of the fact that the two roles of the wild-type retrovirus genome, that is protein encoding and as a template for new genome copies, can be de-coupled (e.g. Soneoka et al 1995 Nucl. Acids Res. 23, 628 and references therein). Protein that is required for the assembly of new virus particles and for enzyme and regulatory functions can be produced by non-genome sequences in, for example, a mammalian packaging cell line (e.g. Miller 1990 Hum. Gene Therapy 1, 5). A genome sequence lacking the protein encoding functions is provided, so that the resulting retroviral vector particles are capable of infecting but not of replicating in a target cell. The genome sequence can also be designed for delivery and integration of a therapeutic gene (Vile and Russel 1995 Brit. Med. Bull 51, 12). Standard methods for producing murine leukaemia virus (MLV)-based vectors, for example, include use of cell lines expressing the gag-pol and env genes (the packaging components) of MLV. These will package a compatible retroviral vector genome introduced by transduction or by transfection with an appropriate plasmid. An alternative method involves simultaneous transfection of gag-pol, env, and vector genome plasmids into suitable cells.

The virus or virus-like particle comprising an agent is loaded into a red blood cell as described in detail above. MEMBRANE TRANSLOCATION SEQUENCES

We further envisage the use of polypeptide sequences or domains which are able to direct proteins, polypeptides, and other molecules (including any agent for delivery) 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. Any one or more of these sequences may be coupled, fused, conjugated or otherwise joined to the agent to be delivered in order to effect, enable or enhance intracellular delivery. Furthermore, any one or more of the sequences may be coupled, etc, to the virus or virus-like particle, instead of, or in addition to being coupled, etc to the agent to be delivered.

Where polypeptides are to be delivered, they may be expressed as fusion proteins with one or more membrane translocation sequences. Thus, we envisage the use of viruses or virus-like particles comprising fusion proteins, where such fusion proteins comprise one or more membrane translocation sequences fused to a polypeptide of interest. The fusion protein may comprise a viral protein, such as a viral capsid protein. Thus, the polypeptide to be delivered may be comprised in the viral coat, for example. Furthermore, the fusion protein may be encoded in and expressed from a viral nucleic acid. Nucleic acids for delivery may comprise sequences encoding membrane translocation sequence(s), in particular, sequences encoding fusion proteins comprising such MTSs.

Use of such membrane translocation sequences enables, effects, or enhances the loading of the red blood cell with the virus or virus-like particle comprising the agent. Furthermore, on release, the presence of the membrane translocation sequence enables or assists, or allows the virus or virus-like particle comprising an agent to enter a target cell. This compliments or replaces the normal infection of the cell by the virus or virus-like particle comprising an agent.

There appears to be no restriction on the type of molecule that can penetrate cell membranes when fused to protein translocation sequences. Therefore the method as described here employing MTS may be used for the in vivo intracellular delivery of a wide variety of agents. For example, Fawell et al. (1994), Proc. Natl. Acad. Sci. USA. 91, 664- 668 demonstrate that fusions can enter tissues in vivo in mice. Pooga et al. (1998), Nat. Biotechnol. 16, 857-861 demonstrate that fusions can penetrate the blood-brain barrier in rats. Many different protein translocation sequences have now been identified that can penetrate the cell membrane (reviewed by Lindgren et al. (2000), Trends Pharma. Sci. 21, 99-103; Morris et al. (2000), Curr. Opin. Biotech. 11, 461-466; Hawiger (1999), Curr. Opin. Chem. Biol. 3, 89-94).

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 are described in further detail below.

HIV-1 -trans-activating prote in (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 β-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. Ill, 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, our invention encompasses 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 RKKR QRR ). 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, the we also disclose 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 - our invention encompasses 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, the we envisage 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 Y. 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. Ill, 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 Penetratin^® 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) EERS Eett. 6, 265-8 show that Penetratin^® traverses a lipid bilayer, further supporting the idea that cell internalization of the third helix of Antp-HD is receptor- and endocytosis-independent. Our invention therefore encompasses 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 Penetratin^® are also described in ΕP 485578. These and other homologues and fragments of these may be used for delivery of agents into cells using the methods and compositions described here. 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 our invention encompasses the use of such truncated and modified forms of Antp-HD and its homologues.

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. Our invention therefore includes 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 NP22 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 ΝAATATRGRSAASRPTERPRAPARSASRPRRPVE 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 in the methods and compositions described here. 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 encompasse 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 AAVALLPAVLLALLAP (Signal-sequence-based peptide II) is derived from the nuclear localisation signal of NF-κB 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 NTVLALGALAGVGVG from the Integrin β₃ 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 β 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 ΝLS peptides originating from nuclear transcription factor kB (ΝF-κB; 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 μM or less. However, concentrations of 50 μM decrease GTPase activity (Pooga et al. (1998), Ann. New York Acad. 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, the invention includes 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. The invention furthermore includes the use of novel short analogues disclosed by Lindgren et al., 2000, Bioconjug Chem ll(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 and higher uptake have been synthesised by Scheller et al. (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), J Biol Chem 111, 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 fusion protein may be conjugated, coupled, etc, to the virus or a viral protein. 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. 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. The membrane translocation sequence may therefore compliment, or take the place of, normal viral infection, to enable intracellular entry of the agent of interest.

The agent(s) and/or the virus or virus-like particle 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. Coupling technologies are well known in the art.

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.

In a highly preferred embodiment, the resulting conjugate comprising the membrane translocation sequence is one which does not elicit an immune response, or one which elicits a minimal immune response, when the conjugate is exposed to the recipient animal. Preferably, the membrane translocation sequence does not elicit, or elicits a minimal, immune response. Thus, preferably, the membrane translocation sequence may be derived from a mammalian source, or is otherwise a mammalian homologue of a membrane translocation sequence as disclosed above. Preferably, therefore, in relation to a human recipient, the membrane translocation sequence comprises a human transportan, a human amphiphilic model peptide, or a human signal-sequence-based peptide. In other words, a signal sequence from any known human protein may be used as the basis for designing a suitable translocation sequence.

In the alternative, the membrane translocation sequence may be a humanised membrane translocation sequence, the term being understood to mean a sequence in which one or more residues of a membrane translocation sequence are substituted with other residues to minimise an immune response.

ASSAYS

In a preferred embodiment of the methods and compositions described here, viruses or virus-like particles comprising agent which are loaded into red blood cells and released retain one or more biological activities, preferably a viral function, of a virus or virus-like particle which is not loaded. Preferred viral functions some or all of which are retained include viral titre, viral infectivity, viral replication, viral packaging, and viral transcription.

Preferably, the virus or virus-like particle retains the function of infectivity. More preferably, the virus or virus-like particle retains all or substantially all biological activities or viral function. These may be assayed as set out below.

Assays are also provided to determine whether any viral function or biological activity has been lost through any of the process described here, for example, loading, sensitisation, pre-sensitisation, release, etc. We therefore disclose an assay comprising the steps of (a) providing a red blood cell; (b) loading the red blood cell with a virus or a virus-like particle comprising the agent; (c) releasing the virus or virus-like particle comprising an agent; (d) determining a biological activity of the released virus or viruslike particle comprising an agent.

Preferred viral functions include some or all of viral titre, viral infectivity, viral replication, viral packaging, and viral transcription. Preferably, the biological activity comprises infectivity. Preferably, the infectivity is determined by exposing the released virus or virus-like particle comprising an agent to a suitable host cell.

POLYMER THERAPEUTICS

The agents may further be delivered attached to polymers, so long as either or both the agent and the polymer is capable of being carried in, associated with, linked to, etc, or generally comprised in a virus or virus-like particle, as described above. 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-hydroxypropyi) 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 10-10% 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 transgenic cells.

NUCLEIC ACID

A nucleic acid of use in the methods and compositions described here (whether as, or to encode, an agent for delivery, an expression vector, etc) 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, condensed particles 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, 1: 450- 460). As used in this document, 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 "synthetic", as used in this document, 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 useful also include sequences encoding proteins, lipoproteins, glycoproteins, phosphoproteins and nucleic acids (e.g., R As 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 etal, 1996, Curr. Opin. Struct. Biol, 6: 527-533). Proteins or polypeptides which can be expressed by nucleic acid molecules delivered according to the methods and compositions described here 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, administered to a tissue of a recipient patient or other vertebrate, and expressed in that tissue. In general, a "therapeutic" should be taken to include any agent, atom, molecule or compound which has a beneficial effect; preferably, such a beneficial effect may be used in treatment or prevention (prophylaxis) of a disease or syndrome or condition, etc. Thus, therapeutic proteins or polypeptides comprise any protein or polypeptide encoded by the nucleic acid sequences listed above.

KITS

We also disclose a number of kits. Some of the kits comprise partially or fully treated red blood cells. Other kits provide a red blood cell, preferably a sensitised red blood cell, a virus or virus-like particle comprising an agent to be loaded and packaging materials therefor (optionally together with instructions for carrying out the methods disclosed here).

A kit designed for the easy delivery of an agent to a recipient vertebrate, whether in a research of clinical setting, is encompassed by the methods and compositions described here. A kit takes one of several forms, as follows:

A kit for the delivery of an agent to a subject vertebrate comprises preferably sensitised red blood cells and the agent and optionally instructions for loading the virus or virus-like particle comprising the agent. Alternatively, the red blood cells are supplied loaded with the virus or virus-like particle comprising the agent for convenience of use by the purchaser. In the latter case, the cells may be supplied in sensitised form, ready for rapid use or pre-sensitised and loaded but needing a final sensitisation step.

The cells of the kit are typically species-specific to the vertebrate of interest, such as a primate, including a human, canine, rodent, mouse, rat, rabbit, sheep, goat, horse, cow, and pig or other, as desired; in other words, the cells are of like species with the intended recipient. The cells of the kit are, additionally, specific to the blood type of the intended recipient organism, as needed. Optionally, the kit comprises one or more buffers for cell sensitisation, pre-sensitisation, washing, re-suspension, dilution and/or administration to a vertebrate. Appropriate buffers are selected from the group that includes low ionic strength saline, physiological buffers such as PBS or Ringer's solution, cell culture medium and blood plasma or lymphatic fluid. The kit additionally comprises packaging materials (such as tubes, vials, bottles, or sealed bags or pouches) for each individual component and an outer packaging, such as a box, canister or cooler, which contains all of the components of the kit. The kit may be shipped refrigerated. Optionally, non-cellular components are supplied at room temperature or frozen, as needed to maintain their activity during storage and shipping. They may be in liquid or dry (i.e., powder) form.

A second kit comprises an agent such as a biological effector molecule, instructions for performing the loading and delivery method and, optionally a sensitising device and buffers therefor (e.g., saline or other physiological salt buffer, culture medium, plasma or lymphatic fluid). In addition, the kit contains appropriate packaging materials, as described above. The individual components may be supplied in liquid or dry (i.e., powder) form, and may be at room temperature, refrigerated or frozen as needed to maintain their activity during storage and shipping. Red blood cells for use with this kit may be obtained independently (for example, they may be harvested from the intended recipient vertebrate).

A preferred aspect is a kit comprising a red blood cell which is loaded with an agent, and packaging materials therefor. Preferably, a kit as described above further comprises an apparatus for applying the sensitising procedure.

Preferably a kit further comprises an immunoglobulin or polyethylene glycol. Preferably the kit further comprises a liquid selected from a buffer, diluent or other excipient. More preferably the liquid is selected from a saline buffer, a physiological buffer and plasma.

We also disclose a physiological composition comprising a red blood cell delivery vector comprising an agent such as a biological effector molecule. The red blood cell is admixed with a pharmaceutically acceptable carrier or diluent, or a physiologically compatible buffer. As used in this document, the term "physiologically compatible buffer" or "physiological buffer" is defined as a liquid composition which, when placed in contact with living cells, permits the cells to remain alive over a period of minutes, hours or days. As such, a physiological buffer is substantially isotonic with the cell, such that cell volume does not change more than 20% due to differences in internal and external ionic strength. Non-limiting examples of physiologically compatible buffers or physiological buffers include dilute saline, which may be buffered (e.g., Hanks' buffered saline or phosphate buffered saline), or other physiological salts (e.g., Ringer's solution), dilute glucose, sucrose or other sugar, dilute glycerol with- or without salts or sugars, cell culture media as are known in the art, serum and plasma. Preferably, the red blood cell of the physiological composition is a human cell.

EXAMPLES

Example 1: Loading of a Virus or Virus Like Particle into a Red Blood Cell

In this Example a virus or virus like particle that encodes and/or has attached a protein or peptide is loaded into a ultrasound sensitive vehicle. We used ultrasound- sensitive human erythrocytes as described previously in WOO 158431. As an alternative, we also loaded virus or virus-like particles into a red blood cell, which is subsequently sensitised.

In this Example the virus or virus like particle comprises a recombinant adenovirus

(Ad), for example as described in Tallone et al 2001, PNAS, 98(14) ρ7910; Davis et al 2001 Mol Biotechnol 18(1) p63; Rubinchik et al, 2000 Gene Ther 7(10) p875 or Tashiro et al, 1999, Hum Gen Ther 10(11) pl845. Briefly the Ads described have been used as vectors in gene transfer and as tools to study the function of the gene transferred or as a therapy. Typically they code for marker proteins such as green fluorescent protein (GFP) and LacZ or fusion proteins that may express both the marker protein and the molecule of functional interest. The Ad vectors are prepared by infection of host cells e.g. HER911 and purified on CsCl gradients (described in Tallone et al 2001, PNAS, 98(14) p7910). Viral particles are loaded into red blood cells that are sensitive to ultrasound using methods described in WO0107011 and WO0158431.

In this experiment lysates are prepared from the loaded and sensitised red cells using ultrasound as described in WOO 107011 and WOO 15843. The lysates are assayed to ensure that the virus or virus like particle is still viable using a transfection assay, as described in Tallone et al 2001, PNAS, 98(14) p7910.

Results 1

We find that viable viral particles are released when assayed using the methods described in Tallone et al 2001, PNAS, 98(14) p7910. Furthermore, we find that the viral particles released are capable of transfecting the host HER911 cells. An assay to detect fluorescence indicates that GFP is expressed in the infected cells. The transfected cells are detected by flow cytometric analysis. Thus, this Example demonstrates that it is possible to load a red blood cell with a virus, whether sensitised before or after loading. The Example also shows that virus released from the loaded red blood cell is biologically active.

Example 2: An in Vitro Assay System for Release/Delivery of a Virus or Virus-Like Particle to a Target Cell Line

This Example describes an assay for activity of virus or virus-like particle loaded into a red blood cell and subsequently released. The assay employs the modified adenovirus vector described in Rubinchik, supra. The adenoviral vector described expressed a fusion of murine FasL and green fluorescent protein (GFP).

The modified adenovirus is loaded into a red blood cell as described in Example 1. Loaded red blood cells are lysed by ultrasound. The lysate is exposed to host target cells (NIH3T3) in the presence of doxycycline. GFP expression is assayed by fluorescence microscopy. Expression of FasL-GFP is assayed using the methods described in Rubinchik, supra.

A second version of the assay makes use of primary cultured cells as host target cells; endothelial cells are obtained from animal tissue using conventional methods and cultured in vitro. However, the target for assaying release or delivery of the loaded entity may also consist of any cell lines or ex vivo tissue in the presence of activator or which expresses an activator for the reporter protein or peptide or molecule of interest. Other assays are set up based on the cell lines/reporters described in Tallone et al 2001, Davis et al 2001 and Tashiro et al, 1999. In each case, cells are assayed in the presence of the relevant activator, for example, tetracycline or phorbol ester or cytokines such as IL-1 or TNF-α. The target cell can be activated before or after the addition of the lysate.

Viral lysates are obtained as described above and exposed to the cultured cells. Uptake and transfection is determined by expression of the green fluorescent protein in the activated target cell, using fluorescence microscopy.

A detailed description of the assay follows: 0.2ml preparations of the Ad loaded and sensitised erythrocytes are exposed to ultrasound at 2.5W/cm² using a tissue mimicking system as described previously (WO0107011). Lysates (typically 0.1-lml aliquots) are added to the target cell line (e.g. grown in tissue culture plates typically 24 or 12 well plates) or added directly to tissue, for example perfusion of a blood vessel. Following incubation e.g. for 12 hours cells are analysed by fluorescent microscopy.

Results 2A: Expression of a Fluorescent Protein Detected by Fluorescence Microscopy

Fluorescent microscopy is used on target cell lines to detect expression of green fluorescent protein. A positive fluorescent signal is detected from the cells, indicating both release of the payload and delivery of this entity into the target cell. The efficiency of transfection is indicated by the number of cells that are fluorescent and/or the level of fluorescence, and we find that a high efficiency of transformation is achieved. Negative controls are represented by i) the addition of lysates of sensitised cells that are not loaded with the virus or virus like particle and ii) the addition of intact sensitised and loaded red blood cells. In both cases no fluorescent signal is detected in the target cells. In cells where expression of green fluorescent protein is influenced by a controllable promoter, e.g. Rubinchik et al, 2000 Gene Ther 7(10) p875, negative controls include: i) the addition of the lysate to target cells that have not been induced to express the promoter, and ii) the addition of lysates of sensitised cells that are not loaded with the virus or virus like particle. In each case no fluorescent signal is detected in the target cell.

The results of this experiment demonstrate that the target cells are transfected with the virus or virus like molecule, when released from loaded red blood cells.

Results 2B: Detection of Expression of a Protein or Peptide Encoded By Virus or Virus Like Particle

Further versions of the assay involve detection of a protein or polypeptide encoded by the virus, for example, beta-galactosidase. The expression of the protein or peptide is under the control of the target cell encoded promoter or in the case of a fusion protein by the addition of e.g. a chemical such as tetracycline. A commercially available cell line such as NIH 3T3 is grown to confluence and lysates added as described in Example 2. The expression of the protein encoded by the virus or virus like particle is assessed by substrate assay or enzyme linked immunoassay (EIA) and measured colorimetrically. The controls for this example are as described in Results 2A.

We find that the protein or peptide encoded by the virus or virus like particle is only expressed by the target cells that are transfected.

Example 3: Release/Delivery of a Virus or Virus Like Particle to a Target in Vivo

We demonstrate that this viral loading and delivery approach can be employed for the localised delivery of the virus or virus like particle in vivo. In this Example, sensitised loaded cells are used to deliver virus or virus like particles encoding a protein or peptide, which in turn are transcribed and translated to allow expression of the protein or peptide in the target cell in vivo. Red blood cells are loaded with a modified adenoviral vector described in Rubinchik, supra, using methods described above.

Ultrasound-sensitive and loaded mouse erythrocytes are prepared and injected into the tail vein of a mouse. The 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 1MHz and a power density of 6 /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 examined using fluorescence microscopy.

Result 3 A: Localised Release/Delivery of Virus or Virus Like Particle Encoding for Green Fluorescent Protein to the Kidney

A strong fluorescent signal is observed microscopically in the ultrasound treated kidney; however no fluorescence is observed in the untreated kidney. These results demonstrate that the virus or virus like payload is released and delivered to a target site in vivo. In addition the virus is capable of transfecting the cell. The stability of transfection is monitored by histological analysis over a time course study.

Result 3B: Localised Release/Delivery of a Virus or Virus Like Particle to Endothelial Cells in vivo

In further assays, the virus or virus like particle encodes a protein or peptide that is under the control of a tissue specific promoter. The Ad product encoded is either expressed on the surface (e.g. E-selectin) or secreted by the target cells (e.g. IL-1).

Following ultrasound treatment, samples of blood are taken at fixed intervals over several hours and/or days. The serum or plasma is assayed using a commercially available kit for the expression of IL-1 (e.g. R&D Systems, UK). The expression of the protein is monitored over time, the efficiency and stability of transfection is determined by the level and duration of expression.

Stable transfection leads to the constitutive expression of the secreted protein, serum or plasma levels are seen to rise over the first few hours following ultrasound treatment and are maintained over days.

In addition tissue from the target site is harvested and stained for the protein or peptide of interest using standard immunohistopathological methods, commercially available validated reagents and kits (e.g. VectaStain Kit). A positive staining signal is observed in cells from the ultrasound treated site, no staining is observed in the surrounding tissue. These results demonstrate that the virus or virus like payload has been released and delivered to a target site in vivo. The stability of transfection is monitored by histological analysis over a time course study.

Example 4: Release/Delivery of a Virus or Virus-Like Particle to a Target in vivo in Pig

This Example demonstrates that the methods and compositions described here can be employed for the localised delivery of a virus or virus like particle, and may therefore be used for the purposes of gene therapy. In a proposed gene therapy regime, sensitised loaded cells are used to deliver nuclear material encoding for a protein or peptide, which in turn is transcribed and translated to allow expression of the protein or peptide in a target cell in vivo.

In this Example, the virus or virus like particle encodes a protein or peptide that is secreted e.g. TNF-α or expressed within the target cell e.g. green fluorescent protein.

This system comprises pigs which are a crossbreed type (Large While x Landrace) of the male sex at least four weeks of age, each weighing 10kg. Venous puncture of the jugular vein of each animal enables 35mls of whole blood to be available for ultrasound sensitisation and loading with virus or virus like particles.

Anaesthesia is induced by injection of pentobarbitone at a dose rate of approx. 25mg/kg bodyweight (Sagatal, Merial). The exterior ileal vein is catheterised and fitted with a 3 -way tap, for sample administration and sampling. Pre-administration samples are collected, prior to the test system receiving the processed packed cells, by slow intravenous injection (5 ml).

Sixty minutes following administration ultrasound is applied to the target site, the surface of the site is liberally covered with Alpha Lube gel (Ultrasonic Scanning gel, BCF Technology Ltd) before application of the head. Ultrasound treatment is at 6W/cm², 1MHz head, pulsed wave; 35% for 3 x 10 min bursts, with a 1 minute rest between each 10 minute burst.

For the detection of secreted proteins, blood samples are collected at fixed time periods following administration of the ultrasound and serum or plasma analysed using commercially available kits.

For the detection of localised tissue expression, the animal is euthanised and tissue is fixed. Tissue sections are analysed by fluorescent microscopy.

Result 4 A: Release/Delivery of a Virus or Virus-Like Particle in Vivo in a Pig

A clear increase in the levels of TNF-α in serum or plasma coincides with the administration of the loaded sensitised red cells and ultrasound treatment. None or basal levels of TNF-α are detected in a control animal, to which sensitised loaded cells are administered but no ultrasound is applied. Result 4B: Release/Delivery of a Virus or Virus-Like Particle in Vivo in a Pig to a Target

Tissue is excised from target and distal sites, the tissue is fixed and sections analysed by fluorescent microscopy. Fluorescent cells are detected in the sections obtained from the target site but not in the tissue from distal sites. No fluorescence is detected in the tissue obtained from the control animals to which sensitised loaded cells are administered but no ultrasound is applied.

Example 5. Delivery of Pathogen-Specific Bacteriophage in vitro

This and the following Example relate to loading and delivery of bacteriophage to a target site.

The increased prevalence of multidrug-resistant bacterial pathogens has motivated a re-examination of phage therapy, i.e. the use of strain-specific lysogenic phage to eliminate bacterial infection (Lederberg, 1996, Proc. Natl. Acad. Sci., 93, 3167-3168; Merril et al, 1996, Proc. Natl. Acad. Sci. 93, 3188-3192). This approach has, in the past, been hindered by factors such as rapid clearance from circulation by the reticuloendothelial system (RES) (Geir et al, 1973, Nature, 246, 221-222).

The advantages of the erythrocyte delivery system described in this document include payload protection from adverse immune responses and/or rapid clearance from circulation. This Example demonstrates that this system may be used to facilitate delivery of bacteriophage to a target pathogen in vitro.

To this end, the pathogenic microorganism, Salmonella. Typhimurium CRM3 is chosen as the target organism and the phage P22 as the therapeutic agent. Phage are isolated and purified using CsCl gradients, essentially as described previously (Merril et al, 1996 Proc. Natl. Acad. Sci. 93, 3188-3192). Preparations are filter sterilised and suspended in phosphate buffered saline and loaded into sensitised pig erythrocytes using the approaches described in WOO 1/07011 and WO0158431. Cell suspensions (6.5xl0⁸ cells/ml) are exposed to ultrasound at a power density of 3 W/cm for 40 sec. using the equipment and tissue-mimicking system described previously (WO01/07011 and WO0158431). Cell lysates are retained for subsequent analysis. Control cell lysates were prepared by hypo-osmitic lysis of unloaded erythrocytes.

The target host organism is grown from a single colony in 150ml of LB medium and harvested by centrifugation as described previously (Merril et al, 1996 Proc. Natl. Acad. Sci. 93, 3188-3192). The cell pellet is re-suspended in phosphate buffered saline to yield a suspension of 10⁶ colony forming units (cfu) per ml. 1ml aliquots of the suspension are dispensed into tubes and varying (50- 500μl) quantities of the erythrocyte lysates are added to these tubes. In addition a control series of tubes are set up in a similar manner except that the phage-containing erythrocyte lysates are replaced by lysates derived from un-loaded erythrocytes. The presence of phage-induced lysis of the target is determined by either visual examination or spreading onto LB agar plates.

Results 5

Following incubation for a period of 12h, the tubes containing the target and the ultrasound-induced lysates derived from the phage-loaded erythrocytes are clear whereas the control tubes were extremely turbid. This demonstrates phage-induced lysis of the target. In addition, when aliquots of the tubes containing the target and the ultrasound- induced lysates derived from the phage-loaded erythrocytes are spread onto LB-agar no growth is detected whereas plates containing the control suspensions exhibit a lawn of bacterial growth. These results demonstrate that pathogen-specific phage may be loaded into the erythrocyte-based delivery vehicle and ultrasound may be employed to facilitate release of the biologically active phage. Example 6. Delivery of Pathogen-Specific Bacteriophage in vivo

This Example demonstrates that a pathogen-specific bacteriophage may be delivered to a pathogenic target in vivo.

An approach similar to that described in Merril et al, 1996 Proc. Natl. Acad. Sci. 93, 3188-3192 is employed except that a pocine system is used as the host. The host is infected with a lethal dose of Sα typhimurium CRM3 by intravenous injection. The lethal dose is determined by titration. Prior to infection, blood is harvested from the host animal and erythrocytes are prepared, loaded with P22 bacteriophage and sensitised as described previously (WO01/07011 and WO0158431).

Loaded and sensitised vehicle is injected into the anaesthetised and infected host.

The animal is treated with ultrasound (5W/cm ) in the carotid/jugular region for 5 min. Prior to and following administration of the loaded erythrocytes, blood samples are harvested to examine the circulating levels of the target pathogen. In this case, the levels of pathogen in circulation are monitored by dilution and direct plating on LB agar plates. Control animals consist of those receiving the loaded vehicle but without ultrasound treatment.

Results 6

In animals that receive ultrasound treatment the colony forming units in circulation are seen to decrease dramatically within 3h of exposure to ultrasound whereas the colony forming units in circulation in animals that did not receive ultrasound treatment continue to increase.

These results of this experiment demonstrate that the erythrocyte-based vehicle will find application in phage-based therapies of pathogenic disease and provide advantage in terms of protection from adverse immunological interference. 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.

Claims

1. A method of preparing a red blood cell vehicle suitable for delivering an agent to a target site in a vertebrate, the method comprising the steps of:

(a) providing a red blood cell; and

(b) loading the red blood cell with a virus or a virus-like particle comprising the agent.

2. A method according to Claim 1, which further comprises the step of sensitising the red blood cell, whether before or after the loading step, to render the red blood cell more susceptible to disruption by exposure to a stimulus than an unsensitised red blood cell.

3. A method of preparing a red blood cell vehicle suitable for delivering an agent to a target site in a vertebrate, the method comprising the steps of: (a) providing a red blood cell loaded with a virus or a virus-like particle comprising an agent; and (b) sensitising the red blood cell.

4. A method of preparing a red blood cell vehicle suitable for delivering an agent to a target site in a vertebrate, the method comprising the steps of: (a) providing a sensitised red blood cell; and (b) loading the red blood cell with a virus or a virus-like particle comprising an agent.

5. A method for delivering an agent to a target site in a vertebrate, the method comprising the steps of:

(a) providing a red blood cell;

(b) loading the red blood cell with a virus or a virus-like particle comprising an agent; (c) sensitising the red blood cell to render it more susceptible to disruption than an unsensitised red blood cell;

(d) introducing the red blood cell into a vertebrate; and

(e) causing the virus or a virus-like particle comprising the agent to be released from the sensitised red blood cell by applying energy to the sensitised red blood cell;

in which steps (b) and (c) may be performed in any order.

6. A red blood cell vehicle suitable for delivery of an agent to a vertebrate, the red blood cell comprising a virus or a virus-like particle comprising an agent.

7. A red blood cell vehicle according to Claim 6, in which the red blood cell is sensitised so that it is rendered more susceptible to disruption by exposure to a stimulus than an unsensitised red blood cell.

8. A method according to any of Claims 2 to 5, or a red blood cell according to Claim

7, in which the red blood cell is sensitised by applying an electric field to the red blood cell.

9. A method or red blood cell according to Claim 8, in which the electric field has a field strength of from about O.lkVolts/cm to about 10 kVolts/cm under in vitro conditions.

10. A method or a red blood cell according to Claim 8 or 9, in which the red blood cell is sensitised by application of an electric pulse for between lμs and 100 milliseconds.

11. A method according to any of Claims 2 to 5 and 8 to 10, or a red blood cell according to any of Claims 7 to 10, in which the sensitised red blood cell is capable of being disrupted by exposure to ultrasound.

12. A method or a red blood cell according to Claim 11 , in which the ultrasound is selected from the group consisting of diagnostic ultrasound, therapeutic ultrasound and a combination of diagnostic and therapeutic ultrasound.

13. A method or a red blood cell according to Claim 11 or 12, in which the applied ultrasound energy source is at a power level of from about 0.05 W/cm² to about 100W/cm².

14. A method or a red blood cell according to any preceding claim, in which the red blood cell vehicle is pre-sensitised so that it is capable of being loaded with a larger amount of agent than a red blood cell which has not been pre-sensitised.

15. A method according to Claim 14, in which the pre-sensitisation comprises exposing the red blood cell to an electric field and/or ultrasound.

16. A method or red blood cell according to any preceding claim, in which the virus or a virus-like particle is capable of penetrating a membrane of a target cell to deliver the agent into an intracellular compartment.

17. A method or red blood cell according to any preceding claim, in which the virus is selected from the group consisting of: an adeno associated virus (AAV), an adenovirus, a baculovirus, a modified Semliki Forest Virus (SFV), a retrovirus, a lentiviruses, an Human Imnunodeficiency Virus (HIV), a herpesvirus, a Herpes Simplex Virus (HSV), a eukaryotic virus, a prokaryotic virus, a bacteriophage, and bacteriophage lambda.

18. A method or red blood cell according to any preceding claim, in which the agent is selected from a group consisting of a biologically active molecule, a protein, a polypeptide, a peptide, a nucleic acid, a virus, a virus-like particle, a nucleotide, a ribonucleotide, a deoxyribonucleotide, a modified deoxyribonucleotide, a heteroduplex, a nanoparticle, 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 carbohydrate, and mixtures, fusions, combinations or conjugates of the above.

19. A method or red blood cell according to any preceding claim, in which the agent comprises a nucleotide sequence comprised in the viral genome, or packaged in a virus- like particle, or is transcribed, reverse-transcribed, translated, or otherwise expressed from, such a nucleotide sequence.

20. A method or red blood cell according to any preceding claim, in which the agent comprises a viral protein, or a fusion protein comprising a viral protein.

21. A method or red blood cell according to any preceding claim, in which the agent is conjugated to, fused to, mixed with or combined with an imaging agent, or in which the virus or a virus-like particle comprises an imaging agent.

22. A red blood cell prepared according to any of Claims 1 to 5 and 8 to 21, or a red blood cell according to any of Claims 6 to 21, for use in the delivery of a therapeutic agent to a target site in a vertebrate.

23. Use of a red blood cell prepared according to any of Claims 1 to 5 and 8 to 21 , or a red blood cell according to any of Claims 6 to 21, in the preparation of a medicament for delivery of a therapeutic agent to a target site in a vertebrate.

24. A kit comprising a red blood cell prepared according to any of Claims 1 to 5 and 8 to 21, or a red blood cell according to any of Claims 6 to 21, a virus or a virus-like particle comprising an agent to be delivered to a target site and suitable for loading into said red blood cell and packaging materials therefor.

25. A pharmaceutical composition comprising a red blood cell prepared according to any of Claims 1 to 5 and 8 to 21, or a red blood cell according to any of Claims 6 to 21, together with a physiologically compatible buffer.

26. A method of loading a red blood cell with an agent, the method comprising the steps of: (a) providing a red blood cell; and (b) exposing the red blood cell to a virus or a virus-like particle comprising an agent.

27. A method according to Claim 26, further comprising allowing the virus or a viruslike particle to infect the red blood cell to load the red blood cell with the agent.

28. Use of a virus or a virus-like particle in a method of delivery of an agent to a vertebrate, in which the method comprises the steps of: (a) providing an agent to be delivered; (b) modifying a virus to produce a virus or a virus-like particle comprising the agent; and (c) loading the virus or a virus-like particle into a red blood cell vehicle.

29. Use according to Claim 28, in which the method comprises the additional steps of: (d) sensitising the red blood cell to render it more susceptible to disruption than an unsensitised red blood cell; (e) introducing the red blood cell into a vertebrate; and (f) causing the virus or a virus-like particle comprising the agent to be released from the sensitised red blood cell by applying energy to the sensitised red blood cell; in which the loading step and the sensitisation step may be performed in any order.

30. Use of a red blood cell prepared according to any of Claims 1 to 5 and 8 to 21 , or a red blood cell according to any of Claims 6 to 21, for the delivery of one or more agents to a vertebrate.

31. A method of treatment or prevention of a disease in a patient, the method comprising administering a red blood cell loaded with a virus or a virus-like particle comprising a therapeutic agent to the patient.

32. Use of a red blood cell prepared according to any of Claims 1 to 5 and 8 to 21, or a red blood cell according to any of Claims 6 to 21, in the preparation of a medicament for delivery of an agent to or at a target site.

33. A method comprising the steps of (a) providing a red blood cell; (b) loading the red blood cell with a virus or a virus-like particle comprising the agent; (c) releasing the virus or virus-like particle comprising an agent; and (d) determining a biological activity or viral function of the released virus or virus-like particle comprising an agent.

34. A method according to Claim 33, in which the biological activity or viral function is compared to a virus or virus-like particle comprising an agent which has not been loaded into a red blood cell.

35. A method according to Claim 33 or 34, in which the viral function is selected from the group consisting of: viral titre, viral infectivity, viral replication, viral packaging, and viral transcription.

36. A method according to Claim 33, 34 or 35, in which viral infectivity is determined by exposing a released virus or virus-like particle comprising an agent to a suitable host cell.

37. An electrosensitised red blood cell loaded with a virus or virus-like particle comprising an agent to be delivered.

38. A kit comprising:

(a) an agent to be delivered, a virus or virus-like particle and a red blood cell, preferably a sensitised red blood cell; or

(b) a virus or virus-like particle comprising an agent and a red blood cell, preferably a sensitised red blood cell; together with packaging materials and optionally instructions for loading or use.