WO2010001175A1

WO2010001175A1 - Modified cells and methods of monitoring their viability

Info

Publication number: WO2010001175A1
Application number: PCT/GB2009/050781
Authority: WO
Inventors: Celestine Santosh
Original assignee: Greater Glasgow Health Board
Priority date: 2008-07-03
Filing date: 2009-07-02
Publication date: 2010-01-07
Also published as: GB0911486D0; GB2461398A; GB0812191D0

Abstract

The invention relates to modified gene therapy or stem cells that express an MRI gene product that is capable of being detected when the cells are viable. The invention also includes methods of monitoring or tracking or detecting the cells post implantation to assess their viability, survival, migration and/or their differentiation status.

Description

MODIFIED CELLS AND METHODS OF MONITORING THEIR VIABILITY

The present invention relates to modified cells comprising at least one reporter gene that expresses a gene product that is capable of being detected when said cells are viable. The invention also includes methods of monitoring or tracking or detecting the modified cells post implantation to assess their viability, survival, migration and/or their differentiation status. The invention includes inter alia methods of making the cells, imaging the cells, enhancing their viability and replication rates post implantation, assisting their regeneration and products therefor.

BACKGROUND

The administration of exogenous stem cells offers promise to regenerate many damaged organs/tissues with successful therapies depending largely on achieving a robust and targeted cell engraftment. It is thought that failure of these cellular therapies can be attributed to any one or more of the following factors; type of stem cell chosen for implantation; the dose of cellular therapeutic; dosing regime; and mode of delivery. The recent ability to directly label stem cells with magnetic resonance (MR) contrast agents provides a simple, straight-forward manner to monitor accurate cell delivery and track stem cells non-invasively in a serial manner. MRI not only provides excellent spatial and contrast resolution but, for humans where it is necessary to repeatedly image and monitor transplanted patients, MRI is a particularly safe method since it does not involve exposing the individual to ionising radiation. Magnetic nuclear imaging (MRI) is currently the only way to non-invasively study the fate of transplanted stem cells in vivo.

A basic and simplified overview of gene therapy involves a culture of cells, some sort of vector, and the newly created genetic material. Most often, cells are extracted from the organism into which they will later be transplanted. A vector is then prepared with the new genetic material, ensuring that the vector itself is not harmful. The cells are then injected with the vector. With the vector in the cell, the genetic material is inserted into the nucleus. The corrected cells are placed inside the donor organism again, typically referred to as an ex vivo gene therapy. Alternatively, the vectors can be injected directly into the body, typically referred to as in vivo gene therapy. Recent efforts as with stem cells have focused on using MRI technology to monitor gene delivery, to enhance gene transfection/transduction, and to track gene expression. The most commonly used MRI technique consists of labelling stem cells or gene therapy cells with superparamagnetic iron oxide (SPIO) nanoparticles such as fluorescein isothiocyanate (FITC)-incorporated silica-coated core-shell SPIO nanoparticles or superparamagnetic gold-coated monocrystalline iron oxide nanoparticles (Au-MION). SIPO labelling is emerging as an ideal probe for non-invasive cell tracking. However, it is not without its disadvantages. For example, the dilution effect of the particles with each cell division, the fact that the presence of an iron signal does not indicate living cells and that, in vivo, free iron in the presence of oxygen has toxic properties as it stimulates the generation of highly reactive oxygen species such as hydroxy radicals means that it has limited utility in humans. A yet further disadvantage is that its low intracellular labelling efficiency has limited the potential usage and has evoked great interest in developing either new improved contrast enhancing labelling strategies or developing alternative strategies altogether.

Possible alternative strategies that could be used to track gene therapy or stem cells post implantation in vivo are the use of molecular probes or reporter genes/probes., each will be discussed in turn hereinafter.

As to molecular probes, these are typically macromolecular carriers such as albumin or polyamidoamine dendrimers conjugated with gadolinium or iron particles, but due to their lack of specificity only Gd or Fe conjugated onto monoclonal-antibodies probes can be used to track stem cells. The use of monoclonal antibodies as carriers present a safety concern in humans given the recent experiences of TGN 1412, a humanized monoclonal antibody (MAb) developed by German firm TeGenero to treat autoimmune diseases and leukaemia where subjects rapidly experienced a massive autoimmune reaction. Accordingly the main disadvantage of a molecular probe based system is in their safety and potential adverse side-effects when they are monoclonal-antibody based or their lack of specificity when based on macromolecular carriers conjugated with Gd or Fe.

The concept of reporter gene is applicable to cell tracking. Surviving cells can be tracked when a promoter of viral origin (e.g., the promoter of cytomegalovirus [CMV]) is used to control the expression of the reporter gene; such a reporter gene is constitutively active as long as the cell is alive and is minimally regulated by physiological processes in the cell. It is known from the prior art to stably transfected human embryonic stem cells with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV-F/t/c) whereby the reporter product can be detected visually. Furthermore it is known to provide human embryonic stem cells containing promoter-reporter systems whereon the promoter can be a PUMA, p21 , CYP3A4 promoter which responds to metabolic or toxicological change and thus drives expression of the reporter gene product which can be detected biochemically. However, none of the reporter gene systems to date are suitable for use with MRI which is considered as the only technology suitable for in vivo tracking of stem cells post implantation.

It is thought that developing the reporter probe is the most promising way forward. This is done by the specific manipulation of the genome (reporter gene) and then identifying its presence in the cell by identifying the protein with the help of an external sensor. This approach has been successfully exploited in animals using optical imaging to visualise bioluminescence and fluorescence. For example in fluorescence imaging, energy from an external source of light is immediately re-emitted at a longer, lower- energy wavelength if, for example, GFP is present. Another commonly used fluorescent reporter gene is the luciferase gene. The disadvantage of such reporter probes for tracking gene therapy or stem cells in vivo in humans is that optical imaging would only be useful for superficial structures and secondly the ethical considerations of introduction of genes from another species into humans.

A basic requirement for a MRI reporter gene is that it is able to produce signal or enough contrast that can distinguish it from the surrounding tissues. The earliest studies to exploit this were the transgenic expression of creatine kinase (CK) from mouse brain in Echerichia coli and subsequently transgenic over expression of CK under a specific promoter in the. However this technique has one major disadvantage as it was based on MRS and not imaging. Subsequently, MR reporter genes that were enzyme-based approach were developed and these had the advantage that they could be imaged. One was the over expression of tyrosine which is an enzyme and part of the melanin synthesis pathway. Melanin has a high affinity for iron and so can be detected by MRI. The tyrosinase approach produces highly reactive oxygen species and this is toxic to cells. Another enzyme based approach used the genetic expression of B- galactosidase (lacZ gene) in the presence of gadoninium based substrate that contains a galactose group. This has disadvantages as it need an exogenous contrast agent for it to work. Subsequently other reporter genes based on metalloproteins have been developed for MRI. The first was through the over expression of the transferring receptor (TfR) on the surface of the cells which allows increased iron transportation and accumulation within the cell. In the last few years two such MRI reporter genes have been developed, the Ferritin gene and MagA gene. However all the above MR reporter techniques have a significant disadvantage. These reporters all indicate their location and expression by the accumulation of iron. If these cells die, the iron accumulated within the cells with still be detectable locally and it is not possible to determine whether the transplanted stem cell or the cells the transfected with genes for therapies are alive/viable.

It is recognised that in order to track and provide more information on their status of gene therapy or stem cells in vivo post implantation using MRI, other reporter strategies need to be found.

In the present invention there is provided a new MRI reporter gene that is capable when integrated into a gene therapy of stem cell of tracking the cell but also, depending on the levels of oxygen, will alter the MRI signals indicating if the cells are alive or dead.

In the present invention, we describe a new approach to the reporter gene route for gene therapy or stem cell tracking. The present invention is based upon the use of MRI reporter genes incorporated into said cells and their unique response to oxygen as a metabolic biosensor.

It is therefore an object of the present invention to provide an improved method of monitoring or tracking or detecting stem cells post implantation by MRI to assess their viability, survival, migration and/or their differentiation status.

BRIEF SUMMARY OF THE DISCLOSURE

According to a first aspect of the invention there is provided a modified cell stably transfected with at least one MRI reporter gene which expresses at least one MRI reporter gene product, the MRI reporter product having a magnetic difference when in its oxy and deoxy forms. A cell is said to be "genetically altered" or "transfected" when a poly nucleotide has been transferred into the cell by any suitable means of artificial manipulation, or where the cell is a progeny of the originally altered cell that has inherited the polynucleotide. A genetic alteration is said to be "stable" if it is inheritable through at least 4 passages of cell culture. Alterations to the cell genome, insertion of a transgene or inactivation of an endogenous gene, are usually stable.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Reference herein to "MRI reporter gene" is intended to include any reporter gene product wherein the oxy and deoxy forms of the expressed protein are magnetically different, that is to say that the difference between the oxy and deoxy forms of the expressed protein are discernable by MR methodology.

MRI is currently the only technology suitable for in vivo imaging of transplanted cells in humans as not only is it non-invasive but it does not detrimentally affect tissues and can give immediate imaging results. MRI uses a powerful magnetic field to align the nuclear magnetization of (usually) hydrogen atoms in water in the body. Radiofrequency fields are used to systematically alter the alignment of this magnetization, causing the hydrogen nuclei to produce a rotating magnetic field detectable by the scanner. This signal can be manipulated by additional magnetic fields to build up enough information to reconstruct an image of the body. With current MRI techniques hydrogen is imaged, however this is non-specific and so by itself it is not possible to detect a specific reporter protein synthesised by the reporter gene. The only way to do so would be indirectly by using a protein which can influence the relaxivity, a term used to define the efficacy of a contrast agent to relax protons. In the present invention, the magnetic properties of the globin reporter gene products are utilised to influence the relaxivity of the hydrogen nuclei.

Preferably, the MRI reporter gene is a globin reporter gene.

Preferably the globin reporter gene is selected from the group comprising a haemoglobin, myoglobin, neuroglobin or cytoglobin reporter gene. In a preferred embodiment the reporter gene is the myoglobin reporter gene.

Myoglobin and haemoglobin belong to an ancient superfamily of haem-associated globin proteins and share the functional characteristics to bind O₂ at the iron atom and so play an important role in metabolism, they also posses important MRI characteristics in that; the oxy and deoxy forms have different magnetic properties. Oxyhaemoglobin is diamagnetic and deoxyhaemoglobin is paramagnetic and so provide different signals on MRI, a property that can be exploited in functional MRI. For example, by using an oxygen challenge this property of haemoglobin can be used to assess tissue metabolism in brain infarction (unpublished data). Myoglobin has similar MRI properties like haemoglobin in that; the oxy and deoxy forms are magnetically different and so can be used to assess cellular oxygenation and so metabolism. Therefore the family of globin proteins can, not only be used as a MRI reporters, but the alteration of signal depending on oxygenation makes them ideal candidates for providing metabolic information and an indication whether the cells/tissues are dead or alive. In the present invention we have developed cells that express myoglobin and results have shown in-vitro that MRI signal changes are detectable with the alteration of oxygen.

Preferably, in one embodiment of the invention the modified cell is a stem cell and more preferably is selected from the group comprising a totipotent, multipotent, pluripotent or induced pluripotent stem cell. It is envisaged that the clinical use of the stem cells of the present invention will involve their ability to form differentiated cells or tissues in the subject into whom they are introduced and as in preferred embodiments of the invention the stem cell is either a multipotent stem cell or pluripotent or induced pluripotent stem cell. Preferably, the stem cell is derived from a human. However, it will be appreciated that the stem cell maybe derived from any species and modified according to the invention to enable tracking of said stem cells in the species under investigation.

Preferably, totipotential stem cells (Embryonic Stem Cells, ESC) are obtained from a zygote or blastomere, multipotential stem cells are obtainable from adult and foetal hematopoeitic stem cells or adult stem cells, pluripotential stem cells are obtainable from cultured ESC and induced pluripotential stem cells are obtainable from adult cells.

In an alternative embodiment of the invention, the modified cell is a gene therapy cell. That is to say a cell that is suitable for and has been engineered for gene therapy purpose. A gene therapy cell may comprise a viral vector such as an adenoviral vector into which is inserted the therapeutic gene of interest and optionally a promoter to drive expression thereof. The present invention is not intended to be limited to the type of gene therapy cell or vector which it contains, merely that the present invention is suitable for use in such cells for the purposes stated herein after.

Preferably, the modified cells of the present invention comprise more than one reporter gene the additional reporter gene can be selected from the other two globin reporter genes or alternatively may comprise a non-MRI reporter gene selected from the group comprising GFP, lacZ, CAT, SEAP, or any other suitable reporter gene. In this embodiment of the invention the stem cells express a further possibly non-MRI detectable reporter protein that is detectable by other techniques known in the art.

Haemoglobin, myoglobin and neuroglobin are considered to be MRI reporter proteins. Because of the differences of the magnetic properties of oxy-form of the globin to that of the deoxy-form of the globin advantageously, an oxygen challenge can be used as a metabolic biosensor. Therefore, the globin reporter gene products of the present invention are MRI reporters but with the use of oxygen can also be considered as bio- reporters of metabolism. Therefore, if the stem cell can express any of the globin proteins it is envisaged that the cells expressing them can be not only located by MRI but also, and more importantly, as oxygen is only utilised by living cells, it is possible to simultaneously assess if the transplanted cells/tissues are alive and healthy. This information cannot be provided by any of the prior art techniques nor by the only other known MRI reporter probe technique, ferritin transgene expression. According to a second aspect of the invention there is provided a modified cell that contains an expression cassette, the expression cassette comprising an MRI reporter gene as herein before described.

As used herein the expression cassette comprises one or more genes and the sequences controlling their expression. At least three components comprise an expression cassette: a promoter sequence, an open reading frame, and a 3' untranslated region that, in eukaryotes, usually contains a polyadenylation site. The cassette is part of vector DNA used for cloning and transformation.

Accordingly, the present invention also includes a cell containing a vector DNA with the elements of the expression cassette and especially the myoglobin gene.

A yet further advantage of the present invention is that the globin reporter gene products themselves may provide additional oxygen storage capacity for the stem cells. Animal studies have shown than only some cells successfully undergo transplantation into the tissues and that this failure may be in part due to the lack of oxygen as the cells are transplanted. It is known that myoglobin is an oxygen storage protein in muscles and it is thought that neuroglobin may provide the same function in the nervous tissues. It has also been shown that the regions that express less neuroglobin, such as the hippocampus (four times less than the cortex) is less tolerant than the cortex and correlates to many diseases such as Alzheimers that preferentially affects the hippocampal region and mesial temporal sclerosis. The present invention may therefore provide a method of improving the viability and replication of the stem cells and their progeny by the additional oxygen storage capacity provided by the globins.

According to a third aspect of the invention there is provided a suspension of modified cells according to either the first or second aspect of the invention, the cells being capable of expressing at least one MRI reporter gene product.

Preferably, the cells are suspended in a fluid medium that is of a physiological pH and that is suitable for introduction into a mammalian body.

Preferably, the suspension of stem cells includes an agent that increases either the transport or the delivery of oxygen to the region/tissue where the gene therapy or stem cells are to be introduced into the mammalian body. In one embodiment of the invention the agent is an erythropoiesis-stimulating substance, preferably selected from the group comprising recombinant human erythropoietin (alpha, beta, omega), darbepoietin-alpha, continuous erythropoiesis receptor activator and hematide. In another embodiment of the invention the agent is a hemoglobin-based oxygen carrier or a fluorocarbon or perfluorocarbon emulsion. Alternatively, allosteric modulators of hemoglobin (RSR-13 and RSR-4) may also be used to increase the transport/ delivery of oxygen.

In a particularly preferred embodiment the agent is a fluorocarbon (FC) or a perfluorocarbons (PFC). FCs and PFCs have numerous applications in the biomedical field because of their unique chemical and biological properties. These compounds are clear, colorless, odorless, nonflammable, biocompatible, and have low reactivity. In addition, they are capable of having dissolved in them large amounts of gases, including oxygen and air, per unit volume. Accordingly, FCs and PFCs have been successfully used as carriers in applications wherein oxygen must be supplied to organs and tissues. It is envisaged that FCs and/or PFCs would ideally compliment a composition or formulation or pharmaceutical comprising a suspension of the gene therapy or stem cells of the present invention.

An "invention formulation", "formulation", "pharmaceutical formulation" or the like means a composition that one can administer to a subject, e.g., human, mammal or other animal, without further manipulations that change the ingredients or the ingredient proportions that are present. Formulations will typically comprise a single formula of the stem cells of the present invention and one or more excipients. Formulations are suitable for human or veterinary applications and would typically have expected characteristics for the formulation, e.g., parenteral formulations for human use would usually be sterile and stored in a suitable closed container.

When referring to mixtures that contain a formula of the stem cells of the present invention, an "invention composition", "composition" or the like means a composition, that is a formulation or that can be an intermediate one can use, e.g., to make a formulation or a formula of stem cells.

Preferably, the suspension of gene therapy or stem cells is for use in transplantation. The suspension of cells may be frozen, cryopreserved or otherwise preserved for transportation and subsequent use.

According to a fourth aspect of the invention there is provided a method of monitoring, tracking, localising or quantifying gene therapy or stem cells in vivo post implantation, the method comprising detecting a magnetic difference by MRI of a signal change in magnetic resonance values between a globin reporter gene product expressed by a gene therapy or stem cell in its oxy form as compared to that MR value in its deoxy form.

Deoxyhaemoglobin and deoxymyoglobins are paramagnetic and therefore will reduce T₂ ^* and oxyhaemoglobin and oxymyoglobin are diamagnetic and do not shorten T₂ ^*. T₂ ^* is a relaxation measurement or spin-spin relaxation measurement, and it occurs when the spins in the high and low energy state exchange energy but do not loose energy to the surrounding lattice. In an idealized system, all protons (hydrogen nuclei) in a given chemical environment within a magnetic field will spin with the same frequency. However, within tissues there are minor differences in the magnetic environment, which leads to a change in the resonance frequencies around the ideal. Over time, this change in the spin frequency will lead to a dispersion of the magnetic spin vectors and so loss of signal. Therefore any alteration from an oxy to deoxy and vice versa would provide a signal change on Susceptibility Weighted Imaging (SWI). This alteration will depend on the oxygenation within the tissues and if necessary can be controlled through respiration. Thus it will be appreciated that a particular advantage of the methods and compositions of the present invention resides in the perceived reduction in MRI imaging strength of the modified cells when in a situation of low oxygen or hypoxia so that the clinician can remedy the situation by either administering more modified/unmodified cells to the patient or by giving them local or systemic increase oxygen supply.

Preferably, the method is repeated over time and the differences in MR values of the oxy and deoxy values of the stem cell MRI reporter gene products can be used to assess the efficacy of the treatment.

It will be appreciated that the methods of the invention are used preferably to assess stem cell viability, survival, migration and/or their differentiation status post implantation.

In practice the method can be repeated several times per day, every day or over several days in order to ascertain the required information. The methods of the present invention conveniently provide a means by which gene therapy or stem cells following transplantation may be tracked so that further medical intervention can be rationally applied if and when necessary.

According to a fifth aspect of the invention there is provided a method of assisted regeneration of gene therapy or stem cells implanted into an individual comprising:

(i) administering to an individual to be treated a therapeutically effective amount of gene therapy or stem cells at least a proportion of which express a MRI reporter gene product;

(ii) assessing differences between magnetic resonance values of the oxy form of the MRI reporter gene product compared to the deoxy form of the MRI reporter gene product; (iii) assessing the viability of said cells; and (iv) depending on whether the said cells are thriving or not, administering a further amount of cells as required.

Preferably, the therapeutically amount of gene therapy or stem cells administered can comprise a total or partial population of modified cells according to the first or second or third aspect of the invention. It will be appreciated that the method of assisted regeneration may in some embodiments comprise a known ratio of unmodified cells compared to modified cells and that viability assessments can be proportionally scaled up to give an overall picture of the state of the transplanted cells.

Preferably, the therapeutically effective amount of modified cells introduced into the mammalian body also includes an agent that increases either the transport or the delivery of oxygen to the region/tissue where the stem cells have been introduced as hereinbefore described. Co-administration of such an agent is a particularly preferred embodiment since a good supply of oxygen to the transplanted cells will enhance their survival potential.

Preferably, step (iv) may be repeated as many times as is required in order to ascertain whether a healthy and viable population of stem cells has been established in the recipient. Replacing diseased cells with healthy cells, or cell therapy, is a promising use of stem cells in the treatment of disease and can be considered as similar to organ transplantation only the treatment consists of transplanting cells instead of organs. In theory, any condition in which there is tissue degeneration can be a potential candidate for stem cell therapies, including Parkinson's disease, spinal cord injury, stroke, burns, heart disease, Type 1 diabetes, osteoarthritis, rheumatoid arthritis, muscular dystrophies and liver diseases. This applies mutatis mutandis to gene therapy cells which may also be implanted so as to correct genetic defects.

In addition, retinal regeneration with stem cells isolated from the eyes can lead to a possible cure for damaged or diseased eyes and may one day help reverse blindness. Bone marrow transplantation (transfer of blood stem cells) is a well-established treatment for blood cancers and other blood disorders.

According to a sixth aspect of the invention there is provided use of the modified cells of the first, second or third aspects of the invention in the treatment of a disease or condition where cells can be used to repair or replace damaged tissues or organs or in the instance of modified gene therapy cells to correct a genetic defect.

For example and without limitation, the stem cells of the present invention can be used as a resource for various, specialized cell types, such as nerve cells, muscle cells, blood cells and skin cells and can be used to treat various diseases.

According to a seventh aspect of the invention there is provided a pharmaceutical composition comprising the modified cells of the first and second aspects of the invention or a suspension of stem cells according to the third aspect of the invention.

According to a eighth aspect of the invention there is provided a method of treatment for a disease selected from the group comprising diabetes, Parkinson's, stroke, cerebal palsy, Alzheimers's heart disease, stroke, rheumatoid arthritis, osteoarthritis and degenerative joint disease, autoimmune disease and visual impairment the method comprising administering a therapeutically effective amount of modified stem cells that express at least one MRI reporter gene product and tracking said implanted stem cells by MRI and assisting their regeneration in situ. The preferred features of each and any aspect of the invention apply mutatis mutandis to each and any other aspects of the invention.

The invention will now be described by way of example only with reference to the following Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a schematic diagram of the myoglobin expression construct.

Figure 2 shows Western blot analysis of myoglobin expressing ES cell clones. Expression was verified on the basis of immunoreactivity against anti-V5 antibody (Invitrogen) and anti-actin antibody was used as a positive control

Figure 3 shows Western blot analysis of differentiated myoglobin-expressing ES cell clones. Clones previous shown to express myoglobin (clones 4, 6 and 8 from Figure 2) were allowed to differentiate for 9 days at which time a Western blot against the V5 epitope was performed.

Figure 4 shows in vitro MRI imaging of control embryonic stem cells. Figure 4A shows the MRI signals at 2 ms (left hand column is the oxygenated sample right hand side is the deoxygenated sample), Figure 4B shows the MRI signals at 8 ms (left hand column is the oxygenated sample right hand side is the deoxygenated sample) and Figure 4C shows the MRI signals at 12 ms (left hand column is the oxygenated sample right hand side is the deoxygenated sample).

Figure 5 shows in vitro MRI imaging of myoglobin expressing embryonic stem cells. Figure 5A shows the MRI signals at 2 ms (left hand column is the oxygenated sample right hand side is the deoxygenated sample), Figure 5B shows the MRI signals at 8 ms (left hand column is the oxygenated sample right hand side is the deoxygenated sample) and Figure 5C shows the MRI signals at 12 ms (left hand column is the oxygenated sample right hand side is the deoxygenated sample).

DETAILED DESCRIPTION

Plasmid DNA Extraction and Electrophoresis An E.coli culture was established that contained the plasmid (pCMV-SPORT6 4396 base pairs) and the myoglobin gene (1 100 base pairs) purchased through Geneservice.com. Plasmid DNA was extracted by taking an amount of the culture sample, centrifuging it and retaining the pellet which contains bacterial cells with genomic and plasmid DNA.. The pellet was re-suspended using 250μl P1 Buffer (RNase A and LyseBlue solutions are added to Buffer P1 ) and then lysed using a detergent and contains SDC. The solutions were mixed by repeated inversion of the tube until the solution turned blue. The cell protein and genomic DNA were precipitated and subjected to a further centrifugation, the supernatant from the earlier step was added to QIA prep spin column, which contains the silicate disc. The plasmid DNA bound to the silicate disc was eluted from the disc by addition of 50μl of water and then spun. The aqueous solution containing the plasmid DNA in 50μl. was then checked by electrophoresis.

In order to assess if the plasmid pcDNA 3.1/His Topo (5523 neucleotides) was expressed correctly electrophoresis was performed. Two restriction enzymes, Sal 1 and Not1 , were used to cleave the plasmid and four 20μl digest samples were prepared: 1. 10μl of DNA from the 50μl obtained from the miniprep; 0.5μl of Sal 1 restriction enzyme; 2.0μl of buffer; 7.5μl of H₂O. 2. 10μl of DNA from the 50μl obtained from the miniprep; 0.5μl of Not 1 restriction enzyme; 2.0μl of buffer; 7.5μl of H₂O.

3. 10μl of DNA from the 50μl obtained from the miniprep; 0.5μl of Sal 1 restriction enzyme; 0.5μl of Not 1 restriction enzyme; 2.0μl of buffer; 7.0μl of H₂O.

4. 10μl of DNA from the 50μl obtained from the miniprep; 2.0μl of buffer; 8.0μl of H₂O

The four digests were incubated for 2 hours at 37 degrees C.

The electrophoresis gel (Agrose Tae Gel) of 1.2% was prepared with Ethidium Bromide and digests were loaded into the wells with loading solution. Electrophoresis was carried out for 20 minutes at 100V. The plasmid pCMV-SPORT6 (4396 base pairs) and Myoglobin gene (1100 base pairs) has a total base pairs 5496 bp, results confirmed that linearization with one restriction enzyme as in samples 1 and 2 results in one fragment with 5496bp whereas in sample 3 with both enzymes resulted in two fragments, one with about 1 100 bp and the other 4396 bp. PCR and Cloning the PCR Product.

To amplify the number of myoglobin genes Polymerase Chain Reaction (PCR) technique was used. The primers used were: 5¹-CAC CAT GGG GCT CAG TGA TG-3¹ (SEQ ID NO:1 ) 5¹-GCC CTG GAA GCC TAG CTC CT-3¹ (SEQ ID NO:2)

The PCR product underwent electrophoresis and results showed a band expected in the position for myoglobin. This sample was excised from the gel and the DNA was extracted using the Qiagen™ gel extraction kit. Before cloning the PCR product onto the transfection vector pcDNA3.1A/5-His-TOPO Adenine (A) was added. Myoglobin ORF was introduced into vector as follows:

4μl DNA with A overhangs, 1 μl expression vextor (pcDNA3.1A/5-His-TOPO), 1 μl Salt, were incubated for 5 minutes at room temperature. 3μl was removed and mixed with E. CoIi (No heat shock for 30 sees at 42⁰C) and then left on ice for a few minutes.

The mixture was then incubated at 37⁰C for 45 minutes for the E. CoIi to start expressing B-Lactamases before plating.

EXAMPLE 1 The steps taken to engineer murine embryonic stem (ES) cells to over-express murine myoglobin are described below. Initially a cDNA copy of murine myoglobin was amplified by reverse transcription-polymerase chain reaction (RT-PCR) the resulting DNA fragment was sub-cloned into the expression vector pcDNA3.1V5His-TOPO (Invitrogen) such that the myoglobin was in-frame with the His and V5 epitope tags (Figure 1 ). The presence of these epiptope tags enabled the myoglobin expression to be verified by western blot. The resulting plasmid was verified by sequencing. In this plasmid the expression of myoglobin is driven by the strong CMV promoter, a polyadenylation signal is also present allowing myoglobin to be expressed at high levels in mammalian cells. The vector also contained a gene conferring resistance to the antibiotic G418 which was used to select for stable transfectants in mammalian cells. The myoglobin expression construct was introduced into the murine ES cells line CGR8 by electropration and stable transfectants selected on the basis of their resistance to G418. Individual clones were picked and screened for myoglobin expression by western blot using antibodies against the V5 epitope (Invitrogen) (Figure 2). Western blot analysis of 8 myoglobin-expressing clones was performed and high expressing clones were identified The stability of myoglobin expression in differentiated clones was also confirmed by examining myoglobin expression nine days after the induction of differentiation by the removal of leukaemia inhibitory factor (LIF) (Figure 3). Results confirm that expression of myoglobin in murine ES cells is stable.

EXAMPLE 2

In vivo measurements of T₂ of the myoclones (murine ES cells expressing myoglobin) were done on a Bruker Biospec 7T system using Multispin Multi Echo sequence.

T₂ of culture medium 109msecs

T₂ of Myoclone cells from cell line 8 at 12.59 hrs 1 1 Omsecs

T₂ of combined Myoclone cells at 13.32 hrs 90msecs

T₂ of combined Myoclone cells at 14.40 hrs 54msecs

T₂ of combined Myoclone cells at 15.30 hrs 59msecs T₂ of combined Myoclone cells at 15.50 hrs 59.1 msecs

T₂ of combined Myoclone cells at 16.22 hrs after oxygenation 75.5msecs

T₂ of combined Myoclone cells at 16.32 hrs after oxygenation 77.5msecs

These results show a reduction of T₂ with time as oxymyoglobin converts to deoxymyoglobin. Following oxygenation of the cells after 15.50 hrs the T₂ increase as the deoxymyoglobin converts to oxymyoglobin.

EXAMPLE 3

T₂ was measured as hereinbefore described for control cells (murine ES cells) and myoclones (murine ES cells expressing myoglobin) on the Bruker Biospec 7T system u sing a Multi Gradient Echo sequence.

T₂ of Control cells at 15.45 hrs 12msecs

T₂ ^* of Myoclones at 15.45 hrs 3msecs T₂ of Control cells at 16.10 hrs after swapping sample positions in magnet 14msec T₂ ^* of Myoclones at 16.10 hrs after swapping sample positions in magnet 5msec

These above results show that the myoclones have a T₂ ^*signal which is different from the control cells. The myclones cells were refreshed at 16.32 hrs with fresh culture medium and T₂ increased to l Omsecs. This indicates that the before the cells were refreshed they were in the deoxymyoglobin state and so had a low T₂ relaxation time between 3msec to 5msec but after the were refreshed deoxymyoglobin altered to the oxymyoglobin the T₂ relaxation time increased to 10msec.

EXAMPLE 4

The MRI signal from myoglobin expressing stem cells dropped by about 37% in conditions of oxygen deprivation but this then increased by about 28%when the cells were oxygenated. This indicates not only that the signal is dependent on the oxygenation state of the cell but also that the some cells can regain their original oxygenated state.

EXAMPLE 5 Using approximately 1 x 10⁶ control embryonic stem cells in each of an oxygenated samples (left hand column of Figures 4 A, B and C) and deoxygenated sample (right hand column of Figures 4A, B and C) in vitro MRI images were taken at different time points. Figures 4A to C show MRI sequences with increasing susceptibility as denoted by the fading from A-C. The results indicate that the MRI images do not demonstrate a difference in the loss of signal between the deoxygenated sample and the oxygenated sample with increasing susceptibility. However, when compared to myoglobin expressing embryonic stem cells (clone E14 myoV5) images taken at the at the same time intervals (Figures 5A-C) the images clearly demonstrate an increasing loss of signal in the deoxygenated samples (right hand columns of Figures 5A-C) with increasing susceptibility and this is much greater than the oxygenated samples (left hand columns of Figures 5A-C) as demonstrated by the marked fading.

In the present invention the strategy for stem cells for CNS transplantation, expressing haemoglobin or myoglobin could be as follows, myoglobin belongs to the same globin superfamily as neuroglobin which is naturally expressed in neurons. Therefore, it is expected that myoglobin would not be deleterious to the function as nervous tissue. The advantages of myoglobin are that like haemoglobin the oxy and deoxy forms are magnetically different. Therefore, we would be able to image, track and demonstrate that the implanted stem cells are alive in vivo and differentiate the transplanted stem cells from the native neurons in vitro. Another advantage to the present invention is a greater success in the survival of the transplanted cells as the myoglobin naturally provides an extra store of oxygen.

As regards stem cell for cardiac tissue replacement, those stem cells expressing haemoglobin as the MRI reporter gene product would be preferred. Haemoglobin has the same evolutionary lineage as myoglobin and therefore its presence within the myocardial cells would not be deleterious. Therefore, it is possible to image, track and demonstrate that the implanted stem cells are alive in vivo and differentiate the transplanted stem cells from the native cells in vitro.

Claims

1. A modified cell stably transfected with at least one MRI reporter gene which expresses at least one MRI reporter gene product, the MRI reporter product having a magnetic difference when in its oxy and deoxy forms.

2. A modified cell that expresses at least one MRI reporter gene product, the MRI reporter product having a magnetic difference when in its oxy and deoxy forms.

3. A cell according to either claim 1 or 2 wherein the MRI reporter gene is a globin reporter gene selected from the group comprising a haemoglobin, myoglobin, cytoglobin or neuroglobin reporter gene.

4. A cell according to claim 3 wherein the reporter gene is a myoglobin reporter gene.

5. A cell according to any preceding claim wherein expression of the MRI reporter gene is under the control of a promoter and optionally wherein the promoter is a CMV promoter.

6. A cell according to any preceding claim that is either a gene therapy cell or a stem cell.

7. A cell according to claim 5 wherein the stem cell is selected from the group comprising a totipotent, multipotent, pluripotent or induced pluripotent stem cell.

8. A cell according to any preceding claim comprising more than one MRI reporter gene and/or a non-MRI reporter gene.

9. A cell according to claim 8 wherein the non-MRI reporter gene is selected from the group comprising GFP, lacZ, CAT and SEAP.

10. A cell that contains an expression cassette, the expression cassette comprising an MRI reporter gene which expresses at least one MRI reporter gene product, the MRI reporter product having a magnetic difference when in its oxy and deoxy forms

1 1. A cell containing a vector DNA that expresses an MRI reporter gene whose products exhibit a magnetic difference when in their oxy and deoxy forms optionally wherein the MRI reporter gene is a myoglobin gene.

12. A suspension of cells according to any preceding claim.

13. A suspension according to claim 12 wherein the cells are suspended in a fluid medium that is of a physiological pH and that is suitable for introduction into a mammalian body.

14 A suspension according to either of claims 12 or 13 further including an agent that increases either the transport or the delivery of oxygen to a region/tissue where the stem cells have been transplanted.

15. A suspension according to claim 14 wherein the agent is selected from the group comprising an erythropoiesis-stimulating substance, a hemoglobin-based oxygen carrier, a fluorocarbon or perfluorocarbon emulsion and an allosteric modulators of hemoglobin.

16. A suspension according to claim 15 wherein the agent is a fluorocarbon or perfluorocarbon emulsion.

17. A suspension according to any one of claims 12 to 16 for transplantation.

18. A method of monitoring, tracking, localising or quantifying gene therapy or stem cells in vivo post implantation, the method comprising detecting a magnetic difference by MRI of a change in magnetic resonance values between a globin reporter gene product expressed by a stem cell in its oxy form as compared to that MR value in its deoxy form.

19. A method according to claim 18 wherein any alteration from an oxy to deoxy and vice versa provides a signal change on Susceptibility Weighted Imaging (SWI).

20. A method according to either claim 18 or 19 wherein the signal change is in T₂

21. A method according to any one of claims 18 to 20 that is repeated over time and the differences in MR values of the oxy and deoxy values of the stem cell MRI reporter gene products are used to assess efficacy of treatment.

22. A method according to any one of claims 18 to 21 for assessing stem cell viability, survival, migration and/or their differentiation status post implantation.

23. A method of assisted regeneration of gene therapy or stem cells implanted into an individual comprising:

(i) administering to an individual to be treated a therapeutically effective amount of modified gene therapy or stem cells at least a proportion of which express a MRI reporter gene product; (ii) assessing differences between magnetic resonance values of the oxy form of the MRI reporter gene product compared to the deoxy form of the MRI reporter gene product;

(iii) assessing the viability of said modified cells; and

(iv) depending on whether the said modified cells are thriving or not administering a further amount of cells as required.

24. A method according to claim 23 wherein the therapeutically amount of modified cells administered comprise either a total or partial population of modified cells.

25. A method according to either claim 23 or 24 further including an agent that increases either the transport or the delivery of oxygen to the region/tissue where the modified cells have been introduced that is co-administered with the modified cells.

26. A method according to any one of claims 23 to 27 wherein step (iv) is repeated as many times as is required in order confirm establishment of a viable population of modified cells in the recipient.

27 A method according to any one of claims 23 to 26 further including any one or more of the features recited in claims 3 to 10.

28. Use of a modified stem cell according to any one of claims 1 to 1 1 or a suspension of modified stem cells according to any one of claims 12 to 17 for the treatment of a disease or condition where stem cells or gene therapy cells can be used to repair or replace damaged tissues or organs or correct a mutational defect.

29. A pharmaceutical composition comprising modified cells according to any one of claims 1 to 1 1 or a suspension of modified cells according to any one of claims 12 to 17.

30. A method of treatment for a disease selected from the group comprising diabetes, Parkinson's, stroke, cerebal palsy, Alzheimers's heart disease, stroke, rheumatoid arthritis, osteoarthritis and degenerative joint disease, autoimmune disease and visual impairment, the method comprising administering a therapeutically effective amount of modified cells that express at least one MRI reporter gene product having a magnetic difference when in its oxy and deoxy forms and tracking said implanted cells by MRI and assisting their regeneration in situ.

31. A method according to claim 30 further including any one or more of the features recited in claims 3 to 1 1.