HK1010139B - Encapsulated cells producing viral particles - Google Patents

Description

Encapsulated cells producing viral particles

The present invention relates to encapsulated cells that produce viral particles, particularly retroviral particles containing a retroviral vector genome carrying a therapeutic gene; methods of making such encapsulated cells; and the use of such encapsulated cells to deliver genes, particularly therapeutic genes, to target organs/cells.

The delivery of therapeutic genes into target cells is critical to gene therapy. If gene therapy can become a routine undertaking, it is of utmost importance to establish systems that can efficiently deliver therapeutic genes to target cells in vivo.

Viral vectors, particularly retroviral vectors, are the most commonly used delivery vectors for gene therapy (Morgan and Anderson, 1993). Most of the currently approved gene therapy protocols employ the in vitro approach of removing cells from a patient, genetically modifying them in vitro, and then returning them to the patient. This approach is complicated and expensive to operate and is limited to advanced technical equipment. Furthermore, this approach is limited to cells that are easy to isolate, culture and reimplant (Gunzburg et al, 1995). Although in vivo delivery of therapeutic genes may provide many advantages, at this stage, this approach is not only ineffective, but also problematic. Due to the low efficiency of gene transfer, a major problem is the need for multiple applications of viral vectors. The requirement for multiple vector transfers not only makes the procedure cumbersome, but also may result in failure due to the immune response directed against the viral particles.

One possible approach to solving these problems is: cells capable of producing viral particles are implanted directly. Implanting cells capable of producing viral particles containing the viral vector genome in situ at or near the target organ may also allow for direct application of the viral vector to the target cell/organ.

Furthermore, if the viral vector virus used is a retroviral vector virus, then such an approach is better than multiple single high dose applications because the chances of the vector virus being present when the target cell undergoes replication and thus the chances of the vector virus infecting the cell are increased. Furthermore, a low level but sustained release of the viral particles may be advantageous to escape the host's immune response to the viral particles.

In order to efficiently transport viral vectors, the virus particle-producing cells should be able to survive in the host for a prolonged period of time after implantation into the host, and virus particles must be produced and released from the cells during this period. These cells survive for a long time when there is no significant immune response, such as after brain implantation (Culver et al, 1992; Ram et al, 1993). However, to be successful in implantation elsewhere in the body, the producer cells must be protected from the immune system.

The long-term effectiveness of this approach is thus dependent on: (1) protecting the cell from the host immune system. The host immune system normally eliminates cells that produce viral particles, especially if the cells that produce viral particles are from a different species (as is often the case with such cells); (2) cells can survive in situ for long periods of time, which may require vascularization (vascularisation).

The work of encapsulating cells in permeable structures that allow the release of certain biologically active molecules but protect the cells producing these molecules from the immune system has been somewhat successful (reviewed in Chang, 1995). Cells genetically modified to produce human growth hormone (hGH) (Tai and Sun, 1993) or secretory human adenosine deaminase (Hughes et al, 1994) have been encysted. In both studies, cells were encapsulated in poly-L-lysine-alginate vesicles, and experiments showed that such cells can survive in culture for a long time with long-term production of enzymes or hormones. Furthermore, studies have shown (Tai and Sun, 1993) that after transplantation of such vesicles into mice, cells survive for one year and continue to produce hGH, demonstrating that the vesicles protect transfected cells from the host immune system.

Other materials have also been reported in the literature to encapsulate cells. Rat and rat kidney cells genetically modified to produce nerve growth factor have been encapsulated in polyacrylonitrile/vinyl chloride and implanted into rat brain. The encapsulated cells survive for at least 6 months and continue to produce NGF (Winn et al, 1994; Deglon et al, 1995).

In addition, hepatocytes have been successfully encapsulated in a polyelectrolyte complex of cellulose sulfate and polydiallylammonium dimethyl (Stange et al, 1993). More than 90% of the encapsulated hepatocytes retain their activity and the encapsulated cells show enhanced metabolic viability compared to hepatocytes grown as monolayers. This does not suggest that the cellulose sulphate/polydiallyldimethylammonium capsule can support the growth of other cell types (e.g. cells capable of producing viral particles) or allow viral particles to escape from such capsules.

The process for the preparation of cellulose sulphate capsules for use in the present invention is described in detail in DE 4021050A 1. The synthesis of cellulose sulphate is also described in this patent application. The comprehensive identification of cellulose sulfate vesicles has been studied in detail in "H.Dautzenberg et al, biomaterials technology, cell and its immobilization Biotechnology (Biomat., Art.cells & Immob.Biotech.), 21(3), 399-. Other cellulose sulphate capsules have been described in GB 2135954. The properties of the cellulose capsules, i.e. the size of the capsules, the size of the pores, the thickness of the walls and the mechanical properties, depend on several factors, such as the physical environment in which the capsules are prepared, the viscosity of the precipitating bath (precipitation bath), its ionic strength, the temperature, the rate of addition of the cell/cellulose sulphate suspension, the composition of the cellulose sulphate, and other parameters described by the Dautzenberg group.

Surprisingly, it has been found that by encapsulating cells in a polyelectrolyte complex, viral particles can be continuously produced from the implanted cells. Although the pore size of this capsule is large enough to allow antibodies and complements known to inactivate viruses (Welsh et al, 1975; Cornetta et al, 1990) to enter the capsule, we have not found evidence of a significant immune or inflammatory response, or necrosis in the vicinity of the implanted capsule. Furthermore, it has been surprisingly found that the vesicles prepared according to the invention migrate well into the host and become rapidly vascularized. Thus, the encapsulated cells according to the present invention can deliver viral vectors carrying therapeutic genes in vivo for a long period of time.

The invention includes in particular the following, alone or in combination:

an encapsulated cell capable of producing a viral particle comprising a core containing cells; and a porous capsule wall surrounding the core, the porous capsule wall being permeable to said viral particles;

the above encapsulated cell wherein the porous capsule wall is comprised of a polyelectrolyte complex formed from an oppositely charged polyelectrolyte;

the encapsulated cells as described above, wherein said porous capsule wall is composed of a polyelectrolyte complex formed from a sulfate group-containing polysaccharide or polysaccharide derivative or a sulfonic acid group-containing synthetic polymer, and a quaternary ammonium group-containing polymer;

the encapsulated cell as described above, wherein the sulfate group-containing polysaccharide or polysaccharide derivative is cellulose sulfate, cellulose acetate sulfate, carboxymethyl cellulose sulfate, dextran sulfate, or starch sulfate; wherein the synthetic polymer containing sulfonic acid groups is a polystyrene sulfonate;

encapsulated cells as described above wherein the quaternary ammonium group containing polymer is polydimethyldiallylammonium or polyvinylbenzyl-trimethylammonium;

the encapsulated cells as described above, wherein the porous capsule wall is composed of a complex of cellulose sulfate and polydiallylammonium dimethyl;

the encapsulated cells as described above have a capsule shape with a diameter of 0.01 to 5mm, preferably 0.1 to 1 mm;

the encapsulated cell as described above, wherein said capsule comprises an inner wall of said capsule formed by a spongy cellulose sulfate matrix and an outer surface of said capsule surrounded by a porous capsule skin; the spongy matrix is filled with cells;

the encapsulated cell has the pore diameter of the surface layer pores of the porous capsule wall of 80-150 nm, preferably 100-120 nm;

the encapsulated cell as described above, wherein the viral particle produced by the encapsulated cell is a retroviral particle comprising a retroviral vector genome;

an encapsulated cell as described above wherein the encapsulated cell producing retroviral particles is a packaging cell line transfected with an expression vector carrying a retroviral vector construct capable of infecting and directing the expression in a target cell of one or more coding sequences carried by the retroviral vector construct; said packaging cell line comprising at least one expression vector carrying a gene encoding a protein required for packaging of the retroviral vector construct;

the encapsulated cell as described above, wherein at least one of said coding sequences is a heterologous peptide encoding a gene selected from the group consisting of a marker gene, a therapeutic gene, an antiviral gene, an antitumor gene, and a cytokine gene;

the above encapsulated cell, wherein the marker gene is selected from the group consisting of marker genomes encoding proteins such as β -galactosidase, neomycin, alcohol dehydrogenase, puromycin, hypoxanthine phosphoribosyl transferase (HPRT), hygromycin, and secreted alkaline phosphatase; wherein the therapeutic gene is selected from the group consisting of genes encoding proteins such as herpes simplex virus thymidine kinase, cytosine deaminase, guanine phosphoribosyl transferase (gpt), cytochrome P450, etc., and cell cycle regulatory genes such as SDI, tumor suppressor genes encoding proteins such as P53, or antiproliferative genes encoding proteins such as melittin, cecropin, or cytokines (e.g., IL-2);

encapsulated cells as described above wherein the packaging cell line is selected from the group consisting of psi-2, psi-crypt, psi-AM, GP + E-86, PA317, and GP + envAM-12;

the encapsulated cell as described above wherein the expression vector transfected into the packaging cell line is pBAG, pLXSN, p125LX, pLX2B1, or pc3/2B1 or a derivative thereof;

a process for preparing the encapsulated cells described above comprises suspending cells capable of producing viral particles in an aqueous polyelectrolyte solution and then introducing the suspension of pre-processed particles into a precipitation bath containing an aqueous oppositely charged polyelectrolyte solution;

a process as above wherein the particles are formed by spraying;

a process as described above, wherein the cells are suspended in an aqueous solution of a sulfate group-containing polysaccharide or polysaccharide derivative, or a sulfonic acid group-containing synthetic polymer;

the process as described above, wherein the sulfate group-containing polysaccharide or polysaccharide derivative is selected from the group consisting of cellulose sulfate, cellulose acetate sulfate, carboxymethyl cellulose sulfate, dextran sulfate, and starch sulfate; wherein the synthetic polymer containing sulfonic acid groups is a polystyrene sulfonate;

a process as described above, wherein the precipitation bath contains an aqueous solution of a quaternary ammonium group-containing polymer;

a process as described above, wherein the polymer containing quaternary ammonium groups is polydimethyldiallylammonium or polyvinylbenzyl-trimethylammonium;

a process as described above, wherein the cells are suspended in an aqueous solution of sodium cellulose sulfate and introduced into a precipitation bath containing an aqueous solution of polydimethyldiallylammonium chloride;

a method as above, wherein the aqueous solution of cellulose sulphate consists of 0.5-50%, preferably 2-5%, sodium cellulose sulphate and 2-10%, preferably 5%, buffered salts of fetal bovine serum;

a process as above, wherein the aqueous solution in the precipitation bath consists of 0.5-50%, preferably 2-10%, or more preferably 3% of a buffered salt of polydimethyldiallylammonium chloride;

encapsulated cells produced by any of the processes described above;

the step of using any of the above encapsulated cells to deliver a gene to a target organ/cell, comprising:

a) culturing the encapsulated cells in a suitable medium, and

b) implanting encapsulated cells into a living animal (including a human);

the use as described above, wherein the target organ/cell is breast or pancreas; and

the above application, wherein the target organ/cell is smooth muscle cells and other cell types surrounding an artery.

It is an object of the present invention to provide encapsulated cells which produce viral particles which, when implanted in a host, permit release of viral particles produced by the cells from the capsules without eliciting a significant host immune or inflammatory response.

It is a further object of the invention to provide a process for producing such encapsulated cells which produces viral particles.

It is another object of the present invention to provide a method for delivering genes, particularly therapeutic genes, to target organs/cells by implanting such encapsulated cells that produce viral particles into a host, and thereby provide for the sustained production and release of viral particles in the target organs or in the vicinity of the target cells.

According to the present invention there is provided encapsulated cells capable of producing viral particles which, when implanted in a host, permit release of viral particles produced by the cells from the capsules without eliciting a significant host immune or inflammatory response.

According to the invention, encapsulated cells can be prepared by: cells capable of producing viral particles are suspended in an aqueous solution of a polyelectrolyte, such as a polysaccharide or polysaccharide derivative containing sulfate groups or a synthetic polymer containing sulfonic acid groups, and the suspension, pre-processed into particles, is then introduced into a precipitation bath containing an aqueous solution of an oppositely charged polyelectrolyte, such as a polymer containing quaternary ammonium groups.

The sulfate group-containing polysaccharide or polysaccharide derivative includes cellulose sulfate, cellulose acetate sulfate, carboxymethyl cellulose sulfate, dextran sulfate, or starch sulfate, in the form of a salt, particularly a sodium salt. Wherein the sulfonic acid group-containing synthetic polymer can be a polystyrene sulfonate, preferably a sodium salt.

The quaternary ammonium group-containing polymer includes polydimethyldiallylammonium or polyvinylbenzyl-trimethylammonium in the form of a salt thereof, preferably a chloride salt.

In a preferred embodiment of the invention, the cells capable of producing viral particles are encapsulated in a complex of cellulose sulphate and polydiallyldimethylammonium.

Such capsules are preferably prepared by suspending the virus particle producing cells in a solution containing 0.5-50%, preferably 2-5% sodium cellulose sulphate and 5% fetal bovine serum, optionally in a buffer solution, and then adding the suspension dropwise with a dispensing system (e.g. an air spray or piezo-electric system) to a precipitation bath containing 0.5-50%, preferably 2-10%, or more preferably around 3% polydimethyldiallylammonium chloride, optionally in a buffer solution, with stirring. The vesicles can be formed within milliseconds and the vesicles containing the cells are placed in the precipitation bath for a further 30 seconds to 5 minutes and then washed. This method is very rapid, ensuring that the cells are not excessively squeezed throughout the procedure (Stange et al, 1993).

The diameter of the capsule according to the invention is variable, between 0.01 and 5mm, but preferably between 0.1 and 1 mm. Thus, the vesicles may contain a different number of cells. Using the encapsulation process according to the invention, up to 10 can be achieved¹⁰Preferably, but preferably, it is 10⁵-10⁷The cells capable of producing virus particles are wrapped in the polyelectrolytesIn a proton complex.

Capsules made of cellulose sulphate and polydiallylammonium dimethyl have very good mechanical properties and the capsules produced are uniform in size and pore size.

The pore diameter of the capsule is 80-150 nm, preferably 100-120 nm.

The encapsulated cells can be maintained in normal cell culture medium (the performance of which depends on the encapsulated cells) at standard humidity, temperature, and CO₂Culturing is carried out under the condition of equal concentration. During the culture process, the viral particles produced from the vesicles are released into the cell culture medium, which can be confirmed by RT-PCR, or by transfecting cell-free supernatant (0.45 μm filter) into the target cells, and then by measuring the activity of the marker protein encoded by the gene carried by the viral vector structure contained in the viral particles. If the marker gene carried by the viral vector is one that confers tolerance to a particular compound on the target cell, the titer of the virus produced by the system can be determined.

After a suitable period of incubation (typically no less than 1 hour, no more than 30 days), such a cell-containing sac can be surgically implanted directly or injected by syringe into various parts of the body.

The viral particles produced by the encapsulated cells according to the invention may be based on any virus useful for gene therapy, including (but not limited to): adenovirus, adeno-associated virus, herpes virus, or retrovirus. See for review "Gunzburg and Salmonons, 1995".

In a preferred embodiment of the invention, the encapsulated cell is a packaging cell line producing retroviral particles containing a genome carrying retroviral vector structures for the marker and/or therapeutic gene.

Retroviral vector systems consist of two components:

1) an expression vector (vector plasmid) carrying a retroviral vector construct, the vector plasmid being a modified retrovirus in which the genes encoding the viral proteins have been replaced by a therapeutic gene (optionally including a marker gene) to be transferred to a target cell. Since replacement of the genes encoding the viral proteins will effectively damage the virus, it is necessary to rescue the modified retrovirus with a second component in the system which provides the modified retrovirus with its missing viral proteins.

The second component is:

2) a cell line that produces large amounts of viral proteins but lacks the ability to produce replication-competent virus. This cell line is called the packaging cell line and is composed of a cell line transfected with a plasmid carrying a gene enabling the packaging of the modified retroviral genome. These plasmids direct synthesis of viral proteins necessary for viral particle production.

To generate the packaged retroviral vector, the vector plasmid is transfected into a packaging cell line. In this case, the modified retroviral genome containing the inserted therapeutic gene and optionally the marker gene is transcribed from the vector plasmid and the resulting modified retroviral genome is packaged into a retroviral particle. Cells infected with such viral particles are incapable of producing viral particles because viral proteins are not present in these cells. However, retroviral vector constructs exist which carry therapeutic and marker genes and are now expressed in infected cells.

WO 94/29437, WO 89/11539, and WO 96/07748 describe other types of retroviral vector systems useful for the purposes of the present invention.

The viral particles produced by the encapsulated cells according to the invention can be constructed so that they contain the genome of a viral vector carrying a gene encoding a marker gene and/or a therapeutic gene.

The marker gene or therapeutic gene carried by the viral vector can be, for example: genes encoding proteins such as β -galactosidase, neomycin, alcohol dehydrogenase, puromycin, hypoxanthine phosphoribosyl transferase (HPRT), hygromycin, and secreted alkaline phosphatase, or therapeutic genes encoding proteins such as herpes simplex virus thymidine kinase, cytosine deacylase, guanine phosphoribosyl transferase (gpt), cytochrome P450, cell cycle regulatory genes such as SDI, tumor suppressor genes encoding proteins such as P53, or antiproliferative genes encoding proteins such as melittin, cecropin, or cytokines (e.g., IL-2).

In a particular embodiment, the invention relates to the use of the encapsulated cells according to the invention in the treatment of tumors.

Many malignancies do not respond well to chemotherapy. In most cases, anticancer drugs for treating tumors are administered systemically, thereby spreading throughout the body of the patient. The need for such systemic high doses of drugs for cancer treatment often entails uncomfortable side effects for the patient. One strategy to address the problem of systemic high concentrations of anticancer drugs is to directly apply or activate the drug in or near the tumor. This is achieved by implanting into the tumour cells or cells in the vicinity thereof encapsulated cells according to the invention which produce viral particles comprising the genome of an engineered virus, particularly a retroviral vector, carrying genes encoding anti-cancer drugs, for example active enzymes capable of converting a prodrug into a cytotoxic agent.

In one embodiment of the invention, encapsulated cells are provided that produce retroviral particles comprising a retroviral vector genome carrying a tumor phase enzyme (e.g., cytochrome P450) gene or a suicide gene (e.g., but not limited to, thymidine kinase that converts non-toxic drugs into one or more toxic metabolites). Such encapsulated cells of the invention can be implanted into or near a tumor (in the pancreas or breast) and thus can be used to treat cancer.

Other targeting of specific cell types may also be performed using target cell-specific regulators and promoters that direct expression of the associated therapeutic gene.

Such target cell-specific regulators and promoters include: for example, pancreas-specific regulators and promoters, including carbonic anhydrase II and β -glucokinase regulators and promoters; lymphocyte-specific regulators and promoters, including immunoglobulin and MMTV lymphocyte-specific regulators and promoters; mammary gland specific regulators and promoters including Whey Acidic Protein (WAP), Murine Mammary Tumor Virus (MMTV) β -lactoglobulin, and casein specific regulators and promoters; and MMTV-specific regulators and promoters that can confer responsiveness to glucocorticoids or direct expression in the mammary gland. Other promoters include: for example, the CD4, CD34, and IL2 promoters. The regulatory element and promoter have a preferred regulatory effect on the expression of the retroviral vector.

Inducible promoters may also be utilized, such as radiation-inducible promoters, for example: the intercellular adhesion molecule-1 (ICAM-1) promoter, the Epidermal Growth Factor Receptor (EGFR) promoter and the Tumor Necrosis (TNF) promoter.

The following examples further illustrate the invention but are not to be construed as limiting it:

example 1

Lipofection (lipofection) of PA317 with pBAG and isolation of G418-resistant cells

Amphibian NIH3T 3-based PA317 packaging cells (Miller and Buttimore, 1986) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum. One day before lipofection, cells were seeded at a density of 3X 10 in a 10cm tissue culture dish⁵And (4) cells. Then 2. mu.g of pBAG vector carrying MLV-based (murine leukemia virus) retrovirus vector (Price et al, 1987),lipofection into packaging cells was performed using lipofectamine (lipofectamine) kit from GIBCO/BRL according to the manufacturer's instructions. The cells were then diluted 1: 10 and cultured in normal medium supplemented with 400. mu.g/ml G418 (GIBCO/BRL). After 14 days of culture, clones of G418-tolerant cells were pooled.

Example 2

Being encapsulated:

will 10⁷The cells were suspended in 1ml of a buffer salt solution containing 2-5% sodium cellulose sulfate and 5% fetal bovine serum, and then the suspension was added dropwise to a precipitation bath of a buffer solution containing 2-3% polydiallylammonium dimethyl by using a dispensing system (air spray system). Within milliseconds, capsule formation can occur, followed by further formation of a more porous inner layer for mechanical support, which consists essentially of cellulose sulfate. The cell-containing vesicles were left in the precipitating bath for 30 seconds to 5 minutes and then washed with DMEM (Stange et al, 1993). Samples were taken under different parameters (i.e. sodium cellulose sulphate concentration, flow rate of the air-spray system, and time in the precipitation bath, as described above) for biological studies. Examples of representative conditions are: e.g. 2.5% sodium cellulose sulphate, 2% polydiallyldimethylammonium in the precipitation bath for 1 minute, or 1.5% sodium cellulose sulphate, 2% polydiallyldimethylammonium in the precipitation bath for 0.5 minute, or 3% sodium cellulose sulphate, 3% polydiallyldimethylammonium in the precipitation bath for 2 minutes. The exact size of the desired capsule, the thickness of the capsule wall, and other characteristics should also be taken into account when choosing the exact parameters.

Example 3

Implantation of the capsule into the mammary gland of mice:

the pouch was inserted into the mammary gland of a 2-month old BALB/c female mouse using a "keyhole" procedure, and a needle was sutured to the entrance site. Up to 6 balloons with a diameter of 0.5-2mm can be inserted per surgical site.

In vitro studies of virus release from the bursa, the structure of the bursa and the effect of implanting the bursa in immunocompetent mice were studied as follows:

A) beta-galactosidase Activity

Infected cells were detected by histochemical staining as described in the literature (Cepko, 1989). Cells, vesicles or tissue sections were washed with pre-cooled PBS and then fixed with 2% paraformaldehyde solution for 20 minutes to 24 hours depending on the specimen thickness. After extensive washing with PBS, the cells, vesicles or tissue sections are placed in a solution containing the substrate X-gal (20mM K)₃FeCN₆，20mM K₄FeCN₆·3H₂O，2mM MgCl₂And 1mg/m 1X-gal) at 37 ℃ for at least 2 hours.

B) Infection with viral infection

6 hours before infection, 4X 10⁴Individual target cells were seeded into 6-well tissue culture plates. The capsules containing the virus-producing cells were placed on top of the target cells and polybrene (8. mu.g/ml) was added to the medium. After 4 hours, the medium was changed to remove residual polybrene. After 5 days, some of the wells were stained for β -galactosidase activity as described above, and the remaining wells were trypsinized, transferred to larger tissue culture dishes and cultured in medium containing 400 μ G/ml G418. G418 tolerant clones were tested after 16 days.

C) RT-PCR analysis

5ml of the sac medium supernatant was ultracentrifuged (240,000 Xg, 1 hour, 4 ℃) to pellet the virus particles. The pellet was suspended in lysis buffer (1% Triton-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, PBS), and RNA was extracted by phenol extraction and ethanol precipitation as described in the literature (Salmonons et al, 1986). The RNA was then reverse transcribed into DNA using the Ready-To-Go T-printed first Strand kit (Pharmacia). PCR amplification was then performed using primers located within the envelope region (env) and R region of the LTR of the MLV-derived BAG vector (FIG. 2). PCR amplification was performed in 100. mu.l reaction containing 500mM KCl, 10mM Tris-HCl (pH 8).3)、1.5mM MgCl₂0.01% (w/v) gelatin, 100. mu.M dNTPs, 40pM primers, and 2.5 units Taq polymerase (Perkinelmer). The reaction was carried out in a DNA amplification apparatus model 9600 from Perkin Elmer under the following conditions: 1 minute at 94 ℃; at 53 ℃ for 2 minutes; 72 ℃ for 3 minutes for 35 cycles. The PCR products were separated by 0.8% agarose gel electrophoresis, transferred to a Zeta Probe Membrane (BIORAD) as described in the literature (Inderaccolo et al, 1995) and hybridized with a 32P-labelled PCR fragment of the 612bp MLV genome generated from the same primers and using pBAG as a template as described in the literature (Inderaccolo et al, 1995). MLV-specific sequences were shown using Fuji (Fuji) phosphoimaging system (BAS 1000).

D) PCR analysis

Genomic DNA (1. mu.g) was amplified by PCR using one primer located within the residual env sequence of the BAG vector and a second primer located in the polyoma region of the plasmid outside the retroviral vector sequence (FIG. 3B). The PCR reaction was carried out as described above, using the following reaction conditions: 1 minute at 94 ℃; 2 minutes at 50 ℃; at 68 ℃ for 3 minutes, for 35 cycles. PCR products and³²a1.5 kb Xbal DNA fragment from pBAG, which is specific for polyoma sequences, was hybridized with P-tag.

F) Electron microscope

Specimens for Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) experiments were rinsed with PBS (pH7.35) and pre-fixed in 1% glutaraldehyde-containing PBS for 15 minutes, then in 2% OsO₄Medium fixation for 15 minutes. The samples were dehydrated with a gradient grade series of ethanol and then divided into two groups. a) CO for SEM sample₂Critical-point-dried and wrapped with 1-3nm platinum (Emscope SC 500; Ashford, England). The wrapped specimen was examined in a 10kV field emission scanning electron microscope (JeoolJSM-6300F; Tokyo, Japan) using an accelerating voltage of 5-10kV in the second mode. b) TEM samples were embedded in Epon and the microtomes were double stained with uranyl acetate and lead citrate and examined in a Zeiss EM-10C (Oberkochen, Germany) transmission electron microscopeAnd (6) observing.

Results

In vitro study of virus release from bursa:

the vesicles obtained from example 2 were stained for cells in the vesicles with substrate X-gal as described in the "beta-galactosidase activity" assay above. The results show that cells express the β -galactosidase gene encoded by pBAG (FIG. 1), and that unencapsulated vector-producing cells also express the β -galactosidase gene (not shown).

To confirm that viral particles can be released from cells within the bursa into the cell culture medium, viral particles were pelleted from the bursa supernatant cultured for various times and RNA was prepared. The RNA was then subjected to RT-PCR analysis using primers complementary to the env and R regions of the virus as described above for "RT-PCR analysis". A PCR fragment (612bp) of the expected size specific for MLV was observed in the bursa culture medium for at least 6 weeks (FIG. 2B, lanes 1-4), and no further analysis was performed after 6 weeks. This fragment was not due to contamination with DNA, since there was no signal if viral RNA was pretreated with RNase prior to RT-PCR (FIG. 2C; lanes 1-4).

To confirm the production and release of infectious virus from the bursa, the bursa was co-cultured with target cells, such as NIH3T3 cells (Jaincall et al, 1969) or CRFK cells (Crandell et al, 1973), as described in the "infection" assay above. After 4 days of co-culture, 1 sample of target cells and 1 sample of encapsulated packaging cells were stained for β -galactosidase activity as described above. The remaining target cells were used to screen for their G418 tolerance. Many co-cultured NIH3T3 cells and CRFK cells were shown to express the β -gal gene (FIG. 3A).

To confirm that the target cells have acquired the β -gal gene by infection, the target cells were subjected to PCR detection. A packaging cell line that can produce BAG is the PA317 cell line (Miller and Buttimore, 1986), which carries the herpes simplex virus thymokinase (HSV-TK) gene, the product of which converts the prodrug ganciclovir (ganciclovir) into a cytotoxic drug. NIH3T3 or CFRK target cells do not normally carry this gene. In an experiment in which co-cultured NIH3T3 and CFRK target cells demonstrating β -gal gene expression were able to tolerate GCV, to confirm that the escape of BAG-producing cells did not occur in this experiment, genomic DNA was extracted from the target cells and its plasmid sequence outside the vector was analyzed by PCR (PCR analysis above). These sequences are present in packaging cells, since the BAG vector is lipofected into these cells in the form of the plasmid pBAG. However, the viruses produced by the packaging cells do not carry these sequences and therefore these sequences are not present in the infected target cells, and FIG. 3B shows that no such plasmid sequences are detected, consistent with infection of these cells with a BAG vector.

The structure of the capsule:

sections of the bursa were prepared and analyzed for structure by electron microscopy. The interior of the sac consists of a spongy matrix, which is filled with cells (fig. 4). Scanning electron microscopy revealed that there were small pores on the surface of the vesicles, which were large enough to allow the retroviral particles to be released from the vesicles, since the white bars represent the average diameter of the retroviral particles (FIG. 4).

In vivo stability in immunocompetent mice:

to determine the in vivo stability of the bursa and whether the bursa or the virus it produces elicits a significant immune response, the bursa was implanted into the mammary glands of two-month-old BALB/c female mice. Mice were sacrificed at various times post-implantation to assess the fate of the bursa and whether infectious virus was produced. At least 6 weeks after implantation, the embedding of the implanted capsule in the mammary fat pad is clearly visible (fig. 5A). Interestingly, in all animals analyzed, vascularization occurred in the vicinity of the bursa (FIG. 5A), presumably as a result of production of angiogenic or growth factors by the packaging cells.

Sections through the pouch and surrounding breast tissue confirmed that blood vessels were found in the immediate vicinity of the pouch (fig. 5B). These sections also showed a layer of connective tissue between the bursa and the mammary cells. There is no evidence of a significant inflammatory or immune response to the bursa or to the virus producing cells it contains.

To confirm that infectious retroviral vector particles have been released from the bursa and have infected the surrounding mammary tissue, some sections of them were analyzed for β -gal expression using X-gal staining. The staining results clearly showed cells expressing the β -gal gene outside the bursa (FIG. 5C).

Example 4

This example describes the construction of a retroviral vector containing the murine cytochrome P4502B 1 gene for intratumoral infection.

The expression vector pLX2B1 (see FIG. 6) was constructed by ligating fragments obtained from the pLX125 plasmid and the pSW1 plasmid (Kedzie et al, 1991). The pLX125 plasmid was linearized with HpaI enzyme and the resulting blunt ends were dephosphorylated with calf intestinal phosphatase. The purified DNA was isolated on a 1% agarose gel and the DNA was cut and prepared using the Qiaquick protocol (Qiagen). Ethanol precipitation of DNA, then DNA heavy suspension in water.

The pSW1 cloning vector was digested with SmaI and HindII to generate two blunt-ended fragments. The digestion mixture was separated on a 1% agarose gel. The shortest fragment (1.5kb) containing the murine cytochrome P4502B 1 cDNA (Fuji-Kuriyama et al, 1982) was cut and eluted using the Qiaquick DNA extraction protocol, ethanol precipitated and resuspended in water.

The SamI/HindII-cut pSW1 fragments of 7.6fMols pLX125 and 24fMols were mixed and ligated with T4-ligase (Boehringer) at 12 ℃ for 3 days. The ligase was inactivated at 65 ℃ for 10 minutes. The DNA was precipitated with 10 volumes of butanol. The DNA pellet was resuspended in water and electroporated into DH 10B-bacteria (Gibco). Ampicillin-resistant clones were screened, DNA was prepared, and digestion experiments were performed with SspBI/SalI, BamHI/SspBI, PvuI, and BamHI. The final correct plasmid was designated pLX2B 1.

Lipofection

One day before lipofection, 3X 10 cells were transfected⁵A single PA317 retroviral packaging cell (Miller and Buttimore, 1986) was seeded into 6cm dishes. On the day of infection, 2. mu.g of pLX2B1 were mixed with 100. mu.l of serum-free medium. Mu.l Lipofectamine (Lipofectamine) (Gibco BRL) was mixed with 100. mu.l serum-free medium at the same time. The plasmid-containing solution was added to the lipofectamine-mixture and incubated for 45 minutes. At 35 min of incubation, the packaging cells were washed once with 2ml serum-free medium. Mu.l of serum-free medium was added to the lipofection-mixture and 1ml of the resulting solution was added to the prepared packaging cells. After 6 hours, 1ml of Dulbecco's modified Eagle's medium containing 10% FCS was added. The next day the cells were trypsinized and diluted 1: 10 before being plated into a 100mm petri dish. After 24 hours, the medium was replaced with the medium containing neomycin analogue G418. Individual cell clones or cell populations are isolated and analyzed for cytochrome P450 expression.

Is encapsulated into a capsule

The resulting packaging cells capable of producing retroviral vectors are packaged into vesicles as described in example 2 above.

Implant

The resulting vesicles were surgically introduced into or near the transplanted or spontaneous tumor of BALB/c or GR mice using a "keyhole" procedure. Approximately 6 balloons with a diameter of 1mm were inserted per surgical site. 1 needle was sutured at the surgical site. Then the mice were treated locally with cyclophosphamide or ifophamide and 100 μ l of 20mg/ml drug was injected directly into the tumor; or systemic treatment, 130mg CPA/kg body weight, intraperitoneal injection, and 40-60mg IFO/kg body weight, intraperitoneal injection, with a treatment period of up to 10 weeks. During this period, the size and apparent shape of the tumor was monitored daily. The mice were then sacrificed and the tissue containing the inserted bursa was removed and prepared into histological sections for optical and electron microscopy. These sections clearly show that the capsule is well implanted, vascularized, and there is no evidence of the presence of lymphocytes indicative of a cellular immune response. In contrast, tumors showed necrosis, with a marked reduction in the size of the tumor during the test, in appearance.

Example 5

This example describes the construction of a stable cell line expressing murine cytochrome P4502B 1.

The pc3/2B1 expression vector was constructed by ligation of fragments obtained from pcDNA3(Invitrogen) and the pSW1 plasmid (Kedzie et al, 1991).

The pcDNA3 plasmid was digested with XhoI/XbaI and the resulting sticky end fragment dephosphorylated with calf intestinal phosphatase. Vector backbone DNA was purified by separation on a 1% agarose gel, and prepared by cutting using Qiaquick protocol (Qiagen). Ethanol precipitation, then water heavy suspension DNA.

The cloning vector pSW1 was digested with XhoI and XbaI to give two fragments. The digestion mixture was separated on a 1% agarose gel. The shortest fragment (1.5kb) containing the murine cytochrome P4502B 1 cDNA (Fuji-Kuriyama et al, 1982) was cleaved and eluted using the Qiaquick DNA extraction protocol, precipitated with ethanol and resuspended in water.

The 8.3fMols pcDNA3 backbone and the 24.8fMols pSW1 XhoI/XbaI-fragment were mixed together and ligated with T4-ligase (Boehringer) at 12 ℃ for 1 day. The ligase was inactivated at 65 ℃ for 10 minutes, and then the DNA was precipitated with 10 volumes of butanol. The DNA pellet was resuspended in water and electroporated into DH 10B-bacteria (Gibco). Ampicillin-resistant clones were screened, DNA was prepared and digestion experiments were performed with EcoRI, BamHI, EcoRV and XhoI. The final correct plasmid was designated pc3/2B 1.

Lipofection

The day before infection, 3X 10⁵Individual NIH3T3 cells were seeded into 35mm dishes. On the day of infection, 2. mu.g of pc3/2B1 was mixed with100 μ l serum-free medium. At the same time, 15. mu.l Lipofectamine (Lipofectamine) was mixed with 100. mu.l serum-free medium. The plasmid-containing solution was added to the lipofectamine-mixture and incubated for 45 minutes. During 35 min of incubation, cells were washed once with 2ml serum-free medium. Mu.l of serum-free medium was added to the lipofection-mixture and 1ml of the resulting solution was added to the prepared cells. After 6 hours, 1ml of DMEM (Glutamax) containing 10% FCS was added. The next day the cells were trypsinized and diluted 1: 10 before being plated into a 100mm petri dish. After 24 hours, the medium was replaced with neomycin-containing medium. After 14 days, neomycin-resistant clones were isolated and tested for the presence and activity of the vector.

Capsules containing these cells were prepared and implanted near the tumor site in mice as described in example 2. Treatment with cyclophosphamide or ifosfamide was evaluated for efficacy as described above.

Illustration of the drawings

FIG. 1: histological staining of pBAG-stably transfected encapsulated PA317 cells confirmed the expression of beta-galactosidase.

FIG. 2: RT-PCR analysis of viral particles released from vesicles.

(2B; lanes 1-4: medium after 2, 3, 5, 6 weeks of sac culture). Cell culture medium of non-encapsulated BAG virus-producing cells served as positive control (lane 5) and culture medium of non-transfected PA317 as negative control (lane 6). If the viral sample is digested with RNase before RT-PCR analysis, no signal is observed, ensuring that the amplified band is from viral RNA (2C; lanes 1-4). Viral RNA prepared from non-encapsulated BAG virus-producing cells without RNase treatment was used as a positive control in RT-PCR (lane 7).

FIG. 3: the encapsulated virus-producing cells were co-cultured with NIH3T3 cells.

(A) The day before the addition of the encapsidation of the packaging cells containing the retroviral vector producer, the target cells are seeded at low density. After several days, both cells and vesicles were histologically stained to determine their β -galactosidase activity.

(B) To confirm that the β -galactosidase expression target cells in a were the result of infection, and not due to escape from virus-producing cells, genomic DNA was extracted from the cells and subjected to PCR analysis.

FIG. 4: the capsule surface, as revealed by scanning electron microscopy, was visualized under high magnification as a pinhole-like structure. White bars indicate 100nm (diameter of the retroviral particle), indicating that these structures may represent small pores through which the virus may be released from the capsule.

FIG. 5: histological analysis of the bursa implanted in the mammary gland of mice. (A)

(B) Light microscope (133 magnification) of a section through one capsule at 4 weeks of implantation into the mammary gland of a mouse

(C) Infection of mouse mammary cells after implantation of a pouch containing BAG vector-generated PA317 cells.

FIG. 6: the structure of pLX2B1 expression vector containing the murine cytochrome P4502B 1 gene, which will be controlled by the U3-MMTV promoter after promoter conversion.

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Claims (36)

1. An encapsulated, encapsulated retroviral packaging cell capable of producing a retroviral particle, said capsule comprising a core containing said cell; and a porous capsule wall surrounding said core and permeable to said viral particles.

2. Encapsulated cells according to claim 1 wherein said porous capsule wall comprises a polyelectrolyte complex formed from an oppositely charged polyelectrolyte.

3. Encapsulated cells according to claim 1 or 2 wherein said porous capsule wall comprises a polyelectrolyte complex formed from a sulfate group-containing polysaccharide or polysaccharide derivative or a sulfonic acid group-containing synthetic polymer, and a quaternary ammonium group-containing polymer.

4. Encapsulated cells according to claim 3 wherein the sulphate group containing polysaccharide or polysaccharide derivative is selected from one or more of the following: cellulose sulfate, cellulose acetate sulfate, carboxymethyl cellulose sulfate, dextran sulfate, or starch sulfate; wherein the synthetic polymer containing sulfonic acid groups is a polystyrene sulfonate.

5. Encapsulated cells according to claim 3 or 4 wherein the polymer containing quaternary ammonium groups is polydimethyldiallylammonium or polyvinylbenzyl-trimethylammonium.

6. Encapsulated cells according to any of claims 1 to 5 wherein the porous capsule wall comprises a complex formed from cellulose sulphate and polydimethyldiallylammonium.

7. Encapsulated cells according to any of claims 1 to 6 in the form of microcapsules having a diameter of 0.01 to 5 mm.

8. Encapsulated cells according to claim 7 in the form of microcapsules of 0.1 to 1mm diameter.

9. Encapsulated cells according to any of claims 1 to 8 wherein said capsule comprises a sponge matrix forming the inner wall of the capsule surrounded by a foraminous wall; the sponge matrix is filled with cells.

10. Encapsulated cells according to any one of claims 1 to 9 wherein the pores on the surface of the porous capsule wall have a pore size of 80 to 150 nm.

11. Encapsulated cells according to claim 10 wherein the pores on the surface of the porous capsule wall have a pore size of 100 to 120 nm.

12. An encapsulated cell as claimed in any one of claims 1 to 11 wherein the retroviral particle produced by the encapsulated cell comprises a retroviral vector genome.

13. Encapsulated cell according to claim 12 wherein the encapsulated cell producing retroviral particles is a packaging cell line transfected with an expression vector carrying a retroviral vector construct capable of infecting and directing the expression in a target cell of one or more coding sequences carried by the retroviral vector construct; the packaging cell line comprises at least one expression vector carrying a gene encoding a protein required for packaging of the retroviral vector construct.

14. Encapsulated cells according to claim 13 wherein at least one of said coding sequences is a gene encoding a heterologous polypeptide selected from the group consisting of a marker gene, a therapeutic gene, an antiviral gene, an antitumor gene, or a cytokine gene.

15. Encapsulated cells according to claim 14 wherein said marker gene is selected from the group consisting of marker genes encoding β -galactosidase, neomycin, alcohol dehydrogenase, puromycin, hypoxanthine phosphoribosyl transferase (HPRT), hygromycin, and secreted alkaline phosphatase; wherein the therapeutic gene is selected from genes encoding herpes simplex virus thymidine kinase, cytosine deaminase, guanine phosphoribosyl transferase (gpt), cytochrome P450, cell cycle regulatory genes, tumor suppressor genes, cytokine genes or anti-proliferative genes.

16. Encapsulated cells according to claim 15 wherein said cell cycle regulatory gene is SDI or said tumor suppressor gene encodes p53 protein or said antiproliferative gene encodes a bacitracin or cecropin, or said cytokine is IL-2.

17. Encapsulated cells according to claim 13 wherein the packaging cell line is selected from the group consisting of psi-2, psi-crypt, psi-AM, GP + E-86, PA317, and GP + envAM-12.

18. Encapsulated cell according to any one of claims 12 to 17 wherein the expression vector is pBAG, pLXSN, p125LX, pLX2B1, or pc3/2B1 or a derivative thereof.

19. A method of preparing encapsulated cells according to any of claims 1 to 18, comprising suspending cells capable of producing viral particles in an aqueous polyelectrolyte solution and then introducing the suspension in the form of preformed particles into a precipitation bath containing an aqueous solution of an oppositely charged polyelectrolyte.

20. The method of claim 19 wherein the particles are formed by spraying.

21. A method according to claim 19 or 20 wherein the cells are suspended in an aqueous solution of a sulphate-containing polysaccharide or polysaccharide derivative or an aqueous solution of a sulphonic acid-containing synthetic polymer.

22. A process according to claim 21 wherein the sulphate group-containing polysaccharide or polysaccharide derivative is selected from one or more of the following: cellulose sulfate, cellulose acetate sulfate, carboxymethyl cellulose sulfate, dextran sulfate, or starch sulfate; wherein the synthetic polymer containing sulfonic acid groups is a polystyrene sulfonate.

23. A method according to claim 19 or 20, wherein the precipitation bath comprises an aqueous solution of a polymer containing quaternary ammonium groups.

24. The method according to claim 23, wherein the polymer containing quaternary ammonium groups is polydimethyldiallylammonium or polyvinylbenzyl-trimethylammonium.

25. A method according to claim 19 or 20, wherein the cells are suspended in an aqueous solution of sodium cellulose sulphate and introduced into a precipitation bath containing an aqueous solution of polydimethyldiallylammonium chloride.

26. The method according to claim 25, wherein the aqueous solution of cellulose sulfate is composed of 0.5-50% sodium cellulose sulfate and 2-10% fetal bovine serum phosphate buffer.

27. The method according to claim 26, wherein the concentration of sodium cellulose sulfate is 2-5% or the concentration of fetal bovine serum is 5%.

28. The process according to claim 25, wherein the aqueous solution in the precipitation bath consists of 0.5-50% of polydiallyldimethylammonium chloride in phosphate buffer.

29. The method according to claim 28, wherein the aqueous solution in the precipitation bath consists of 2-10% polydiallyldimethylammonium chloride in phosphate buffer.

30. The method according to claim 29, wherein the aqueous solution in the precipitation bath consists of 3% phosphate buffer of polydimethyldiallylammonium chloride.

31. Encapsulated cells according to any of claims 1 to 18, produced according to the method of any of claims 19 to 30.

32. Use of the encapsulated cells of any one of claims 1 to 18 or 31 for the preparation of a pharmaceutical composition.

33. A pharmaceutical composition comprising the encapsulated cells of any one of claims 1-18 or 31.

34. The pharmaceutical composition of claim 33, in a dosage form suitable for injection and/or suitable for implantation into a target organ/cell and/or in proximity to a target organ/cell.

35. The pharmaceutical composition of claim 34, wherein said target organ/cell is a cancerous tissue.

36. The pharmaceutical composition of claim 34 or 35, wherein the target organ/cell is a smooth muscle cell of breast, pancreas, or periarterial.