WO1999011761A1 - Suspension culture of retroviral producer cells - Google Patents

Abstract

A suspension culture capable of producing a retrovirus or a recombinant retroviral vector is described. Preferably, the retroviral vector comprises at least one NOI.

Description

SUSPENSION CULTURE OF RETOV IRAL P RODUCER CELLS

The present invention relates to a process. In particular, the present invention relates to a process for producing retroviral vectors.

It is well known that retroviruses have potential for delivering nucleotide sequences of interest (NOIs) into cells for in vitro and/or in vivo applications, such as gene therapy.

Gene therapy may include any one or more of: the addition, the replacement, the deletion, the supplementation, the manipulation etc. of one or more nucleotide sequences in, for example, one or more targeted sites - such as targeted cells. If the targeted sites are targeted cells, then the cells may be part of a tissue or an organ. General teachings on gene therapy may be found in Molecular Biology (Ed Robert Meyers, Pub VCH, such as pages 556-558).

By way of further example, gene therapy can also provide a means by which any one or more of: a nucleotide sequence, such as a gene, can be applied to replace or supplement a defective gene; a pathogenic nucleotide sequence, such as a gene, or expression product thereof can be eliminated; a nucleotide sequence, such as a gene, or expression product thereof, can be added or introduced in order, for example, to create a more favourable phenotype; a nucleotide sequence, such as a gene, or expression product thereof can be added or introduced, for example, for selection purposes (i.e. to select transformed cells and the like over non-transformed cells); cells can be manipulated at the molecular level to treat, cure or prevent disease conditions - such as cancer (Schmidt-Wolf and Schmidt-Wolf, 1994, Annals of Hematology 69;273-279) or other disease conditions, such as immune, cardiovascular, neurological, inflammatory or infectious disorders; antigens can be manipulated and/ or introduced to elicit an immune response, such as genetic vaccination.

In recent years, retroviruses have been proposed for use in gene therapy. Indeed, Maulik and Patel 1997 (Molecular Biotechnology, Therapeutic Applications and Strategies, Published by Wiley-Liss, Chapter 2 page 41) state that:

"The unique life cycles of retroviruses and their compact genome (which makes engineering of the constructs practically easier) make them ideal vectors for gene therapy. "

When used in, for example, gene therapy applications, such retroviruses are usually called retroviral vectors or recombinant retroviral vectors. Essentially, retroviruses are RNA viruses with a life cycle different to that of lytic viruses. In this regard, a retrovirus is an infectious entity that replicates through a DNA intermediate. When a retrovirus infects a cell, its genome is converted to a DNA form by a reverse transcriptase enzyme. The DNA copy serves as a template for the production of new RNA genomes and virally encoded proteins necessary for the assembly of infectious viral particles.

There are many retroviruses and examples include: murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV).

A detailed list of retroviruses may be found in Coffin et al ("Retroviruses" 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758- 763).

Details on the genomic structure of some retroviruses may be found in the art. By way of example, details on HIV may be found from the NCBI Genbank (i.e. Genome Accession No. AF033819). Essentially, all wild type retroviruses contain three major coding domains, gag, pol, env, which code for essential virion proteins. Nevertheless, retroviruses may be broadly divided into two categories: namely, "simple" and "complex" . These categories are distinguishable by the organisation of their genomes. Simple retroviruses usually carry only elementary information. In contrast, complex retroviruses also code for additional regulatory proteins derived from multiple spliced messages.

Retroviruses may even be further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are the lentiviruses and the spumaviruses. A review of these retroviruses is presented in "Retroviruses" (1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 1-25).

All oncogenic members except the human T-cell leukemia virus-bovine leukemia virus group (HTLV-BLV) are simple retroviruses. HTLV, BLV and the lentiviruses and spumaviruses are complex. Some of the best studied oncogenic retroviruses are Rous sarcoma virus (RSV), mouse mammary tumour virus (MMTV) and murine leukemia virus (MLV) and the human T-cell leukemia virus (HTLV).

The lentivirus group can be split even further into "primate" and "non-primate" . Examples of primate lentiviruses include the human immunodeficiency virus (HIV), the causative agent of human acquired-immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV). The non-primate lentiviral group includes the prototype "slow virus" visna/maedi virus (VMV), as well as the related caprine arthritis- encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

A distinction between the lentivirus family and other types of retroviruses is that lentiviruses have the capability to infect both dividing and non-dividing cells (Lewis et al 1992 EMBO. J 11: 3053-3058; Lewis and Emerman 1994 J. Virol. 68: 510-516). In contrast, other retroviruses - such as MLV - are unable to infect non-dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.

During the process of infection, a retrovirus initially attaches to a specific cell surface receptor. On entry into the susceptible host cell, the retroviral RNA genome is then copied to DNA by the virally encoded reverse transcriptase which is carried inside the parent virus. This DNA is transported to the host cell nucleus where it subsequently integrates into the host genome. At this stage, it is typically referred to as the provirus. The provirus is stable in the host chromosome during cell division and is transcribed like other cellular proteins. The provirus encodes the proteins and packaging machinery required to make more virus, which can leave the cell by a process sometimes called "budding" . In a defective retroviral vector genome gag, pol and env may be absent or not functional.

As mentioned earlier, retroviruses have been proposed as a delivery system (otherwise expressed as a delivery vehicle or delivery vector) for inter alia the transfer of a NOI, or a plurality of NOIs, to one or more sites of interest. The transfer can occur in vitro, ex vivo, in vivo, or combinations thereof. Typically, recombinant retroviral vectors do not comprise the entire viral protein encoding regions. In this regard, typically, all or some of those regions are replaced with one or more NOIs. These recombinant retroviral vectors are usually unable to propagate themselves. The recombinant retroviral vectors, however, usually comprise sufficient regions that enable the vectors to integrate into a target genome. Propagation of the vectors is often achieved by use of a packaging cell line or a helper cell line. When used in this fashion, the retroviruses are typically called retroviral vectors or recombinant retroviral vectors. Retroviral vectors have even been exploited to study various aspects of the retrovirus life cycle, including receptor usage, reverse transcription and RNA packaging (reviewed by Miller, 1992 Curr Top Microbiol Immunol 158: 1-24).

Further introductory teachings on recombinant retroviral vectors may be found in Maulik and Patel 1997 {ibid, Chapter 2 pages 41-47). In a typical recombinant retroviral vector for use in gene therapy, at least part of one or more of the gag, pol and env protein coding regions may be removed from the virus. This makes the retroviral vector replication-defective. The removed portions may even be replaced by a NOI in order to generate a virus capable of integrating its genome into a host genome but wherein the modified viral genome is unable to propagate itself due to a lack of structural proteins. When integrated in the host genome, expression of the NOI occurs - resulting in, for example, a therapeutic and/or a diagnostic effect. Thus, the transfer of a NOI into a site of interest is typically achieved by: integrating the NOI into the recombinant viral vector; packaging the modified viral vector into a virion coat; and allowing transduction of a site of interest - such as a targeted cell or a targeted cell population.

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

In some instances, propagation and isolation may entail isolation of the retroviral gag, pol and env genes and their separate introduction into a host cell to produce a "packaging cell line" . The packaging cell line, or "helper " genome as it is sometimes called, produces the proteins required for packaging retroviral DNA but it cannot bring about encapsidation due to the lack of an essential region. However, when a recombinant vector carrying an NOI or NOIs (as replacement for gag, pol and env) and the essential region is introduced into the packaging cell line, the helper proteins can package this recombinant vector to create a "producer" line which produces the recombinant virus stock. This producer line releases infectious vector particles containing the full length RNA genome packaged in trans with the gag, pol and env gene products from the helper genome. These particles can transduce target cells via specific cell surface receptors to introduce the NOI into the genome of the cells. The recombinant virus whose genome lacks all genes required to make viral proteins can tranduce only once and cannot propagate. These viral vectors which are only capable of a single round of transduction of target cells are known as replication defective vectors. Hence, the NOI is introduced into the host/ tar get cell genome without the generation of potentially harmful retrovirus. A summary of the available packaging lines is presented in "Retroviruses" (1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 449).

The design of retroviral packaging cell lines has evolved to address the problem of inter alia the spontaneous production of helper virus that was frequently encountered with early designs. As recombination is greatly facilitated by homology, reducing or eliminating homology between the genomes of the vector and the helper has reduced the problem of helper virus production. More recently, packaging cells have been developed in which the gag, pol and env viral coding regions are carried on separate expression plasmids that are independently transfected into a packaging cell line so that three recombinant events are required for wild type viral production. This reduces the potential for production of a replication-competent virus. This strategy is sometimes referred to as the three plasmid transfection method (Soneoka et al 1995 Nucl. Acids Res. 23: 628-633).

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

With regard to vector titre, the practical uses of retroviral vectors have been limited largely by the titres of transducing particles which can be attained in in vitro culture

7 (typically not more than 10 particles/ml). In this regard, reference may be made again to Maulik and Patel 1997 (ibid, Chapter 2 page 42) where the authors state:

"It is sometimes difficult to generate high-tit er recombinant virus because insertion of the foreign gene [i.e. the NOI] can cause instability or adversely affect production of vector stocks ."

The sensitivity of many enveloped viruses to traditional biochemical and physicochemical techniques for concentrating and purifying viruses has also posed a problem. By way of example, several methods for concentration of retroviral vectors have been developed, including the use of centrifugation (Fekete and Cepko 1993 Mol Cell Biol 13: 2604-2613), hollow fibre filtration (Paul et al 1993 Hum Gene Ther 4: 609-615) and tangential flow filtration (Kotani et al 1994 Hum Gene Ther 5: 19-28). Although a 20-fold increase in viral titre can be achieved, the relative fragility of retroviral Env protein limits the ability to concentrate retroviral vectors and concentrating the virus usually results in a poor recovery of infectious virions. While this problem can be overcome by substitution of the retroviral Env protein with the more stable VSV-G protein which allows for more effective vector concentration with better yields, it suffers from the drawback that the VSV-G protein is quite toxic to cells.

Although helper-virus free vector titres of 10⁷ cfu/ml are obtainable with currently available vectors, experiments can often be done with much lower-titre vector stocks. However, for practical reasons, high-titre virus is desirable, especially when a large number of cells must be infected. In addition, high titres are a requirement for transduction of a large percentage of certain cell types. For example, the frequency of human hematopoietic progenitor cell infection is strongly dependent on vector titre, and useful frequencies of infection occur only with very high-titre stocks (Hock and Miller 1986 Nature 320: 275-277; Hogge and Humphries 1987 Blood 69: 611- 617). In these cases, it is not sufficient simply to expose the cells to a larger volume of virus to compensate for a low virus titre. On the contrary, in some cases, the concentration of infectious vector virions may be critical to promote efficient transduction.

Through our studies, we have now determined the reason why it was difficult to generate high-titer recombinant virus. In this regard, recombinant retroviral vectors have until now always been prepared by a flat bed process. This process is sometimes referred to as a monolayer culture. In this particular preparative technique, the recombinant retroviruses are grown on plates or dishes, such as on petri dishes. We have found that the inherent nature of this particular preparative technique itself prevents the production of high-titre recombinant virus.

Thus, according to a first aspect of the present invention there is provided a suspension culture capable of producing a retrovirus.

According to a second aspect of the present invention there is provided a suspension culture capable of producing a recombinant retroviral vector.

According to a third aspect of the present invention there is provided a suspension culture capable of producing a recombinant retroviral vector, wherein the retroviral vector is subsequently used for gene therapy.

Preferably, the suspension culture is not, or hardly, stirred during at least cell multiplication or growth. Preferably, the suspension culture is exposed to a higher CO₂ environment than atmospheric CO₂ levels.

Preferably, the suspension culture is exposed to CO levels greater than 5 %, more preferably greater than 6 % , more preferably greater than 7 % , more preferably greater than 8%, more preferably greater than 9% , more preferably about 10%.

According to a fourth aspect of the present invention there is provided a producer cell, a retrovirus or a retroviral vector obtained from the suspension culture of the present invention.

According to a fifth aspect of the present invention there is provided a producer cell, a retrovirus or a retroviral vector obtained from the suspension culture of the present invention for use in gene therapy.

According to a sixth aspect of the present invention there is provided the use of a producer cell, a retrovirus or a retroviral vector obtained from the suspension culture of the present invention in the manufacture of a medicament for use in gene therapy.

According to a seventh aspect of the present invention there is provided a method of treatment which comprises administering to a subject a producer cell, a retrovirus or a retroviral vector obtained from the suspension culture of the present invention, wherein the retrovirus or the retroviral vector has a therapeutic effect for the subject.

According to an eighth aspect of the present invention there is provided a process comprising culturing the suspension culture of the present invention; and isolating and/or purifying therefrom a retrovirus or a retroviral vector.

According to a ninth aspect of the present invention there is provided an insertion technique for inserting at least one NOI into a nucleotide site of interest, the technique comprising delivering a recombinant retroviral vector according to the present invention to the nucleotide site of interest.

According to a tenth aspect of the invention, there is provided a method for administering a retrovirus or a retroviral vector to a subject comprising administering to the subject a producer cell capable of producing a retrovirus or a retrovirus vector.

Preferably, the producer cell is capable of growing in a non-adhesive and/or a non- invasive manner.

The producer cell may be an in vivo producer cell in the body of an individual to be treated or it may be a cell cultured in vitro such as a tissue culture cell line. Suitable producer cell lines include mammalian cells such as murine fibroblast derived cell lines or other human cell lines.

Alternatively, the producer cell may be a cell derived from the individual to be treated such as a monocyte, macrophage, blood cell or fibroblast. The cell may be isolated from an individual and the vector components administered ex vivo followed by re- administration of the autologous producer cells. Alternatively the packaging and vector components may be administered to the producer cell in vivo. Methods for introducing retroviral packaging and vector components into cells of an individual are known in the art. For example, one approach is to introduce the different DNA sequences that are required to produce a retroviral vector particle e.g. the env coding sequence, the gag- pol coding sequence and the defective retroviral genome into the cell simultaneously by transient triple transfection (Landau & Littman 1992 J. Virol. 66, 5110; Soneoka et al 1995 Nucleic Acids Res 23:628-633).

Thus, the present invention is based on the surprising finding that it is possible to produce a stock of a retrovirus or a retroviral vector by means of suspension culture. Moreover, the present invention is based on the even more highly surprising finding that it is possible to produce a reasonably high titre stock of a retrovirus or a retroviral vector by means of suspension culture.

The in vitro suspension culture of the present invention is advantageous in that it enables workers to generate high-titer recombinant virus. This is particularly important as it is now possible to ultimately deliver a higher concentration of recombinant retroviral vector - and hence a higher concentration of a NOI - to a site of interest, particularly an in vivo site of interest. This, for reasons that are clearly apparent, is highly advantageous.

In addition, workers can now prepare larger volumes of culture medium comprising retroviruses and/ or recombinant retroviral vectors. This is again highly advantageous.

The suspension culture cells of the present invention are preferably capable of growing in a non-adhesive and non-invasive manner. These properties may be attained during development of cells capable of growing in suspension culture. Cells according to the invention may be administered to organisms, thereby administering the retrovirus or retroviral vector produced by the cells. The cells of the invention may be better able to distribute themselves throughout the organism, and moreover may reach more remote organ systems.

Preferably, producer cells according to the invention may be transfected with marker genes, such as those commonly used in suspension cell culture. Examples of selectable markers which have been used successfully in retroviral vectors include but are not limited to the bacterial neomycin and hygromycin phosphotransferase genes which confer resistance to G418 and hygromycin respectively (Palmer et al 1987 Proc Natl Acad Sci 84: 1055-1059; Yang et al 1987 Mol Cell Biol 7: 3923-3928); a mutant mouse dihydrofolate reductase gene (dhfr) which confers resistance to methotrexate (Miller et al 1985 Mol Cell Biol 5: 431-437); the bacterial gpt gene which allows cells to grow in medium containing mycophenolic acid, xanthine and aminopterin (Mann et al 1983 Cell 33: 153-159); the bacterial hisD gene which allows cells to grow in medium without histidine but containing histidinol (Danos and Mulligan 1988 Proc Natl Acad Sci 85: 6460-6464); the multidrug resistance gene (mdr) which confers resistance to a variety of drugs (Guild et al 1988 Proc Natl Acad Sci 85: 1595-1599; Pastan et al 1988 Proc Natl Acad Sci 85: 4486-4490) and the bacterial genes which confer resistance to puromycin or phleomycin (Morgenstern and Land 1990 Nucleic Acid Res 18: 3587-3596).

In a particularly advantageous embodiment, the cells are equipped with a mechanism for programmed or inducible cell death, for instance by pretreatment of the cells with mitomycin C or by gamma irradiation prior to administration, or by introduction of a drug induced suicide pathway, such as an enzyme/prodrug pathway, for example the thymidine kinase/gancyclovir pathway. The invention thus provides for the continuous production of virus, allowing several cycles of infection before being destroyed, optionally by a suicide system.

Preferably, the retroviral vector comprises at least one NOI.

Hence, according to a preferred aspect of the present invention there is provided a suspension culture capable of producing a recombinant retroviral vector, wherein the retroviral vector comprises at least one NOI.

Preferably, the NOI is for subsequent use in or for gene therapy application(s).

Preferably, the retrovirus or the retroviral vector are in a high concentration. The term "high concentration" preferably means in a concentration of at least 1 x 10⁶ i.u./ml. In a preferred embodiment, the term means titres of at least about 5 x 10⁹ i.u./ml. In a highly preferred embodiment, the term means titres in excess of 1 x 10¹⁰ i.u./ml. The retrovirus or the retroviral vector obtained from the suspension culture of the present invention may be administered to a subject by any appropriate means and to any one or more suitable sites.

Suitable target sites for the vector system according to the invention include but are not limited to haematopoietic cells (including monocytes, macrophages, lymphocytes, granulocytes or progenitor cells of any of these); endothelial cells; tumour cells; stromal cells; astrocytes or glial cells; muscle cells; and epithelial cells, slowly dividing and rapidly dividing tumour cells. Alternatively the target cell may be a growth- arrested cell capable of undergoing cell division such as a cell in a central portion of a tumour mass or a stem cell such as a haematopoietic stem cell or a CD34-positive cell. As a further alternative, the target cell may be a precursor of a differentiated cell such as a monocyte precursor, a CD33-positive cell, or a myeloid precursor. As a further alternative, the target cell may be a differentiated cell such as a neuron, astrocyte, glial cell, microglial cell, macrophage, monocyte, epithelial cell, endothelial cell or hepatocyte. Target cells may be transduced either in vitro after isolation from a human individual or may be transduced directly in vivo.

The vector of the present invention may be a delivered to a target site by a viral or a non-viral vector.

As it is well known in the art, a vector is a tool that allows or faciliates the transfer of an entity from one environment to another. By way of example, some vectors used in recombinant DNA techniques allow entities, such as a segment of DNA (such as a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell. Optionally, once within the target cell, the vector may then serve to maintain the heterologous DNA within the cell or may act as a unit of DNA replication. Examples of vectors used in recombinant DNA techniques include plasmids, chromosomes, artificial chromosomes or viruses. Non-viral delivery systems include but are not limted to DNA transfection methods. Here, transfection includes a process using a non-viral vector to deliver a gene to a target mammalian cell.

Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated, cationic facial amphiphiles (CFAs) (Nature Biotechnology 1996 14; 556), and combinations thereof.

Preferably, the recombinant retroviral vector of the present invention according to the present invention is prepared by means of a method that includes the use of electroporation.

Viral delivery systems include but are not limited to adenovirus vector, an adeno- associated viral (AAV) vector, a herpes viral vector, retroviral vector, lentiviral vector, baculoviral vector. Other examples of vectors include ex vivo delivery systems, which include but are not limited to DNA transfection methods such as electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection.

For some applications, it may be feasible to take samples from the suspension culture and then directly administer those samples.

Preferably, the suspension producer cells are non-adhesive, in the sense that they do not readily adhere to each other and/or to the sides of the culture vessel. This means that the cells are even more amenable to suspension culture.

Examples of suitable retroviruses for the suspension culture of the present invention include amphotropic murine leukaemia virus, such as that produced by the producer cell line TELCeB/AF-7, or any other retrovirus known for use in gene therapy applications. Examples of suitable NOIs that may be inserted by the recombinant retroviral vector of the present invention include but are not limited to any one or more of marker gene segments, nucleic acids encoding proteins, biologically active nucleic acids and nucleic acids encoding proteins responsible for producing biologically active molecules of interest. When included, such coding sequences may be typically operatively linked to a suitable promoter or promoters, Representative examples of such NOIs include but are not limited to lac z, sequences encoding cytokines, chemokines, hormones, antibodies, engineered immunoglobulin-like molecules, a single chain antibody, fusion proteins, enzymes, immune co-stimulatory molecules, immunomodulatory molecules, anti-sense RNA, a transdominant negative mutant of a target protein, a toxin, a conditional toxin, an antigen, a tumour suppressor protein and growth factors, membrane proteins, vasoactive proteins and peptides, anti-viral proteins and ribozymes, and derivatives therof (such as with an associated reporter group), gene segments coding for therapeutic proteins such as the cystic fibrosis transmembrane regulator gene (CFTR), coagulation factors such as Factor VIII or Factor IX, α-1 antitrypsin, glycogen degrading enzymes, insulin, adenosine deaminase.

Other examples of an NOI include proteins that are toxic or deleterious to abnormal cells within the body. These include but are not limited to a prodrug or an enzyme capable of converting a prodrug into its active form such as a pro-drug activating enzyme to a tumour site for the treatment of a cancer. In each case, a suitable pro-drug is used in the treatment of the individual (such as a patient) in combination with the appropriate pro-drug activating enzyme. An appropriate pro-drug is administered in conjunction with the vector. Examples of pro-drugs include: etoposide phosphate (with alkaline phosphatase, Senter et al 1988 Proc Natl Acad Sci 85: 4842-4846); 5-fluorocytosine (with cytosine deaminase, Mullen et al 1994 Cancer Res 54: 1503- 1506); Doxorubicin-N-p-hydroxyphenoxyacetamide (with Penicillin-V-Amidase, Kerr et al 1990 Cancer Immunol Immunother 31: 202-206); Para-N-bis(2-chloroethyl) aminobenzoyl glutamate (with carboxypeptidase G2); Cephalosporin nitrogen mustard carbamates (with β-lactamase); SR4233 (with P450 Reducase); Ganciclovir (with HSV thymidine kinase, Borrelli et al 1988 Proc Natl Acad Sci 85: 7572-7576); mustard pro- drugs with nitroreductase (Friedlos et al 1997 J Med Chem 40: 1270-1275) and Cyclophosphamide (with P450 Chen et al 1996 Cancer Res 56: 1331-1340), a cytotoxic agent, a transcription factor or other regulatory molecule and other molecules with applications in therapy, especially gene therapy.

With the insertion technique according to the present invention, preferably the nucleotide site of interest is a genomic site. More preferably, the nucleotide site of interest is an animal genomic site.

In a preferred embodiment, the subject of the present invention is an animal, preferably a human.

The retrovirus or the retroviral vector obtained from the suspension culture of the present invention may be administered either alone or in admixture with one or more pharmaceutically acceptable excipient, diluent or carrier or combination thereof.

The present invention also provides a pharmaceutical composition for treating an individual by gene therapy, wherein the composition comprises a therapeutically effective amount of the retroviral vector of the present invention comprising one or more deliverable therapeutic and//or diagnostic NOI(s) or a viral particle produced by or obtained from same. The pharmaceutical composition may be for human or animal usage. Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular individual.

The composition may optionally comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), and other carrier agents that may aid or increase the viral entry into the target site (such as for example a lipid delivery system).

Where appropriate, the pharmaceutical compositions can be administered by any one or more of: inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

The delivery of one or more therapeutic genes by a vector system according to the present invention may be used alone or in combination with other treatments or components of the treatment.

For example, the retroviral vector of the present invention may be used to deliver one or more NOI(s) useful in the treatment of the disorders listed in WO-A-98/05635. For ease of reference, part of that list is now provided: cancer, inflammation or inflammatory disease, dermatological disorders, fever, cardiovascular effects, haemorrhage, coagulation and acute phase response, cachexia, anorexia, acute infection, HIV infection, shock states, graft-versus-host reactions, autoimmune disease, reperfusion injury, meningitis, migraine and aspirin-dependent anti-thrombosis; tumour growth, invasion and spread, angiogenesis, metastases, malignant, ascites and malignant pleural effusion; cerebral ischaemia, ischaemic heart disease, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration, Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn's disease and ulcerative colitis; periodontitis, gingivitis; psoriasis, atopic dermatitis, chronic ulcers, epidermolysis bullosa; corneal ulceration, retinopathy and surgical wound healing; rhinitis, allergic conjunctivitis, eczema, anaphylaxis; restenosis, congestive heart failure, endometriosis, atherosclerosis or endosclerosis.

In addition, or in the alternative, the retroviral vector of the present invention may be used to deliver one or more NOI(s) useful in the treatment of disorders listed in WO-A- 98/07859. For ease of reference, part of that list is now provided: cytokine and cell proliferation/differentiation activity; immunosuppressant or immunostimulant activity (e.g. for treating immune deficiency, including infection with human immune deficiency virus; regulation of lymphocyte growth; treating cancer and many autoimmune diseases, and to prevent transplant rejection or induce tumour immunity); regulation of haematopoiesis, e.g. treatment of myeloid or lymphoid diseases; promoting growth of bone, cartilage, tendon, ligament and nerve tissue, e.g. for healing wounds, treatment of burns, ulcers and periodontal disease and neurodegeneration; inhibition or activation of follicle-stimulating hormone (modulation of fertility); chemotactic/chemokinetic activity (e.g. for mobilising specific cell types to sites of injury or infection); haemostatic and thrombolytic activity (e.g. for treating haemophilia and stroke); antiinflammatory activity (for treating e.g. septic shock or Crohn's disease); as antimicrobials; modulators of e.g. metabolism or behaviour; as analgesics; treating specific deficiency disorders; in treatment of e.g. psoriasis, in human or veterinary medicine.

In addition, or in the alternative, the retroviral vector of the present invention may be used to deliver one or more NOI(s) useful in the treatment of disorders listed in WO-A- 98/09985. For ease of reference, part of that list is now provided: macrophage inhibitory and/or T cell inhibitory activity and thus, anti-inflammatory activity; anti- immune activity, i.e. inhibitory effects against a cellular and/or humoral immune response, including a response not associated with inflammation; inhibit the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, as well as up-regulated fas receptor expression in T cells; inhibit unwanted immune reaction and inflammation including arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or other oto-rhino-laryngological diseases, dermatitis or other dermal diseases, periodontal diseases or other dental diseases, orchitis or epididimo-orchitis, infertility, orchidal trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, habitual abortion, eclampsia, pre-eclampsia and other immune and/or inflammatory -related gynaecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e.g. retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreo-retinopathies, acute ischaemic optic neuropathy, excessive scarring, e.g. following glaucoma filtration operation, immune and/or inflammation reaction against ocular implants and other immune and inflammatory-related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders where, both in the central nervous system (CNS) or in any other organ, immune and/or inflammation suppression would be beneficial, Parkinson's disease, complication and/or side effects from treatment of Parkinson's disease, AIDS-related dementia complex HIV-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of stokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora, myasthenia gravis, pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery, bone marrow transplantation or other transplantation complications and/or side effects, inflammatory and/or immune complications and side effects of gene therapy, e.g. due to infection with a viral carrier, or inflammation associated with AIDS, to suppress or inhibit a humoral and/or cellular immune response, to treat or ameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia, by reducing the amount of monocytes or lymphocytes, for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.

Other aspects of the present invention will become apparent from the following commentary. In this regard, the present invention will be further described by way of example in which reference is made to the following Figures:

Figure 1 which is a photographic representation of WIL-E suspension producer cells

In slightly more detail: Fig. l represents an Infection assay of WIL-E Suspension Producer Cells

Figure la, lb) WIL-E clone B9 (enhanced). NIH3T3 cells shown were incubated with filtered supernatant obtained from WIL-E B9.1 producer cells, with 5μg/ml DOGS (Promega). Viral infection was titred at 3 x 10exp4 i.u./ml.

Figure lc) WIL-E (pooled). NIH3T3 cells shown were incubated with filtered supernatant obtained from pooled WIL-E producer cells, with 5μg/ml DOGS (Promega). Viral infection was titred at 7 x 10exp2 i.u./ml. Figure Id) TELCeB/AF-7 positive control. NIH3T3 cells shown were incubated with filtered supernatant of TELCeB/AF-7 retrovirus producer cells (provided by Y. Takeuchi, Institute of Cancer Research), with 5μg/ml DOGS (Promega). Vial infection was titred at 1 x 10exp7 i.u./ml.

Figure le) Wil-2/TELCeB/AF-7 negative control. NIH3T3 cells shown were incubated with filtered supernatant of Wil-2/TELCeB/AF-7, with 5μg/ml DOGS (Promega). Wil-2/TELCeB/AF-7 was obtained by infecting Wil-2 cells which does not express any viral components, with the amphotropic high producer cell line TELCeB/AF-7 (provided by Y. Takeuchi, Institute of Cancer Research) expressing MLV-A lacZ virus. This is performed to confirm the absence of any replication competent retrovirus which may arise during co-culturing.

Figure If) NIH3T3 negative control. NIH3T3 cells shown were incubated with filtered supernatant of NIH3T3. Cells supernatant was incubated with 5μg/ml DOGS (Promega).

METHODS

The following commentary describes the generation of a retrovirus producer cell line which exists as a suspension culture. Here we describe the methods used to generate lacZ retrovirus from WIL-E cells - which are cells with Moloney murine leukaemia virus gag/pol components and env for ecotropic infection. We also discuss the possible uses of such a cell line.

WIL-E is derived from the Wil-2 lymphoblastoid cell line which exist naturally in suspension and is not restricted by surface contact inhibition as observed in conventional retrovirus producer cell lines. This has the potential to enable the large- scale production of retroviral particles at titres which exceed those currently available from producer cell lines. Generation of an Ecotropic packaging cell line using human lymphoblastoid suspension culture cells

Materials, Methods and Results

MLV-Env and MLV-Gag-Pol used in this study were kindly gifted to our laboratory by Cossett et al on plasmids pFB3LPh (kindly donated by Y Takeuchi, Chester Beatty Laboratories, London) and pCEb (also kindly donated by Y Takeuchi, Chester Beaty Laboratories) conferring phleomycin resistance and Blasticidin resistance of transfected cells, respectively.

Transfection of plasmid DNA by electroporation

1 x 10 Wil-2 immortal human lymphoblastoid suspension culture cells were pelleted by centrifugation and resuspended in culture medium consisting of 25% preconditioned and 75% fresh RPMI 1640 supplemented with 15% Foetal Bovine Serum, 2% L- Glutamine, 2% Penicillin-Streptomycin.

30 μg of FB3LPh DNA was added to the cells in a pre-chilled Gene Pulser cuvette (BioRad). After incubating on ice for 10 minutes, the cuvette was loaded onto a Gene Pulser II system (BioRad). Electroporation was performed at 960 mF/250 volts. Cells were then left at RT for 20 minutes, then suspended in 10 ml fresh medium.

A stepwise programme with different antibiotic drug concentration was performed to establish a survival curve that determines the optimal concentration that is required to select for successfully transfected cells.

Selection of MLV-Env expressing clones was made using 15 μg/ml phleomycin. The resulting cells were cloned in 96 well plates after diluting to 40 cells per dish. The Wil-2 B4/Env cells were then electroporated with pCeB as described above. 20 μg/ml Blasticidin S was determined to select for Blasticidin S resistance in pCeB transfected cells to generate Wil-2 B4/Env/Gag-Pol cells which are pooled and not of clonal origin. The cells were placed in Blasticidin selection which was increased stepwise to produce stable cells in this drug.

Infection by Amphotropic virus carrying the lacZ genome

Wil-2 B4/Env/Gag-Pol cells were infected with the MLV lacZ virus generated by co- culturing with a TELCeB/MOF-1 cell line under co-culturing conditions which produces amphotropic retrovirus. TELCeB were grown at 37 °C in DMEM (supplemented with 10% foetal calf serum) to 90% confluency, 1 x 10⁷ Wil-2 B4/Env/Gag-Pol producer cells were then placed with the TELCeB/MOF-1 producer cells plus 5 μg/ml DOGs (Promega). DOGs has the following structure:

DOGs

After co-culturing for 24 hours at 37 °C the MLV lacZ infected Wil-2 B4/Env/Gag-Pol producer cells (designated as Wil-E cells) were removed, pelleted at 1000 rpm for five minutes and resuspended in fresh medium. To avoid contamination of Wil-E suspension culture producer cells with co-cultured TELCeB/MOF-1 mono-layer producer cells, repeated passaging was performed by transferring the suspension culturing cells to horizontally placed flasks to remove mono-layer TELCeB/MOF-1 cells which will seed on the culture dish while the WIL-E cells remain in suspension. Following a 24 hour period to allow for transgene expression, an aliquot of cells was examined for lacZ expression. Approximately 40% of Wil-2 B4/Env/Gag-Pol cells were successfully infected by this method. Co-culturing of Wil-2 B4/Env/Gag-Pol was repeated until 70% infection was achieved.

Alternatively, Wil-2 B4/Env/Gag-Pol cells were infected with the MLV lacZ virus generated by the amphotropic retrovirus producer cell line TELCeB/AF-7 cell line under co-culturing conditions. TELCeB were grown at 37 °C in DMEM (supplemented with 10% foetal calf serum) to 90% confluency, 1 x 10⁷ Wil-2 B4/Env/Gag-Pol producer cells were then placed with the TELCeB/AF-7 producer cells plus 5 μg/ml DOGs (Promega). DOGs has the structure as outlined above.

After co-culturing for 24 hours at 37 °C the MLV lacZ infected Wil-2 B4/Env/Gag-Pol producer cells (designated as Wil-E cells) were removed, pelleted at 1000 rpm for five minutes and resuspended in fresh medium. To avoid contamination of Wil-E suspension culture producer cells with co-cultured TELCeB/AF-7 mono-layer producer cells, repeated passaging was performed by transferring the suspension culturing cells to horizontally placed flasks to remove mono-layer TELCeB/AF-7 cells which will seed on the culture dish while the WIL-E cells remain in suspension. Following a 24 hour period to allow for transgene expression, an aliquot of cells was examined for lacZ expression. Approximately 40% of Wil-2 B4/Env/Gag-Pol cells were successfully infected by this method. Co-culturing of Wil-2 B4/Env/Gag-Pol was repeated until 70% infection was achieved.

Infection assay to detect Wil-E MLV lacZ producer cells

Wil-E cells were grown in culture to 5 x 10⁷ cells in a 30 ml, 12 ml of cells were pelleted and the supernatant was removed and filtered through a 0.8 μm filter. After the addition of 5 μg/ml DOGs (Promega) and incubation at 1/2 hour RT, the virus/amphiphile was added to 100 mm dish containing 5 x 10⁷ NIH3T3 cells. After 24 hours infection, the supernatant was removed and the cells were refed. Following a further 24 hours period to allow for lacZ expression, the cells were assayed for enzymatic activity of lacZ. 190 lacZ positive cells were detected with no blue cells on the negative control dish. Controls consisted of positive ecotropic lacZ virus from monolayer producer cells and a negative control of supernatant from Wil-2 cells previously infected by amphotropic lacZ virus which were not expected to be able to make virus particles since gag/pol and env components are absent.

In another experiment, Wil-E cells were grown in culture to 3 x 10⁷ cells in a 30 ml, 12 ml of cells were pelleted and the supernatant was removed and filtered through a 0.8 μm filter. After the addition of 5 μg/ml DOGs (Promega) and incubation at 1/2 hour RT, the virus/amphiphile was added to 100 mm dish containing 5 x 10⁶ NIH3T3 cells. After 24 hours infection, the supernatant was removed and the cells were refed. Following a further 24 hours period to allow for lacZ expression, the cells were assayed for enzymatic activity of lacZ. LacZ positive cells detected were then counted and a titre of 7 x 10 i.u./ml was obtained. No blue cells were observed on the negative control dish. Controls consisted of positive amphotropic lacZ virus from mono-layer producer cells TELCeB/AF-7 and a negative control of supernatant from Wil-2 cells previously infected by amphotropic lacZ virus (designated as Wil- 2/TElCeB/AF-7) which were not expected to be able to make virus particles since gag/pol and env components are absent.

Clonal selection and titre enhancement of WIL-E producer cells

Pooled WIL-E producer cells were cloned in 96-well plates after diluting to 40 cells per dish. 5 clones were obtained and western blotting and LacZ staining were performed to confirm the expression of gag-pol, env and LacZ viral components. An infection asssay was then performed with each individual clone on NIH3T3 cells. Viral titres of up to 7 x 10³ i.u./ml were obtained. To further enhance the titre of WIL-E producer cell lines, co-culturing using the monolayer amphotropic producer cell line TELCeB/AF-7 was performed as described above to increase the copy numbers of lacZ in WIL-E cells. TELCeB/AF-7 cells were treated with Mitomycin C prior to co-culturing to ensure that WIL-E cells are not contaminated with any surviving TELCeB/AF-7 producer cells in suspension. A viral titre of 3.1 x 10⁴ i.u./ml has been achieved from cells which we have termed WIL-E B9.1 after this enhancement procedure. The following table and figures summarises the results obtained.

Table 1. Summary of determined viral titres of the Suspension Producer cells WIL-E

Summary

We produced WIL-2 derived cells (now called WIL-E) which initially received ecotropic virus envelope on plasmid. Cells were selected in 15 μg/ml phleomycin. Clones of phleomycin resistant cells were isolated and assayed for high envelope production by Western blotting to isolate clone WIL-2/B4 env. This clone was subsequently expanded in culture to receive the gag/pol plasmid pCeB with the necessary blasticidin selection. Pools of Ψl -2/ 4env/gag/pol blasticidin phleomycin resistant cells were then infected with amphotropic lacZ containing retrovirus under co- culture conditions to ensure the infection of every cell (now called WIL-E). These cells were then used to produce lacZ retrovirus to infect NIH3T3 cells. We were easily able to identify blue NIH3T3 cells which had been infected with retrovirus generated from WIL-E cells. The following steps summarise the procedures used to generate titres of virus from WIL-E cells which exceed other producer cell lines currently available (presently at between lxlO⁵ and lxlO⁷ i.uJml). These include:

1. Repeat co-culturing with Mitomycin C treated TELCeB/AF-7 to further increase the number of virus genomes present in WIL-E cells which has been shown to enhance titre.

2. Re-electroporation of gag-pol and env expressing plasmids to increase expression of viral components.

3. Step two is carried out in conjunction with ongoing cloning and screening of a high producer cell line.

4. Optimal culture conditions for WIL-E cells are used to optimise viral production in suspension.

5. We are currently growing WIL-E cells in large volumes to show production of high titre virus following established concentration methods. Suspension culmre cells are ideally suited to this process which removes virus containing culture medium for processing whilst continually re-feeding cells with fresh medium.

Discussion

The suspension culture in accordance with the present invention allows retrovirus stocks to be generated without the limitations of mono-layer culture.

As mentioned above, the limitations of mono-layer culture collectively inhibit the volume of retrovirus that can be produced in culmre and therefore the titres of virus. Moreover, even with some of the additional techniques already published to date wherein retrovirus titres can be increased by centrifugation or simply by filtration, mono-layer cultures still require a large surface area to expand cell populations in order that enough retrovirus can be produced.

In contrast to the prior methodology, however, the suspension culture of the present invention enables cells to grow according to the volume of the culture medium and are not reliant on surface area. By way of example, the cells used in this study can be grown to a concentration of 5 x 10⁶/ml prior to passage. These cells, WIL-2, also prefer an acid environment which enables high concentration of cells per ml. Since suspension culture cells can be easily adapted to industrial levels in large aerated vessels, cell numbers and ultimately volume of virus can be increased dramatically.

With the latest methodologies for filtration of viruses from contaminating cellular debris and components of serum which includes hollow fibre filtration and ultrafiltration centrifugation such high volumes of virus supernatant can be reduced substantially (as shown in this laboratory) to increase virus titre in a linear fashion. Using 300,000 Kd mw cut-off filters we are able to concentrate virus titre by one order of magnitude to almost 1 x 10⁹ i.u./ml. In addition to this, so far we have not found any toxicity on NIH3T3 cells when titrating these viruses. We believe, therefore, that virus titre can be improved without possible cytotoxicity.

In a further embodiment, it is possible to combine hollow fibre filtration of virus particles directly from suspension culture cells growing in flasks adapted to pass virus containing supernatant directly to filtration apparatus. This operation would obviously suit industrial processes.

Retrovirus particles are notoriously fragile. Each virion is coated with glycoproteins which are vital for attachment to host cells for infection. Recent work by Dornburg et al. (J. Virol. 71: 720-725, 1997) has shown that harsh methods of concentration such as ultracentrifugation destroy virus envelopes making them non-specific in their ability to infect cells. In a preferred embodiment, the present invention, however, avoids harsh processes which may damage virus particles since specificity is an important property of any gene therapy vector. Preferably, therefore, it is suitable to grow the suspension culture cells without stirring. Also, WIL-2 cells appear to grow well in 4% CO₂ environment. This is a further preferred embodiment of the present invention.

In our studies we have found that under the preferred culmre conditions we have been able to establish almost about 100% survival of the cloned cells. This factor is important to avoid loss of cells in the production process and ultimately virus.

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

Claims

1. A suspension culmre capable of producing a retrovirus.

2. A suspension culmre capable of producing a recombinant retroviral vector.

3. A suspension culmre capable of producing a recombinant retroviral vector, wherein the retroviral vector comprises at least one NOI.

4. A producer cell, a retrovirus or a retroviral vector obtained from the suspension cultore of any one of claims 1 to 3.

5. A producer cell, a retrovirus or a retroviral vector obtained from the suspension culmre of any one of claims 1 to 3 for use in gene therapy.

6. The use of a producer cell, a retrovirus or a retroviral vector obtained from the suspension culture of any one of claims 1 to 3 in the manufacture of a medicament for use in gene therapy.

7. A process comprising culturing the suspension culmre of any one of claims 1 to 3; and isolating and/or purifying therefrom a retrovirus or a retroviral vector.

8. A method of treatment which comprises administering to a subject a producer cell, a retrovirus or a retroviral vector obtained from the suspension culture of any one of claims 1 to 3, wherein the retrovirus or the retroviral vector has a therapeutic effect for the subject.

9. An insertion technique for inserting at least one NOI into a nucleotide site of interest, the technique comprising delivering a recombinant retroviral vector according to claim 3, or any claim dependent thereon, to the nucleotide site of interest.

10. A method for administering a retrovirus or a retroviral vector to a subject comprising administering to the subject a producer cell capable of producing a retrovirus or a retrovirus vector.

11. A method according to claim 10 wherein the producer cell is capable of growing in a non-adhesive and/or a non-invasive manner.

12. A suspension culmre or a retrovirus or a retroviral vector or a use or a method or a process or an insertion technique substantially as described herein.