GB2560933A - Transduced cell formulation - Google Patents
Transduced cell formulation Download PDFInfo
- Publication number
- GB2560933A GB2560933A GB1704968.5A GB201704968A GB2560933A GB 2560933 A GB2560933 A GB 2560933A GB 201704968 A GB201704968 A GB 201704968A GB 2560933 A GB2560933 A GB 2560933A
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- cells
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
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- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Developmental Biology & Embryology (AREA)
- Diabetes (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to a composition comprising a high concentration of transduced cells, in particular transduced stem cells for use in methods of gene therapy. The composition comprises transduced cells, particularly haematopoietic stem cells, at a concentration of at least 0.5 x 108 cells/mL. The cells may be transduced with a lentiviral vector and may contain a transgene encoding human beta globin. The compositions may comprise a cryoprotectant. Methods for preparing said transduced cell compositions are also disclosed, wherein the methods can involve suspending transduced cells at a concentration of least 0.5 x 108 cells/mL, optionally in a cryoformulation comprising DMSO, and optionally HSA and/or saline. The compositions can be administered to a patient in methods of gene therapy.
Description
(71) Applicant(s):
GlaxoSmithKline Intellectual Property Development Limited
980 Great West Road, Brentford, Middlesex,
TW8 9GS, United Kingdom (72) Inventor(s):
Chaminda Salgado (74) Agent and/or Address for Service:
GlaxoSmithKline
Gunnels Wood Road, STEVENAGE, Hertfordshire, SG1 2NY, United Kingdom (56) Documents Cited:
EP 3002011 A1 WO 2003/002155 A1 CN 102792947 A
WO 2008/147057 A1 CN 103263440 A
Blood, Vol. 83, No. 9, May 1994, SD Rowley, et al., Effect of Cell Concentration on Bone Marrow and
Peripheral Blood Stem Cell Cryopreservation, pages 2731-2736.
Human gene therapy, Vol. 20, No. 6, June 2009, J Hayakawa, et al., Transient in vivo beta-globin production after lentiviral gene transfer to hematopoietic stem cells in the nonhuman primate., pages 563 - 572.
Blood, Vol. 113, No. 23, June 2009, H Zhao, et al., Amelioration of murine beta-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both gamma-globin and the MGMT drug-resistance gene., pages 5747 - 5756.
Carbohydrate polymers, Vol. 91, No. 1, Jan. 2013, Liang Xianxiang, et al., Direct saccharification and ethanol fermentation of cello-oligosaccharides with recombinant yeast, pages 157-161.
(58) Field of Search:
INT CLC12N
Other: EPODOC, WPI, BIOSIS, MEDLINE (54) Title of the Invention: Transduced cell formulation
Abstract Title: Compositions comprising high concentrations of transduced cells (57) The invention relates to a composition comprising a high concentration of transduced cells, in particular transduced stem cells for use in methods of gene therapy. The composition comprises transduced cells, particularly haematopoietic stem cells, at a concentration of at least 0.5 x 108ceIls/mL. The cells may be transduced with a lentiviral vector and may contain a transgene encoding human beta globin. The compositions may comprise a cryoprotectant. Methods for preparing said transduced cell compositions are also disclosed, wherein the methods can involve suspending transduced cells at a concentration of least 0.5 x 108cells/mL, optionally in a cryoformulation comprising DMSO, and optionally HSA and/or saline. The compositions can be administered to a patient in methods of gene therapy.
TRANSDUCED CELL FORMULATION
FIELD OF THE INVENTION
The invention relates to formulations of transduced cells, in particular transduced haematopoietic stem cells or T cells, which may be used in methods of gene therapy.
BACKGROUND TO THE INVENTION
Gene therapy involves the transfer of genetic information into patient tissues and organs in order for diseased genes to be eliminated or their normal functions rescued. This can be performed either by direct transfer of genes into the patient (e.g. via viral vectors or liposomes) or by administering living cells containing the genes of interest.
The concept of stem cell gene therapy is based on the genetic modification of stem cells (autologous or allogenic) which are administered to the patient. These persist long-term in the body with the hope that they will generate large numbers of progeny containing the genetic modification. This ensures a continuous supply of corrected cells for the rest of the patient's lifetime. HSCs are particularly attractive targets for gene therapy since their genetic modification will be passed to all the blood cell lineages as they differentiate. Traditionally, HSCs were obtained from the bone marrow, however, HSCs can also be obtained from peripheral blood, or blood taken from the placenta at birth (cord blood).
Another key focus of cellular gene therapies has been on the modification of T cells to include chimeric antigen receptors (CARs) or modified T cell receptors (TCRs). These are being developed to treat cancers in patients that are resistant to conventionally available therapies and use a patient's own immune cells to combat the disease. The immune cells are genetically engineered ex vivo to express a CAR (CAR-T cells) specific to a tumour antigen, and the cells are subsequently transferred back to the patient.
Current cell concentrations used in methods of gene therapy comprise around 1 χ 106 cells/mL. However, this generates large drug product volumes containing high amounts of excipients which have been associated with negative side effects. It is therefore desirable to minimise the amount of any excipients being dosed to patients.
In particular, dimethyl sulfoxide (DMSO) is a low molecular weight, cell-permeable cryoprotectant that is routinely used for cryopreservation of bone marrow, cord blood and other blood products intended for transplantation. However, the use of DMSO in cryopreserved stem cell transplants is associated with adverse effects following infusion which are more commonly mild symptoms e.g. fever, chills and a garlic odour, but occasionally more severe (Pereira-Cunha etal., (2015) Vox Sang., 108 (1), 72-81). For this reason, there is a keen interest in ways of reducing the amount of excipients administered to patients.
Rowley etal. (1994) Blood, 83(9), 2731-2736 previously described the effect of cell concentration on bone marrow and peripheral blood stem cell cryopreservation. However, this paper relates to traditional methods of autologous bone marrow transplantation, not the effect on transduced cells to be used in stem cell gene therapy techniques.
Transplantation of a sufficiently high number of modified HSCs is needed in order for stem cell gene therapy to be successful. However, there needs to be a careful balance between the administration of a sufficiently high concentration of cells and minimising the side effects associated with administering large volumes of excipients. Accordingly, there is a need in the art to provide an improved transduced cell formulation to be used in methods of gene therapy.
SUMMARY OF THE INVENTION
According to a first aspect of the invention, there is provided a composition comprising transduced cells at a concentration of at least 0.5 χ 108 cells/mL
According to a further aspect of the invention, there is provided a method for preparing a transduced cell composition, comprising:
(a) obtaining a plurality of transduced cells; and (b) suspending the transduced cells in a medium at a concentration of at least 0.5 χ 108 cells/mL.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel etal., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc. which are incorporated herein by reference in their entirety) and chemical methods. All patents and publications referred to herein are incorporated by reference in their entirety.
The term comprising encompasses including or consisting e.g. a composition comprising X may consist exclusively of X or may include something additional e.g. X + Y.
The term consisting essentially of limits the scope of the feature to the specified materials or steps and those that do not materially affect the basic characteristic(s) of the claimed feature.
The term consisting of excludes the presence of any additional component(s).
The term about as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, including ±5%, ±1%, and ±0.1% from the specified value.
The term haematopoietic cell refers to blood cells, such as any of the kinds of cell normally found circulating in the blood. Examples of haematopoietic cells include red blood cells (e.g. erythrocytes) and white blood cells (e.g. T cells, B cells, and natural killer cells). For the avoidance of doubt, it will be understood that this term includes haematopoietic stem cells.
The term haematopoietic stem cell or HSC refers to stem cells that give rise to blood cells through the process of haematopoiesis. They are derived from mesoderm and located in the red bone marrow, which is contained in the core of most bones. HSCs give rise to both myeloid (i.e. monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, and megakaryocytes or platelets) and lymphoid (i.e. T cells, B cells, and natural killer cells) lineages of blood cells.
The term cryoprotective formulation refers to a formulation or medium in which cells are suspended when frozen. In particular, the formulation is used to protect the cells/tissue from freezing damage. It will be understood that the term cryoprotective formulation can be used interchangeably with cryoprotective medium.
The term Human Serum Albumin or HSA refers to a type of serum albumin found in human blood. Albumin is an essential protein which: transports hormones, fatty acids, and other compounds in the blood by acting as a carrier protein; buffers pH; and maintains oncotic pressure.
The term Dimethyl Sulfoxide or DMSO refers to an organosulfur compound with the formula (CH3)2SO. It is commonly used as a polar aprotic solvent.
The term vector refers to a vehicle which is able to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. Examples of vectors include plasmids and viral vectors, such as retroviral and lentiviral vectors, which are of particular interest in the present application. Lentiviral vectors, such as those based upon Human Immunodeficiency Virus Type 1 (HIV-1) are widely used as they are able to integrate into non-proliferating cells. Viral vectors can be made replication defective by splitting the viral genome into separate parts, e.g., by placing on separate plasmids. For example, the so-called first generation of lentiviral vectors, developed by the Salk Institute for Biological Studies, was built as a three-plasmid expression system consisting of a packaging expression cassette, the envelope expression cassette and the vector expression cassette. The packaging plasmid contains the entire gag-pol sequences, the regulatory (tat and rev) and the accessory (vif, vpr, vpu, nef) sequences. The envelope plasmid holds the Vesicular stomatitis virus glycoprotein (VSVg) in substitution for the native HIV-1 envelope protein, under the control of a cytomegalovirus (CMV) promoter. The third plasmid (the transfer plasmid) carries the Long Terminal Repeats (LTRs), encapsulation sequence (ψ), the Rev Response Element (RRE) sequence and the CMV promoter to express the transgene inside the host cell.
The second lentiviral vector generation was characterized by the deletion of the virulence sequences vpr, vif, vpu and nef. The packaging vector was reduced to gag, pol, tat and rev genes, therefore increasing the safety of the system.
To improve the lentiviral system, the third-generation vectors have been designed by removing the tat gene from the packaging construct and inactivating the LTR from the vector cassette, therefore reducing problems related to insertional mutagenesis effects.
The various lentivirus generations are described in the following references: First generation: Naldini etal. (1996) Science 272(5259): 263-7; Second generation: Zufferey etal. (1997) Nat. Biotechnol. 15(9): 871-5; Third generation: Dull etal. (1998) J. Virol. 72(11): 8463-7, all of which are incorporated herein by reference in their entirety. A review on the development of lentiviral vectors can be found in Sakuma etal. (2012) Biochem. J. 443(3): 603-18 and PicangoCastro etal. (2008) Exp. Opin. Therap. Patents 18(5):525-539.
The terms transfection, transformation and transduction as used herein, may be used to describe the insertion of the vector into the target cell. Insertion of a vector is usually called transformation for bacterial cells and transfection for eukaryotic cells, although insertion of a viral vector may also be called transduction. References to transduced cells refer to cells where foreign/heterologous DNA has been introduced into a cell, in particular by a viral vector.
The term transgene refers to heterologous or foreign DNA which is not present or not sufficiently expressed in the host cell (i.e. the haematopoietic cell) in which it is introduced. This may include, for example, when a target gene is not expressed correctly in the host cell, therefore a corrected version of the target gene is introduced as the transgene. Therefore, the transgene may be a gene of potential therapeutic interest. The transgene may have been obtained from another cell type, or another species, or prepared synthetically. Alternatively, the transgene may have been obtained from the host cell, but operably linked to regulatory regions which are different to those present in the native gene. Alternatively, the transgene may be a different allele or variant of a gene present in the host cell.
The term autologous as used herein, refers to cells from the same subject. The term allogeneic as used herein, refers to cells of the same species that differ genetically to the cell in comparison.
The terms individual, subject and patient are used herein interchangeably. In one embodiment, the subject is a mammal, such as a mouse, a primate, for example a marmoset or monkey, or a human. In a further embodiment, the subject is a human.
FORMULATIONS
According to a first aspect of the invention, there is provided a composition comprising transduced cells at a concentration of at least 0.5 x 108 cells/mL.
The present inventors have developed compositions comprising high concentrations of transduced cells because minimising volume by concentrating cells will help to reduce the volume of excipients administered to patients. This is desirable in methods of gene therapy where administration of large volumes of excipients has been associated with negative side effects. Furthermore, high concentrations of cells are thought to be more stable and maintain cell viability due to increased cell proximity. Therefore, there is potentially another advantage for the use of high concentration formulations of transduced cells.
In one embodiment, the transduced cells are at a concentration of at least 0.7 x 108 cells/mL. In one embodiment, the transduced cells are at a concentration of at least about 0.8 x 108 cells/mL, such as about 0.9 x 108, 1 x 108, 1.2 x 108, 1.5 x 108, 1.7 x 108, 2 x 108, 2.5 x 108, 3 x 108, 3.5 x 108, 4 x 108, 4.5 x 108, 5 x 108, 5.5 x 108, 6 x 108, 6.5 x 108, 7 x 108, 7.5 x 108, 8 x 108, 8.5 x 108 or 9 x 108 cells/mL.
In one embodiment, the transduced cells are at a concentration of about 1 x 108cells/mL. In an alternative embodiment, the transduced cells are at a concentration of about 2 x 108cells/mL. In one embodiment, the transduced cells are at a concentration of between about 0.5 x 108cells/mL to about 2 x 108 cells/mL. In a further embodiment, the transduced cells are at a concentration of between about 1 x 108cells/mL to about 2 x 108cells/mL.
TRANSDUCED CELLS
In one embodiment, the transduced cells are stem cells or haematopoietic cells.
In one embodiment, the stem cells are haematopoietic stem cells. In a further embodiment, the haematopoietic stem cells are CD34+ haematopoietic stem cells, CD34+CD38- haematopoietic stem cells or a mixture thereof. In a yet further embodiment, the haematopoietic stem cells are CD34+ haematopoietic stem cells.
In one embodiment, the haematopoietic stem cells are obtained from bone marrow, peripheral blood or cord blood (also referred to as umbilical cord blood). In a further embodiment, the haematopoietic stem cells are obtained from bone marrow.
In one embodiment, the haematopoietic cells are white blood cells, such as lymphocytes. In a further embodiment, the haematopoietic cells are lymphocytes, such as B cells, T cells, Natural Killer cells or a mixture thereof. In a yet further embodiment, the haematopoietic cells are T cells or Natural Killer cells.
In one embodiment, the cells are mammalian cells. In a further embodiment, the mammalian cells are human cells.
In one embodiment, the cells are allogeneic or autologous. It will be understood that autologous refers to cells obtained from the patient themselves, whereas allogeneic refers to cells obtained from a donor. Autologous cells have the advantage that they are compatible with the patient and therefore avoid any immunological compatibility problems leading to graft-versus-host disease (GvHD). It will be understood that in order to prevent the allogeneic cells from being rejected by the patient, they would either need to be derived from a compatible donor or modified to ensure no antigens are present on the cell surface which would initiate an unwanted immune response.
In one embodiment, the cells are transduced with a viral vector. In a further embodiment, the cells are transduced with a retroviral vector, such as a lentiviral vector.
In one embodiment, the retroviral vector is derived from, or selected from, a lentivirus, alpha-retrovirus, gamma-retrovirus or foamy-retrovirus, such as a lentivirus or gamma-retrovirus, in particular a lentivirus. In a further embodiment, the retroviral vector particle is a lentivirus selected from the group consisting of HIV-1, HIV-2, SIV, FIV, EIAV and Visna. Lentiviruses are able to infect non-dividing (i.e. quiescent) cells which makes them attractive vectors for gene therapy. In a yet further embodiment, the retroviral vector is HIV-1 or is derived from HIV-1. The genomic structure of some retroviruses may be found in the art. For example, details on HIV-1 may be found from the NCBI Genbank (Genome Accession No. AF033819). HIV-1 is one of the best understood retroviruses and is therefore often used as a viral vector.
In one embodiment, the cells contain a transgene. Therefore, in one embodiment, the viral vector comprises a transgene (i.e. a heterologous nucleic acid coding sequence). This transgene may be a therapeutically active gene which encodes a gene product which may be used to treat or ameliorate a target disease. The transgene may encode, for example, a protein (for example an enzyme), an antisense RNA, a ribozyme, a toxin, an antigen (which may be used to induce antibodies or helper T-cells or cytotoxic T-cells) or an antibody (such as a single chain antibody). Recent methods of gene therapy have involved the introduction of modified T cell receptor or chimeric antigen receptors (CARs) into haematopoietic cells in order to improve or initiate specific target recognition. Therefore, in one embodiment, the transgene encodes a modified T cell receptor or a chimeric antigen receptor (CAR).
In a one embodiment, the transgene encodes a protein. In a further embodiment, the transgene encodes haemoglobin (such as beta globin), adenosine deaminase (ADA), arylsulfatase A (ARSA), Wiskott-Aldrich syndrome protein (WASp), phagocyte NADPH oxidase, galactosylceramidase, or alpha-L-iduronidase, or functional fragments or derivatives thereof.
The aim of gene therapy is to modify the genetic material of living cells for therapeutic purposes, and it involves the insertion of a functional gene into a cell to achieve a therapeutic effect. For example, haematopoietic stem cell (HSCs) may be extracted from the patient and purified by selecting for CD34 expressing cells (CD34+). Those cells can be cultured with cytokines and growth factors, and then transfected with a viral vector containing the transgene encoding the normally functioning protein, and then given back to the patient. These cells take root in the person's bone marrow, replicating and creating cells that mature and create normally functioning protein, thereby resolving the problem.
Therefore, in one embodiment, the transduced cells described herein may be used in ex vivo gene therapy. The term ex vivo gene therapy refers to the in vitro transduction (e.g. by retroviral transduction) of cells to form transduced cells prior to introducing them into a patient. Therefore, the transduced cells described herein may be used in methods of gene therapy because they contain the corrected gene. In particular, the transduced stem cells described herein are useful in methods of gene therapy because all progeny from the stem cells will contain the corrected gene. The transduced cells can therefore be used for treatment of a mammalian subject, such as a human subject, suffering from a condition including but not limited to, inherited disorders, cancer, and certain viral infections.
Beta Thalassemia (also called Beta Thai) is an inherited blood disorder characterized by reduced levels of functional haemoglobin caused by a mutation in the HBB gene. Therefore, it will be understood that if the cell is transduced with a viral vector containing a transgene encoding functional haemoglobin (e.g. beta globin), then this transduced cell may be used in the treatment of Beta Thalassemia. Therefore, in one embodiment, the transduced cell contains a transgene encoding functional haemoglobin or a fragment or derivative thereof. In a further embodiment, the transduced cell contains the HBB gene. In a further embodiment, the transduced cell is for use in the treatment of Beta Thalassemia.
Adenosine deaminase deficiency (also called ADA deficiency or ADA-SCID) is a metabolic disorder that causes immunodeficiency due to a lack of the enzyme adenosine deaminase (ADA). Therefore, it will be understood that if the cell is transduced with a viral vector containing a transgene encoding adenosine deaminase, then this transduced cell may be used in the treatment of ADA-SCID. Therefore, in one embodiment, the transduced cell contains a transgene encoding adenosine deaminase or a fragment or derivative thereof. In a further embodiment, the transduced cell is for use in the treatment of ADA-SCID. In a yet further embodiment, the transduced cell is Strimvelis™ (autologous CD34+ enriched cell fraction that contains CD34+ cells transduced with retroviral vector that encodes for the human ADA cDNA sequence).
Metachromatic leukodystrophy (also called MLD or Arylsulfatase A deficiency) is a lysosomal storage disease caused by a deficiency of the enzyme arylsulfatase A (ARSA). Therefore, it will be understood that if the cell is transduced with a viral vector containing a transgene encoding arylsulfatase A, then this transduced cell may be used in the treatment of MLD. Therefore, in one embodiment, the transduced cell contains a transgene encoding arylsulfatase A or a fragment or derivative thereof. In a further embodiment, the transduced cell contains the ARSA gene. In a further embodiment, the transduced cell is for use in the treatment of MLD.
Wiskott-Aldrich syndrome (also called WAS or eczema-thrombocytopenia-immunodeficiency syndrome) is a X-linked recessive disease caused by mutations in the WASp gene. Therefore, it will be understood that if the cell is transduced with a viral vector containing a transgene encoding a functional WASp gene, then this transduced cell may be used in the treatment of WAS. Therefore, in one embodiment, the transduced cell contains a transgene encoding Wiskott-Aldrich syndrome protein (WASp) or a fragment or derivative thereof. In a further embodiment, the transduced cell contains the WASp gene. In a further embodiment, the transduced cell is for use in the treatment of WAS.
Chronic granulomatous disease (also called CGD) is caused by mutations in any one of five different genes which leads to a defect in an enzyme called phagocyte NADPH oxidase. Certain white blood cells use this enzyme to produce hydrogen peroxide, which these cells need in order to kill certain bacteria and fungi. Therefore, it will be understood that if the cell is transduced with a viral vector containing a transgene encoding phagocyte NADPH oxidase, then this transduced cell may be used in the treatment of CGD. Therefore, in one embodiment, the transduced cell contains a transgene encoding phagocyte NADPH oxidase or a fragment or derivative thereof. In a further embodiment, the transduced cell is for use in the treatment of CGD.
Globoid cell leukodystrophy (also called GCL, galactosylceramide lipidosis or Krabbe disease) is caused by mutations in the GALC gene which causes a deficiency of an enzyme called galactosylceramidase. This affects the growth ofthe nerve's protective myelin sheath and causes severe degeneration of motor skills. Therefore, it will be understood that if the cell is transduced with a viral vector containing a transgene encoding galactosylceramidase, then this transduced cell may be used in the treatment of GCL. Therefore, in one embodiment, the transduced cell contains a transgene encoding galactosylceramidase or a fragment or derivative thereof. In a further embodiment, the transduced cell contains the GALCgene. In a further embodiment, the transduced cell is for use in the treatment of GCL.
Mucopolysaccharidosis Type I (also called MPS Type I) is a form of MPS (i.e. an inability to metabolize complex carbohydrates known as mucopolysaccharides into simpler molecules) caused by a deficiency ofthe enzyme alpha-L-iduronidase. Therefore, it will be understood that if the cell is transduced with a viral vector containing a transgene encoding alpha-L-iduronidase, then this transduced cell may be used in the treatment of MPS Type I. Therefore, in one embodiment, the transduced cell contains a transgene encoding alpha-L-iduronidase or a fragment or derivative thereof. In a further embodiment, the transduced cell is for use in the treatment of MPS Type I.
CRYOPRESERVATION
In one embodiment, the composition is cryopreserved, i.e. the composition is frozen.
Cryopreservation involves freezing the cells at extremely low temperatures (typically -80°C in a mechanical freezer or -196°C in liquid- or vapour-phase nitrogen) and can give a shelf life of months to years. In most stem cell gene therapy techniques, it is necessary to cryopreserve collected cells to allow for cell conditioning regimens requiring multiple days, as well as storage of HSCs for future use.
During freezing, water is removed from the cytoplasm of the cell as ice forms outside the cell. This increases the concentration of solutes within the cell through hyper-osmosis which can lead to dehydration and pH changes which may be damaging to the cell (Motta eta/., (2014) Cryobiology, 68, (3) 343-348). This can also result in cell volume changes which can cause mechanical damage to the cells. Formation of ice crystals during freezing, or re-crystallisation during thawing, can also cause mechanical damage to the cell through pressure or puncture of the cell membrane. Furthermore, low temperatures may alter the physical-chemical structure of cell membranes e.g. lipid complexes are denatured.
In an attempt to minimize the effects of ice crystal formation, cells are typically frozen in a medium with cryoprotectants. There are two main types of cryoprotectant used to minimise freezing-related damage to cells: intracellular cryoprotectants penetrate the cell membrane, and work to prevent intracellular ice crystal formation and membrane rupture; while extracellular cryoprotectants cannot penetrate the cell membrane unless helped by an additional reagent, and work by lowering the hyperosmotic effect. Therefore, in one embodiment, the composition comprises a cryoprotectant. In a further embodiment, the composition comprises a cryoprotective formulation. Thus, according to a further aspect of the invention, there is provided a cryoprotective formulation comprising transduced cells at a concentration of at least 0.5 χ 108 cells/mL, such as about 1 χ 108 cells/mL.
Different types and combinations of cryoprotectants have been found to be effective to preserve specific types of cells. For example, human bone marrow committed stem cells have been shown to be preserved by a cryoprotectant combination of dextran, glycerol, and dimethyl sulfoxide. Most cryoprotective formulations comprise dimethyl sulfoxide (DMSO).
Therefore, in one embodiment, the composition comprises DMSO. In a further embodiment, the composition comprises 1% to 10%, 2% to 8%, 3% to 7% by volume of DMSO, such as 4% to 6%, 4% to 5% or 5% to 6% by volume of DMSO. In one embodiment, the composition comprises about 5% to about 10% by volume of DMSO. In a further embodiment, the cryoprotective formulation comprises about 5% by volume of DMSO.
Human serum albumin (HSA) is the most abundant circulating protein in the plasma, which transports hormones, fatty acids, and other compounds, buffers pH, and maintains oncotic pressure. HSA has also been found to have antioxidant properties due to its ability to trap free-radicals (Roche etal., (2008) FEBS Lett., 582(13), 1783-1787). Therefore, in one embodiment, the composition comprises 4% to 10%, 5% to 9%, such as 6% to 8%, 6% to 7% or 7% to 8% by volume of HSA.
In a further embodiment, the cryoprotective formulation comprises about 7% by volume of HSA.
The composition may contain auxiliary substances, such as water, saline, pH buffering agents, carriers or excipients, other stabilizers and/or buffers or other reagents that enhance the viability of the haematopoietic cells following the freezing and thawing process.
In one embodiment, the composition is formulated in saline solution, i.e. 0.9% NaCI. Saline solution is suitable for administration to patients, therefore the advantage of formulating the formulation in saline solution is that it allows direct infusion into patients.
In one embodiment, the composition is for administration to a mammal. In one embodiment, the mammal is a human or mouse. Therefore, in one embodiment, the composition is for administration to a human (i.e. a patient).
The compositions described herein may be administered by injection or continuous infusion (examples include, but are not limited to, intravenous, intrabone, intraperitoneal, intradermal, subcutaneous, intramuscular and intraportal). In one embodiment, the composition is suitable for intrabone or intravenous administration.
Intrabone administration (also known as intraosseous infusion) is the process of injecting directly into the marrow of a bone. The composition described herein provides a high concentration of transduced cells per mL, therefore a smaller volume of the composition needs to be administered in order for the stem cell gene therapy to be successful. This is especially well suited for intrabone administration which has a finite space for such infusion, hence the administration of lower volumes is preferred.
Cryoformulations of cells containing large amounts of DMSO have been associated with considerable toxicity during infusion (Rowley etal. (1994) Blood). Therefore, the majority of protocols described in the art requiring intrabone injection, require that the cell preparation is washed (e.g. with a saline solution) prior to infusion. The composition described herein provides a higher concentration of transduced cells, without an increased amount of excipients or DMSO, therefore the compositions described herein will have fewer side effects.
METHODS
According to a further aspect of the invention, there is provided a method for preparing a transduced cell composition, comprising:
(a) obtaining a plurality of transduced cells; and (b) suspending the transduced cells in a medium at a concentration of at least 0.5 χ 108 cells/mL.
In one embodiment, step (a) comprises: (i) obtaining a plurality of cells; and (ii) transducing the cells with a viral vector. The transducing methods of step (ii) may be performed by methods well known in the art. For example, the method may additionally comprise cell processing steps, such as cell analysis and/or expansion. Analysis may comprise analysing the cells for immunophenotype, clonogenic potential, cell count and/or cell viability. Expansion may comprise culturing cells for several days (e.g. 2-3 days) in various factors to encourage cell expansion.
In one embodiment, the cells are obtained from a patient (i.e. autologous) or a donor (i.e. allogeneic). In a further embodiment, the cells are obtained from a patient (i.e. autologous).
In one embodiment, the cells are transduced with a viral vector. As described herein, the viral vector may be, for example, a retroviral or lentiviral vector.
In one embodiment, the medium is a cryoformulation (such as the cryoformulation as described hereinbefore). In a further embodiment, the cryoformulation comprises DMSO and optionally HSA and/or saline. In a yet further embodiment, the cryoformulation comprises 5% DMSO and 7% HSA in 0.9 % saline.
In one embodiment, the method additionally comprises freezing the composition.
There are many standard methods known in the art which can be used to freeze the cells, e.g. immersing containers holding the suspension of step (b) in a solid carbon dioxide and alcohol mixture, or in liquid nitrogen, or placed directly in a freezer set at a desired temperature. In one embodiment, the suspension (i.e. obtained in step (b)) is frozen in step (c) with a programmed freezer (i.e. controlled rate freezer). Controlled rate freezers are commercially available and well known in the art, for example the EF600M controlled rate freezer (Aysmptote). Such controlled rate freezers can be used to both freeze and thaw a suspension.
In one embodiment, the suspension is frozen in step (c) at a temperature from about -200°C to a temperature of about -35°C. In a further embodiment, the suspension is frozen in step (c) at a temperature of less than -80°C, such as less than -130°C.
In one embodiment, the transduced cells are frozen at a cell concentration of at least about 1 x 108cells/mL.
In one embodiment, the method additionally comprises: (d) thawing the frozen suspension. There are many standard methods known in the art which can be used to thaw the frozen suspension, e.g. by allowing the suspension to thaw slowly at room temperature, or by immersing the frozen suspension in a liquid, e.g. a water-bath set at a temperature of about 37°C. Cells can also be thawed by mixing the suspension with a thawed medium.
High concentrations of cells are thought to be more stable and maintain cell viability due to increased cell proximity. Therefore, in one embodiment, there is provide a method of stabilising a transduced cell composition using the methods described herein. This is both beneficial in the context of long term storage and also during freeze-thaw methods.
In one embodiment, the method additionally comprises administering the transduced cell composition to a patient. If the suspension is frozen, the method additionally comprises: administering the thawed suspension to a patient. In one embodiment, the thawed suspension is washed prior to the administering step.
In one embodiment, the transduced cell composition is administered to the patient in an effective amount, i.e. an amount sufficient to induce or reduce the desired phenotype. Effective doses and treatment regimes for administering the composition ofthe present invention may be dependent on factors such as the age, weight and health status of the patient and disease to be treated. Such factors are within the purview of the attending physician.
In one embodiment, the patient is a human. In a further embodiment, the human may be at any stage of development at the time of administration, e.g. infantile, juvenile or adult.
In one embodiment, the transduced cell composition is administered to the patient via intrabone administration or intravenously.
According to a further aspect of the invention, there is provided a frozen suspension obtained by the method described herein.
According to a further aspect of the invention, there is provided a thawed suspension obtained by the method described herein.
The compositions described herein can be used to treat patients using methods of gene therapy. Therefore, according to a further aspect of the invention, there is provided a method of treatment comprising:
(a) obtaining a plurality of transduced cells;
(b) suspending the transduced cells in a medium at a concentration of at least about 1 x 108 cells/mL; and (c) administering the suspension obtained in step (b) to a patient.
It will be understood that the embodiments described herein for the composition, equally apply to the methods described herein.
USES
According to a further aspect of the invention, there is provided a use of the composition described herein in a method of therapy, in particular in a method of gene therapy.
According to a further aspect of the invention, there is provided the composition described herein for use in therapy. In one embodiment, the therapy comprises ex vivo gene therapy. The transduced stem cells described herein are useful in methods of gene therapy because all progeny from the stem cells will contain the corrected gene. The transduced cells can therefore be used for treatment of a mammalian subject, such as a human subject, suffering from a condition including but not limited to, inherited disorders, cancer, and certain viral infections.
It will be understood that the embodiments described herein may be applied to all aspects of the invention. Furthermore, all publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth.
Claims (20)
1. A composition comprising transduced cells at a concentration of at least 0.5 χ 108 cells/mL.
2. The composition of claim 1, wherein the cells are stem cells or T cells.
3. The composition of claim 2, wherein the stem cells are haematopoietic stem cells.
4. The composition of any one of claims 1 to 3, wherein the cells are allogeneic or autologous.
5. The composition of any one of claims 1 to 4, wherein the cells are transduced with a lentiviral vector.
6. The composition of any one of claims 1 to 5, wherein the cells contain a transgene encoding human beta globin or a fragment or derivative thereof.
7. The composition of any one of claims 1 to 6, which additionally comprises a cryoprotectant.
8. The composition of any one of claims 1 to 7, which is for administration to a mammal.
9. The composition of any one of claims 1 to 8, which is suitable for intrabone or intravenous administration.
10. The composition of any one of claims 1 to 9, wherein the transduced cells at a concentration of between about 1 χ 108 cells/mL and about 2 χ 108 cells/mL.
11. A method for preparing a transduced cell composition, comprising:
(a) obtaining a plurality of transduced cells; and (b) suspending the transduced cells in a medium at a concentration of at least 0.5 χ 108 cells/mL.
12. The method of claim 11, wherein step (a) comprises: (i) obtaining a plurality of cells; and (ii) transducing the cells with a viral vector.
13. The method of claim 11 or claim 12, wherein the cells are obtained from the patient or a donor.
14. The method of any one of claims 11 to 13, wherein the cells are transduced with a lentiviral vector.
15. The method of any one of claims 11 to 14, wherein the medium is a cryoformulation.
16. The method of claim 15, wherein the cryoformulation comprises DMSO and optionally HSA and/or saline.
17. The method of claim 16, wherein the cryoformulation comprises 5% by volume of dimethyl 10 sulfoxide (DMSO) and about 7% by volume of human serum albumin (HSA) formulated in 0.9 % saline solution.
18. The method of any one of claims 15 to 17, which additionally comprises freezing the composition.
19. The method of any one of claims 11 to 18, additionally comprising administering the composition to a patient.
20. The method of claim 19, wherein the composition is administered to the patient via 20 intrabone administration or intravenously.
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| GB1704968.5A GB2560933A (en) | 2017-03-28 | 2017-03-28 | Transduced cell formulation |
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| GB1704968.5A GB2560933A (en) | 2017-03-28 | 2017-03-28 | Transduced cell formulation |
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| GB201704968D0 GB201704968D0 (en) | 2017-05-10 |
| GB2560933A true GB2560933A (en) | 2018-10-03 |
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| GB1704968.5A Withdrawn GB2560933A (en) | 2017-03-28 | 2017-03-28 | Transduced cell formulation |
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| GB (1) | GB2560933A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110583622A (en) * | 2019-08-30 | 2019-12-20 | 依科赛生物科技（太仓）有限公司 | T cell serum-free freezing medium and use method thereof |
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| WO2003002155A1 (en) * | 2001-06-29 | 2003-01-09 | Sloan Kettering Institute For Cancer Research | Vector encoding human globlin gene and use thereof in treatment of hemoglobinopathies |
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| WO2003002155A1 (en) * | 2001-06-29 | 2003-01-09 | Sloan Kettering Institute For Cancer Research | Vector encoding human globlin gene and use thereof in treatment of hemoglobinopathies |
| WO2008147057A1 (en) * | 2007-05-25 | 2008-12-04 | Rnl Bio Co., Ltd | Composition for treating ischemic limb disease comprising stem cells derived from adipose tissue |
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| CN110583622A (en) * | 2019-08-30 | 2019-12-20 | 依科赛生物科技（太仓）有限公司 | T cell serum-free freezing medium and use method thereof |
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| Publication number | Publication date |
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| GB201704968D0 (en) | 2017-05-10 |
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