Patent Grant
11959099
This application is a U.S. national stage filing under 35 U.S.C. § 371 from International Application No. PCT/GB2018/000064, filed on 11 Apr. 2018, and published as WO2018/189501 on 18 Oct. 2018, which claims the benefit under 35 U.S.C. 119 to United Kingdom Application No. GB 1705955.1, filed on 13 Apr. 2017, the benefit of priority of each of which is claimed herein, and which applications and publication are hereby incorporated herein by reference in their entirety.
This invention relates to a method of culturing cells.
The development and maintenance of the organisms requires many complex interactions between cells and extracellular matrix (ECM) components. Control of the cellular microenvironment is also important for assuring functionality of tissue engineered organ substitutes. The use of ECM mimics, which utilize collagen gel compaction, electromagnetic fields, electrospinning of nanofibers, mechanical stimulation and microstructured culture plates for artificial guidance of cells, have all been explored.
The inventors have found that a polyhedron-based delivery system which releases growth factor (Polyhedra Delivery System, PODS) provides a stable physiologically relevant gradient of growth factor. The inventors' work investigates the activity of a growth factor gradient generated by a PODS which is set up by sustained release over a period of time, and is able to direct changes in relevant growth factor sensitive cells. The inventors have surprisingly found that using a PODS in this way allows differentiated cells with desired properties, such as directional growth, to be produced in the absence of extracellular matrix (ECM). Part of their work concerns preparation of an unbranched chain of neurons connected by axons.
Accordingly a first aspect of the invention provides a method of altering cells comprising culturing them in a growth factor gradient, wherein said gradient is provided by a polyhedra delivery system (PODS) that releases the growth factor to set up the gradient.
A second aspect of the present invention provides PODS as a therapeutic agent for use in a method of therapy.
A third aspect of the invention provides cells altered by the methods of the invention, optionally with a delivery vehicle, for use in a method of therapy.
The terms ‘PODS’, ‘microcrystal’ and ‘crystal’ are used interchangeably herein. However it should be understood that in embodiments which refer to ‘microcrystal’ or ‘crystal’ non-crystalline forms of PODS can also be used.
When used in culture systems growth factors can be rapidly and homogenously diffused, but the imbalance and asymmetry of growth factors is important for the development of certain cells. The invention shows these complex phenomena can be reproduced in vitro by a PODS that releases a growth factor. The invention concerns altering cells by means of a gradient of growth factor generated by a PODS. The altering of the cells which occurs in the method of the invention may be due to one part of the cell being in contact with the growth factor at a different concentration from another part of the cell. It may be due to one cell being in communication or contact with another cell in contact with the growth factor at a different concentration. Through this mechanism the gradient is able to impart certain characteristic to the cell which would not be possible where the growth factor was present at a homogenous concentration, for example at the same concentration at each point of the cell surface. Further the fact that the gradient is unchanging is also an important in imparting the desired characteristic to the cell.
Culturing
The invention concerns culturing cells, which typically comprises placing them in conditions where they grow and/or differentiate. The conditions may be in vivo or in vitro, such as in a human or animal body.
Whilst the features of the invention will be described with reference to its in vitro embodiments these features may also apply to the in vivo embodiments as appropriate.
Culturing in vitro is typically in an aqueous medium. Optionally the culturing takes place in a medium comprising aqueous gel. The culturing may take place in a vessel, optionally a dish. The culturing may or may not take place on a coverslip, optionally in a culture medium in contact with a coverslip.
The culturing may be in any suitable system, for example a 2-dimensional or 3-dimensional system. The culturing may in static conditions, for example in which there is no flow of medium and/or there is no change in medium. In preferred embodiments medium is not changed for at least 10 hours, for example for at least 20, 30, 50, 100 or 500 hours.
The culturing will preferably take place in a medium in which is able to sustain the cells and/or allow them to grow. The medium typically comprises one or more nutrients. The medium may or may not be serum-free. The medium may comprise DMEM or neurobasal.
Cells
As used herein the term cells typically refers to eukaryotic cells, preferably human or animal cells, such as mammalian or avian cells. Optionally the cells described herein are neuronal cells. Preferably the cells are PC12 cells. Before culturing according to the methods of the invention the cells are typically undifferentiated cell such as stem cells or pluripotent precursor cells. The cells are typically non-specialized cells, or cells that are not mature, or cells that can undergo further stages in development. The shape of the cells before culturing is typically amorphous or spherical. The arrangement of the cells before culturing is typically incoherent, or without a well-defined order. The cells which are cultured may be continuous cell lines (e.g. with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g. non-transformed cells), or any other cell population maintained in vitro. The cells may be isolated, purified or partially purified cells.
Neurons are preferred cells, and the method of the invention may result in formation of sympathetic neurons.
The cells are typically responsive to the growth factor. They may be of the same species as the growth factor. They may be cells associated with any condition mentioned herein and/or they may be cells which can be used to treat any condition mentioned herein. Whenever ‘treatment’ or ‘treating’ is mentioned it includes ‘preventative’ or ‘prophylactic’ uses. The cells may be associated with or responsible for any of the activities mentioned in herein or in Table 1.
Growth Factor
As used herein a growth factor is typically an extracellular protein capable of stimulating or inhibiting cellular growth, proliferation and cellular differentiation. Thus the growth factor may be a hormone or cytokine. The growth factor may be a natural or artificial one, and may for example be a homologue and/or fragment of a natural growth factor which retains growth factor activity. It may be a homologue and/or fragment of any specific growth factor mentioned herein, for example in Table 1. It may be a eukaryotic, preferably human or animal, such as mammalian or avian, growth factor.
The growth factor can be a neurotrophin. The expression ‘Neurotrophin’ is used interchangeably herein with the expression ‘Neurotrophic growth factor’. Neurotrophins are the family of biomolecules that support the growth, survival and differentiation of both developing and mature neurons. The growth factor can be one or more members of one of the three main families, such as neurotrophin family (for example, nerve growth factor NGF, neurotrophin-3 NT-3, brain-derived neurotrophic factor BDNF, neurotrophin 4 NT-4 (also known as NT-5), ciliary neurotrophic factor (CNTF) family (CNTF, Leukemia inhibitory factor LIF, Interleukin-6), and GDNF (Glial cell-line neurotrophic factor) family (for example GDNF, CDNF, Artemin, Neuturin, Persephin). The growth factor may be a semaphorin or slit molecule.
Neurons can be cultured using gradients of neurotrophic growth factors. Nerve growth factor (NGF) can promote myelination and/or differentiation of neurons and/or axon growth and/or dendrite formation and/or elongation of the neuron perpendicular to the direction of the gradient and/or intermediate filament growth and/or tau protein expression and/or microtubule growth.
The growth factor can be a bone growth factor. A bone growth factor is a growth factor that stimulates the growth of bone tissue. Bone growth factors include bone morphogenetic proteins (BMPs), insulin-like growth factor (IGF-1), insulin-like growth factor-2 (IGF-2), transforming growth factor beta (TGF-b), fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), parathyroid hormone-related peptide (PTHrP), bone morphogenetic proteins (BMPs), and certain members of the growth differentiation factor (GDF) group of proteins. Preferred GDF proteins include:
GDF1—Studies in rodents suggest that this protein is involved in the establishment of left-right asymmetry in early embryogenesis and in neural development in later embryogenesis.
GDF2—This protein regulates cartilage and bone development, angiogenesis and differentiation of cholinergic central nervous system neurons.
GDF5—This protein regulates the development of numerous tissue and cell types, including cartilage, joints, brown fat, teeth, and the growth of neuronal axons and dendrites
GDF7—This protein may play a role in the differentiation of tendon cells and spinal cord interneurons.
GDF10—This promotes neural repair after stroke.
GDF11—This protein plays a role in the development of the nervous and other organ systems, and may regulate aging.
The growth factor is preferably one which exerts an effect via microtubules, and or intermediate filaments/and or microfilaments, such as neuronal growth factors, or neurotrophins, for example NGF.
Table 1 describes preferred growth factors and exemplified constructs for expressing them, one or more of which may be used in the methods of the invention.
The growth factor in the gradient will be in purified or substantially purified form.
Differentiation and/or Proliferation
Differentiation is typically the process whereby cells become specialized in order to perform a specific function. Through culturing according to the method of the invention cells can alter by acquiring specialized structural and/or functional features.
Proliferation is typically a process that results in an increase in the number of cells. Proliferation can be regulated by the slope (change in concentration per unit distance) of a gradient and/or by the direction of gradient.
Changes that Typically Occur in the Method of the Invention
The cells may alter in one or more structural features including:
Change of type: Cells can change to a different type in response to a gradient of growth factor. Cells can become a first type at one part of the gradient. Cells can become a second type at a second part of the gradient. For example for Wnt in mammals at the highest concentration establishes the posterior region whilst in areas of lowest concentration establishes the anterior region.
Change in arrangement or long range order: The cells can become more or less prevalent at one part of the gradient in comparison to uncultured cells or cultured cells grown in an equivalent uniform concentration of growth factor. The cells can become more or less prevalent at one part of the gradient in comparison to cells grown at a second part of the gradient. In one embodiment the cells are completely absent from certain points of the gradient. The cells or prominent cellular features, for example projections, such as neurites, optionally in axons or dendrites, can align in a direction which is well defined with respect to the direction of the gradient, for example along the gradient, or perpendicular to the gradient.
In one embodiment the cells align and/or attach to other (for example as described in the Examples). Such cells may align at a distance of 50 to 150 microns from the PODS, such as at a distance of 80 to 120 microns. In one embodiment there may be only a single set of aligned and/or attached cells (for example forming a circle), or there may be no aligned and/or attached cells outside the above distance ranges. The alignment is preferably autonomous, in that the gradient is the only cause of the alignment.
Change of shape: Cells can change shape optionally becoming elongated in shape, or can alter so that there is an increase in the cell surface to volume ratio. In an elongated cell the ratio of longest to shortest dimension is typically at least 2 to 1, at least 5 to 1, at least 10 to 1, at least 20 to 1, at least 100 to 1, at least 500 to 1, and optionally less than 100,000 to 1 or less than 10,000 to 1.
Change in expression profile: Cells can upregulate expression of proteins not expressed or minimally expressed compared to uncultured cells or cultured cells grown in equivalent uniform concentration of growth factor. Cells can downregulate expression of proteins not expressed or minimally expressed compared to uncultured cells or cultured cells grown in equivalent uniform concentration of growth factor. Cells can upregulate expression of one or more proteins and downregulate expression of one or more different proteins not expressed or minimally expressed compared to uncultured cells or cultured cells grown in equivalent uniform concentration of growth factor. Expression of proteins relevant to the structure of the cells may be upregulated, optionally proteins in filaments such as microfilaments, neurofilaments, microtubules, associated with microtubules such as tau. Expression of receptors can be upregulated. In one embodiment specific markers are expressed when the cells are altered.
Change in physical features: A change in the microtubules and/or microtubule associated proteins and/or intermediate filaments, for example tau and neurofilaments can occur, such as alignment of the microtubules or intermediate filaments, optionally neurofilaments, with respect to the gradient of growth factor, for example alignment of the longest dimension of the cell along the gradient of the growth factor or alignment of the shortest dimension of the cell along the gradient of the growth factor.
Cells cultured according to the methods of the invention may form lines (e.g. straight, curved or circular lines) of connecting cells, optionally where the line of cells is orientated perpendicular to the direction of the growth factor gradient.
Nerve cells, or neurons, cultured according to the method of the invention are preferably elongated in shape, and/or unbranched and/or have neurite projections at polar positions. Nerve cells may have axons and/or a growth cone/and or dendrites.
The Gradient
In the method of the invention the growth factor is not present at a homogenous concentration. Instead it is in the form of a gradient. As used herein, the gradient is typically the change in the value of growth factor concentration per unit distance in a particular direction, normally determined by sustained release by the PODS and diffusion through the medium. The concentration is preferably highest at the PODS and decreases with distance away from the PODS. The direction of the gradient is from the/a region containing PODS towards the/a region containing no PODS. For a circular arrangement of PODS in 2D or 3D the direction of the gradient is radially outwards from the PODS. For a linear arrangement of PODS the direction of the gradient is perpendicular to the axis along which the PODS are arranged. The gradient is provided by the PODS which, for example, break down and release growth factor in situ.
Growth factor is typically slowly released from the microcrystals field (which is the location where the PODS are), preferably resulting in a steady physiologically relevant gradient in growth factor at the periphery of the field. This microenvironment can result in the alteration of the cells in one or more ways as described in the paragraphs above. Typically for neurons, culturing in a growth factor gradient results in alteration of the cells in all of the ways described in the paragraphs above. The altering for neurons cultured in a growth factor gradient includes induction of differentiation and can induce alignment of cells, for example nerve cells, preferably PC12 cells.
Gradients will normally occur when the PODS is confined to a small part of the culture space. An important property of a PODS is that it typically generates short gradients. Short gradients are important for reducing off-target effects caused by high concentrations of growth factor diffusing to the surrounding area, for example to neighbouring tissues in therapeutic use. The concentration of growth factor in the gradients described herein is typically at a maximum in the microcrystals field. The concentration reduces as the distance from the microcrystals field increases, typically dropping to half of the maximum value at up to 5 microns, up to 10 microns, or up to 30 microns, or up to 60 microns, or up to 100 microns, or up to 150 microns from the microcrystals field. The PODS usually provide a ‘short range’ gradient, and in certain situations problems can arise with long range gradients. In one embodiment the gradient has a maximum size of up to 300 microns, such as up to 200 microns.
The gradients of the invention will be established in up to a day. The gradient may persist/last for at least 12 hours, 1 day, 5 days, 14 days, 30 days or 90 days. In a preferred embodiment the gradient is unchanging, or substantially unchanging. For example the concentration of the growth factor does not decrease by more than 10% over 12 hours, over 1 day or over 2 days at 10 microns from the PODS.
Microcrystals
Viruses such as cypoviruses and baculoviruses produce microcrystals which are crystals of the protein polyhedrin, also known as polyhedra. The microcrystals of the invention, or polyhedra, are typically regular arrays of the polyhedrin protein assembled in a cubic crystal lattice. The microcrystals are typically cubic in shape, or optionally form as irregular crystals. The microcrystals of the invention are typically isolated from cells, such as insect cells, which have been infected with virus, preferably baculovirus. The microcrystals typically range in size from 0.1 to 10 micron, for example 2 to 5 micron (measured as the maximum distance inside the microcrystal between different surfaces or the maximum length of an edge). The microcrystals typically comprise, for example encapsulate, one or more types of growth factor.
PODS and Polyhedra
The PODS used in the invention is typically in the form of, or comprises, microcrystals which comprise a structural protein and a growth factor. Each microcrystal (PODS) comprises more than one copy of the structural protein and growth factor, and typically comprises 107 to 109, for example 108 polyhedron proteins and/or 107 to 109, for example 108 growth factor molecules. Typically each PODS comprises 5×107 to 5×108 polyhedron proteins and/or 5×107 to 5×108 growth factor molecules.
The structural protein is typically capable of forming polyhedra, and is preferably a natural or artificial polyhedrin protein. The polyhedrin protein is typically of viral origin, for example a virus of the genus Cypovirus (CPV) or Baculovirus, such as a Bombyx mori cypovirus polyhedrin. Polyhedra are typically in the form of microcrystals which are preferably cubic crystals. Optionally PODS used in the methods of the invention have a maximum dimension (defined by distance between surfaces or length of an edge) of 1 micron, 2 micron, 1-4 micron, up to 10 micron, up to 15 micron or up to 20 micron. The polyhedrin protein typically has homology to all or a part of any such protein described herein.
Characteristics of the Crystal
Typically a crystal unit cell has a dimension of 100 angstroms, i.e. 1×10−8 m. In a 1 micron crystal, a cube with each edge 10−6 m long, there are 100 unit cells along each edge, and so a 1 micron crystal has 100×100×100 unit cells, i.e. 106 unit cells. Each unit cell typically contains 24 copies of polyhedrin, so a 1 μm crystal has 24×106, or 2.4×107 copies of polyhedron, a density of 2.4×107 per μm3. That means a 2 μm crystal has 2×2×2×2.4×107=19.2×107=approx. 2×108 copies of polyhedrin.
H1 tag: this replaces H1 in the polyhedrin. Assuming a 1 in 4 incorporation to minimise disrupting the crystal (H1 forms bunches of four), this would give 6 copies of H1-Growth factor per micron crystal, 6×106 per μm3, and so typically so a 1 μm3 sized PODS contains 6×106 per μm3 copies of growth factor.
VP3 tag: potentially 1 VP3 binding site per polyhedrin molecule, so up to 2.4×107 per μm3, and 2.4×107 copies in a 1 μm crystal.
Typically a crystal comprises 107 to 109 molecules of growth factor, such as about 108 molecules.
Attaching, Encapsulating or Packaging Growth Factor
The PODS of the invention comprises growth factor. This is typically immobilised and/or attached to the PODS in manner that allows release to provide the gradient. In a preferred embodiment the growth factor comprises a tag that is used to attach it to the PODS.
The growth factor can be targeted for packaging in a polyhedra by attachment of the tag to the growth factor. The growth factor polypeptide can typically comprise the growth factor amino acid sequence and the amino acid sequence of part of the polyhedrin protein, preferably helix 1 (H1), even more preferably a sequence with at least 80% homology to an H1 sequence mentioned herein. The growth factor polypeptide can comprise the growth factor amino acid sequence and the amino acid sequence of part of the baculovirus coat protein, preferably VP3, even more preferably a sequence with at least 80% homology to a VP3 sequence disclosed herein. A short VP3 sequence is preferred to minimise any disruptive effect on the PODS.
PODS incorporating growth factor can be prepared by coexpressing polyhedrin protein and polypeptide comprising a targeting portion and a growth factor portion. Cells can be inoculated with recombinant baculovirus expressing polyhedrin to generate empty polyhedra. Cells can be coinfected with recombinant baculovirus expressing polyhedrin and recombinant baculovirus expressing a polypeptide comprising the growth factor and a tag which targets the growth factor for packaging, for example VP3 or H1. PODS comprising polyhedrin and the polypeptide can be prepared. Cells can be coinfected with recombinant baculovirus expressing polyhedrin and one or more different recombinant baculoviruses each expressing a different polypeptide comprising the growth factor and a tag which targets the growth factor for packaging.
PODS comprising polyhedrin and one or more different polypeptides, encoding one or more different growth factors, can be prepared. The different polypeptides can have the same tag or different tags.
The polyhedrin protein can be Bombyx mori cypovirus polyhedrin protein. The recombinant baculovirus can be AcCP-H29 expressing Bombyx mori cypovirus polyhedrin. The cells used to produce the PODS, or growth factor-encapsulated polyhedral, can be Spodoptera frugiperda IPLB-SF21-AE cells (Sf cells). The PODS can be isolated from the cells, optionally insect cells, by centrifugation.
The PODS can be prepared in an aqueous suspension. The concentration of the PODS suspension can be more than 104 PODS per microliter volume, typically from 104 to 107 PODS per microliter volume, preferably from 5×104 to 5×106 or 105 to 106 PODS per microliter volume. PODS can be applied to (contacted with) the culture system/medium at these concentrations. These can be used in therapy at concentrations of 105 to 107 crystals per microliter, for example at 3×106 crystals per microliter.
The Microcrystals (PODS) Field
The microcrystals field is the location of the microcrystals (that set up the gradient). The term ‘microcrystals field’ includes the feature of a ‘PODS field’ where the PODS are not in crystalline form.
The geometry and size of the microcrystals field may depend on the growth factors. The microcrystals field can be a circular, linear or rectangular field. The shape of the field can be customized for a particular use, for example it can be a curved, including S-shaped, or it can be a shape consisting of straight lines, such as a square, hexagon or an octagon. The microcrystals field can comprise 104-108, preferably 105-107 or 5×105-106 microcrystals in total, or this range of microcrystals in 20 mm2. The field can comprise up to 105 microcrystals, or up to 106 microcrystals, for example in 20 mm2. The microcrystals field can comprise one or more shape elements, for example one or more lines. Each shape element can comprise these stated ranges of microcrystals, for example 104-105 microcrystals, or up to 105 microcrystals, or up to 106 microcrystals.
A microcrystals field can comprise one or more lines or shapes. The lines can be straight lines. The lines and/or shapes may be spaced so that the concentration of growth factor reaches substantially zero between the lines and/or shapes. Adjacent lines or shapes may each comprise the same growth factor. Adjacent lines or shapes may each comprise a different growth factor. The lines and/or shapes may intersect. For example, the lines may form a regular grid pattern. One or more lines or shapes may comprise one or more different growth factors. In a preferred embodiment the field is a 0.5 to 10 mm diameter, for example 1 to 8 or 2 to 4 mm diameter circular field.
A grid structure is envisaged in which individual lines of the grid can contain different growth factor combinations. Adjacent and intersecting grid lines can provide areas where complex gradients will develop, allowing the creation of a wide range of defined and addressable culture conditions in a small area. The lines of the grid can be placed up to 0.1 mm, up to 1 mm, up to 5 mm, from 0.1 mm to 1 mm, from 1 mm to 5 mm, apart.
In the methods herein, the microcrystals field may be applied to a surface, for example the surface of a coverslip, the surface of a cell culture vessel, or the surface of a delivery vehicle such as hydrogel. The microcrystals field may be applied by hand, for example using a micropipette, or may be applied using a robot or other methods of surface deposition including lithography. The microcrystals field can be dried after application to a surface, for example for more than 3 hours. The microcrystals field may be extruded into hydrogel for example using a syringe. The use of a hydrogel or other carrier can increase the duration of the gradient. Synthetic hydrogels or natural materials, preferably collagen, can be used as a delivery vehicle. A field of PODS as described herein can be provided in a delivery vehicle such as a layer of hydrogel which can be manipulated, for example to provide a gradient of growth factor in a defined direction along a pre-determined path. For example, in one embodiment the methods provide an insulated line of PODS spanning an injury to encourage axons to navigate across the injury.
For neuronal growth factors a linear pattern is envisaged. There are any number of different geometries that may be effective. One possibility is creating 3D sandwiches of cells and PODS. A microcrystal field comprising parallel lines of PODS (0.1 to 1 mm apart) can be created. A layer of soft hydrogel or other delivery system vehicle could be layered on this and optionally seeded with neuronal cells or neuronal precursor cells. A stiffer layer of hydrogel can be layered above. After the gel has set, the gel can be manipulated to change the 3D disposition of the lines of PODS. For example, the structure could be rolled to create a “Swiss roll” of cells and PODS. The nerve cells would form linear, unbranched connections perpendicular to the gradient, and therefore along the lines of PODS. This entire structure comprising PODS and cells could be used as an implant, for example to restore nerves that had been lost due to neurodegeneration or injury.
PODS are typically denser than water, and can sink to the bottom of aqueous cell culture medium in a few seconds to within 10 minutes. PODS may be held in place in a culture system by gravity. The microcrystals may be added to a drop of cell culture medium suspended from a surface, optionally example a plastic surface, for example a coverslip. This would establish a gradient across the drop with cells at the bottom experiencing the highest concentrations.
Any suitable may be used to place or attach the PODS. In one embodiment the PODS is attached via a micropatterning system, for example using a photoactivatable reagent and a UV illumination system.
In one embodiment the method of the invention does not comprise a component that mimics an extracellular matrix, for example it might not comprise collagen gel compaction, an electromagnetic field, electrospinning of cells, mechanical stimulation and microstructured culture plates.
For the avoidance of doubt any feature described herein, such as specific growth factors or specific properties of cells may be excluded in certain embodiments of the invention.
Medical Use
The invention includes setting up a gradient for use in therapy. Therefore the invention includes administering the PODS for preventing or treating a condition, such as any condition mentioned herein.
Preferred conditions include growth factor deficiency, degeneration, injury or nerve damage. The PODS may be used in the manufacture of a medicament for preventing or treating a condition. Thus the PODS may be used in therapy, optionally together with a delivery vehicle, for example collagen or a gel (such as a hydrogel). The PODS may be delivered using a scaffold. Examples of diseases that can be treated by the PODS are mentioned herein, including in Table 1.
In preferred embodiments:
Cells prepared using the method of the invention may be used in therapy, for example to treat any condition mentioned herein, such as degeneration, injury or nerve damage. In a preferred embodiment nerve cells prepared in a gradient of neurotrophic growth factors, can be used in a method of treatment for nerve injury or for neurodegeneration.
Homologues and Fragments
Homologues of polypeptide sequences are referred to herein (for example growth factors and tags). Such homologues typically have at least 70% homology, preferably at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% homology, for example over a region of at least 10, 15, 20, 30, 100 or more contiguous amino acids. The homology may be calculated on the basis of amino acid identity (sometimes referred to as “hard homology”). Preferred homologues retain activity, for example growth factor or tag activity.
The UWGCG Package provides the BESTFIT program which can be used to calculate homology and/or % sequence identity (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p 387-395). The PILEUP and BLAST algorithms can be used to calculate homology and/or % sequence identity and/or line up sequences, such as identifying equivalent or corresponding sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.
Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W5 T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both sequences.
The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
The homologous sequence typically differs by 1, 2, 3, 4 or more amino acids, such as less than 10, 15 or 20 amino acids (which may be substitutions, deletions or insertions of amino acids). These changes may be measured across any of the regions mentioned above in relation to calculating homology and/or % sequence identity.
Fragments of any of the polypeptides mentioned herein can be used. Typically such fragments retain the original activity, for example growth factor or tag activity. The fragments typically comprise at least 60%, for example at least 70%, 80%, 90% or 95% of the original sequence.
Therapeutic Agents
Therapeutic agents (PODS and cells) and uses are mentioned herein. The invention provides such agents for use in preventing or treating the relevant condition. This may comprise administering to an individual in need a therapeutically effective amount of the agent. The invention provides use of the agent in the manufacture of a medicament to prevent or treat the disease.
The formulation of the agent will depend upon the nature of the agent. The agent will be provided in the form of a pharmaceutical composition containing the agent and a pharmaceutically acceptable carrier or diluent. Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. The agent may be formulated for parenteral, intravenous, intramuscular, subcutaneous, transdermal or oral administration. Preferably administration can be by injection, nasal (a spray or dry powder), by inhaler or as eye drops.
The dose of an agent may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the individual to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular agent. A suitable dose may however be from 0.1 to 100 mg/kg body weight such as 1 to 40 mg/kg body weight, for example, to be taken from 1 to 3 times daily.
The requisite cDNA for selected growth factors (see Table 1) was purchased from commercial suppliers. Using standard cloning techniques, growth factors sequences were cloned and subsequently subcloned into transfection the plasmids pDEST/VP3 and/or pDEST/H1), resulting in the production of transfer vectors encoding a growth factor sequence fused to VP3 or H1 tags, C-terminally or N-terminally, respectively. Then, Spodoptera frugiperda cells were co-transfected with growth factor transfection vectors and a linear version of non-recombinant parent baculovirus DNA. The successful homologues recombination within insect cells inserted growth factor sequences into the baculovirus genome and so created a recombinant baculovirus variant that allowed the co-expression of a tagged growth factor protein and the polyhedrin protein, both under the control of the polyhedrin promoter for maximum efficiency.
Recombinant baculovirus was amplified as required and growth factor polyhedra were subsequently expressed in suspension insect cell cultures using a shaking incubator for 7-10 days at 27° C. The rapid progress of the cubic growth factor polyhedra expression was monitored by microscope. After culturing for 7-10 days, polyhedra containing cells were harvested by centrifugation and cell pellets stored at −20° C.
Growth factor polyhedra were isolated from insect cells by cell lysis and next purified by at least 3 rounds of PBS washes (from commercial suppliers, pH 7.6-7.9), followed by centrifugation at 3000×g, 4° C., or until a pure product has been achieved, where pure polyhedrin is characterised by a milky-white appearance in aqueous liquid, depending on the concentration of polyhedra, and was readily assessed under a standard bright field microscope with a 20× and 40× magnification. Lastly, growth factor polyhedra were counted and stored at 4° C. in PBS (pH 7.6-7-9).
The cDNA encoding the NGF ORF was purchased from Toyobo in an entry vector. The full-length (241 amino acids) and mature (120 amino acids) form NGF were cloned and subcloned into each destination vector (pDEST/VP3, and pDEST/H1), resulting in production of the transfer vectors encoding the full-length or mature form of NGF fused with VP3 or H1 tags (pTransH1/full NGF, pTransH1/mature NGF, pTransVP3/full NGF, and pTransVP3/mature NGF). These transfer vectors were co-transfected into Sf21 and/or Sf9 insect cells with BaculoGold™ baculovirus linearized DNA. After incubation for 5 days at 27° C., recombinant baculoviruses expressing the full-length and mature forms of NGF with VP3 or H1 tags were harvested and stored at 4° C.
Full-length or mature NGF was fused with polyhedron-targeting tags (H1 or VP3) to obtain NGF-encapsulated polyhedra (PODS NGF) (
To generate empty polyhedra, Spodoptera frugiperda IPLB-5F21-AE cells (Sf cells) were inoculated with recombinant baculovirus AcCP-H29S expressing Bombyx mori cypovirus polyhedrin under the control of the baculovirus polyhedrin promoter. For production of NGF polyhedra (PODS NGF), Sf cells were co-infected with AcCP-H29S and another recombinant baculovirus expressing recombinant NGF fused with the VP3 or H1 tags. The infected cells were cultured for 10 days at 27° C. and then the cells were harvested in a conical tube by centrifugation. The cell pellet was resuspended in phosphate-buffered saline (PBS; pH 7.2) and treated with an ultrasonic homogenizer at 6% power for 30 sec. The cell homogenate was centrifuged at 1500×g at 4° C. and the supernatant was removed. These treatments were repeated and the purification was complete. The polyhedron suspension was adjusted to 5×104 or 1×105 numbers per microliter volume and stored at 4° C. in distilled water containing 100 units/ml penicillin and 100 μg/ml streptomycin.
One microliter of PODS NGF suspension (5×104 or 1×105 crystals/μl) was manually spotted onto a gelatin-coated cover slip using a micropipette to create a circular field of approximately 2-4 mm in diameter of either PODS full-length NGF or PODS mature NGF (
Alignment and axon extension of PC12 cells surrounding PODS NGF field was also observed by scanning electron microscopy (SEM).
PC12 Cell Culture.
The PC12 DMEM cell culture medium was discarded and 0.02% EDTA solution was added. After the EDTA solution was discarded, 1 ml of 0.25% trypsin-EDTA was added and incubated for 1 min. One millilitre of DMEM culture medium was added and the cell suspension was centrifuged at 1,200 rpm for 2 min. After the supernatant was discarded, cells were suspended in 1 ml of serum-free DMEM and the number of cells was counted. Cells (7×104 cells/well) were then seeded into a well. Half of the medium (serum-free DMEM) was gently exchanged after 3 days. Images of cell alignment were obtained via scanning electron microscopy (SEM) on the 5th day following cell seeding.
Preparation of Cells for SEM Imaging.
After the cell medium was gently discarded, cells were fixed by 4% paraformaldehyde phosphate buffer solution, 1% osmium tetroxide and 1% tannic. Dehydration was carried out by immersing the cover slips in a series of ethanol solutions of increasing concentrations until 100% dehydration was achieved. Cover slips were covered with hexamethyldisilazane and allowed to dry overnight. Images of cell alignment were obtained via SEM after gold-sputtering (200 Å).
Immunocytochemistry.
For immunofluorescence, cells on the cover slips were fixed for 30 min at room temp with 4% paraformaldehyde. After three washes with PBS for 5 min each, the cells were permeabilized with 0.3% Triton X-100 in PBS for 15 min at room temperature. After three washes with PBS for 5 min each, the cells were incubated in blocking buffer (3% FBS in PBS) for 1 h at room temperature and then incubated with primary antibodies (anti-tau antibody (Merck Millipore) or anti-neurofilament heavy polypeptide antibody (SIGMA)) for overnight at 4° C. After three washes with PBS for 10 min each, the cells were incubated with FITC-conjugated secondary antibody (goat anti-mouse IgG antibody (Invitrogen)) for 1 h at room temperature. Cover slips were then washed three times with PBS and finally mounted on microscope slides in mounting medium with propidium Iodide (Invitrogen) for nuclei staining. Stained cells were observed using an Olympus Fluoview FV1000-IX81 confocal microscope.
These results indicate that PODS NGF field regulates the direction of alignment and axon extension of PC12 cells. We confirmed the differentiation of PC12 cells using tau and neurofilament antibodies. Tau and neurofilament proteins were observed in the aligned PC12 cells (
To determine the extent of the gradient, PODS NGF were mixed with polyhedra encapsulating enhanced green fluorescent protein (PODS EGFP), and subsequently spotted on a cover slip. PC12 cells were then seeded and incubated with PODS NGF and PODS EGFP, and the fluorescence emission on the periphery of the aligned PC12 cells was measured (
| Number | Date | Country | Kind |
|---|---|---|---|
| 1705955 | Apr 2017 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2018/000064 | 4/11/2018 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/189501 | 10/18/2018 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 6005081 | Burton | Dec 1999 | A |
| 6835567 | Sah | Dec 2004 | B1 |
| Number | Date | Country |
|---|---|---|
| WO-2014130449 | Aug 2014 | WO |
| WO-2014130449 | Aug 2014 | WO |
| WO-2018189501 | Oct 2018 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20200277570 A1 | Sep 2020 | US |
