Composition for non-viral transfection into a cell and use in cancer treatment

Abstract

The present invention relates to compositions and pharmaceutical compositions for non-viral transfection into a cell. The invention also relates to use of these and other compositions as a medicament; vaccine or specifically in a method for treating cancer. Lastly, the invention also relates to in vitro use of the compositions for transfecting cells; an in vitro method for transfecting a cell with nucleic acid and kits comprising the transfection components.

Description

[0001] P153160PC00

[0002] Improved cancer treatment

[0003] Field of the Invention

[0004] The present invention relates to compositions and pharmaceutical compositions for non-viral transfection into a cell. The invention also relates to use of these and other compositions as a medicament; vaccine or specifically in a method for treating cancer. Lastly, the invention also relates to in vitro use of the compositions for transfecting cells; an in vitro method for transfecting a cell with nucleic acid and kits comprising the transfection components.

[0005] Background

[0006] Treating cancer with radiotherapy and / or chemotherapy prolongs the survival of patients with solid tumours. However, relapse often occurs due to tumour drug resistance mechanisms. One of these mechanisms is limited access to the core of the tumour mass which is where tumour stem cells are found. Delivery vehicles for delivery cancer therapeutics often lack efficacy in penetrating to this core giving the solid tumour capacity to evolve resistance. Summary of the Invention

[0007] The inventors have addressed this need by providing an improved delivery composition which can penetrate deeply into tumours and deliver cargo effective at killing cancer cells deep within tumours. The inventors have surprisingly found that the novel compositions claimed not only are capable of deep penetration into tumours but also are able to transfect and kill cancer stem cells (as shown in examples 7-13) which are often located deep within the tumour in hypoxic core regions which helps the sternness of these cells and also helps shield these cells from chemotherapy and radiotherapy. The compositions therefore offer a new and effective treatment for cancer, in particular relapsed cancers.

[0008] In a first aspect the invention provides a composition for non-viral transfection into a cell, the composition comprising:

[0009] a) a lipid component comprising a cationic lipid and DOPE;

[0010] b) i) a first targeting peptide sequence which binds to a tenascin protein and / or a nestin protein; and

[0011] ii) a second polycationic nucleic acid-binding peptide sequence.

[0012] In a further aspect, the invention provides a pharmaceutical composition comprising the above composition and one or more pharmaceutical excipients.P153160PC00

[0013] In a further aspect, the invention provides in vitro use of the above composition for transfecting cells, optionally cancer cells, optionally cancer stem cells.

[0014] In a further aspect, the invention provides an in vitro method for transfecting a cell with a nucleic acid, the method comprising contacting the cell with the composition, the composition further comprising a nucleic acid, to obtain a transfected cell, optionally wherein the cell is a cancer cell, optionally wherein the cell is a cancer stem cell.

[0015] In a further aspect, the invention provides a kit comprising:

[0016] a) a lipid component comprising a cationic lipid and DOPE;

[0017] b) i) a first targeting peptide sequence which binds to a tenascin

[0018] protein and / or a nestin protein; and

[0019] ii) a second polycationic nucleic acid-binding peptide sequence.

[0020] In a further aspect, the invention provides the above composition or pharmaceutical composition for use as a medicament or a vaccine, optionally wherein the vaccine is a cancer vaccine.

[0021] In a further aspect, the invention provides any of the above compositions or pharmaceutical compositions for use in a method of treating cancer, optionally comprising a nucleic acid encoding a suicide gene, optionally wherein the method further comprises administering a compound capable of being converted by the suicide gene into a toxic product causing cell death.

[0022] In a second aspect, the invention provides a composition comprising:

[0023] a) a lipid component comprising a cationic lipid and DOPE;

[0024] b) i) a first targeting peptide sequence which binds to an extracellular tumour antigen expressed by cancer stem cells; and

[0025] ii) a second polycationic nucleic acid-binding peptide sequence

[0026] wherein the composition further comprises a nucleic acid, for use in a method of treating cancer in a subject, wherein the composition transfects and kills one or more cancer stem cells.

[0027] In a further aspect, the invention provides in vitro use of a composition for transfecting cancer stem cells, the composition comprising:

[0028] a) a lipid component comprising a cationic lipid and DOPE;P153160PC00

[0029] b) i) a first targeting peptide sequence which binds to an extracellular tumour antigen expressed by cancer stem cells; and

[0030] ii) a second polycationic nucleic acid-binding peptide sequence.

[0031] In a further aspect, the invention provides an in vitro method for transfecting a cancer stem cell with a nucleic acid to obtain a transfected cancer stem cell, the method comprising contacting the cancer stem cell with a composition comprising:

[0032] a) a lipid component comprising a cationic lipid and DOPE;

[0033] b) i) a first targeting peptide sequence which binds to an extracellular tumour antigen expressed by cancer stem cells; and

[0034] ii) a second polycationic nucleic acid-binding peptide sequence,

[0035] wherein the composition further comprises the nucleic acid.

[0036] In a third aspect, the invention provides a composition for non-viral transfection into a cell, the composition comprising:

[0037] a) a lipid component comprising a cationic lipid and DOPE;

[0038] b) i) a first targeting peptide sequence which binds to a tenascin protein and / or a nestin protein; and

[0039] ii) a second polycationic nucleic acid-binding component.

[0040] In a fourth aspect, the invention provides a composition comprising:

[0041] a) a lipid component comprising a cationic lipid and DOPE;

[0042] b) i) a first targeting peptide sequence which binds to an extracellular tumour antigen expressed by cancer stem cells; and

[0043] ii) a second polycationic nucleic acid-binding component;

[0044] wherein the composition further comprises a nucleic acid, for use in a method of treating cancer in a subject, wherein the composition transfects and kills one or more cancer stem cells.

[0045] The alternative compositions comprised in the third and fourth aspects may be substituted into any of the further aspects above, i.e. pharmaceutical compositions, in vitro uses, kits, medical uses or in vitro methods.P153160PC00

[0046] Detailed description

[0047] Non-viral

[0048] By non-viral is meant the composition is not formed of a viral (protein) capsid (i.e. does not contain such a capsid) but instead is based on a composition of lipid(s) and amino acid sequence(s) in which nucleic acid can be delivered.

[0049] Cell

[0050] The cell transfected may be any cell, for example a cancer cell. For example, a cancer cell expressing a tenascin protein on its surface which can be targeted by the composition.

[0051] Transfection into a cell means delivery of a cargo (e.g. a nucleic acid as described below) by the composition into the interior of the cell.

[0052] The cell may be a cancer stem cell.

[0053] Composition

[0054] The composition may also be referred to as a complex between the lipid component and amino acid sequences (and optionally nucleic acid cargo and / or other cargo). That is, a non-viral transfection complex. By complex is meant the components (lipid component and amino acid sequence(s) and optionally further nucleic acid cargo) are non-covalently associated. The composition or complex may also be referred to as a non-viral vector. That is, a particle to deliver genetic material into cells without the aid of viral protein capsid packaging of the genetic material. The composition may also be referred to as a nanoparticle composition (as shown in Figure 20).

[0055] Amino acid sequences

[0056] The composition comprises:

[0057] 1) A first targeting peptide sequence. By targeting is meant that the amino acid sequence binds to (targets or interacts with) for example a tenascin protein.

[0058] 2) A second nucleic acid binding peptide sequence.

[0059] Alternatively, the composition may comprise only one targeting sequence as above.

[0060] The amino acid sequences (peptide(s)) are non-naturally occurring. They may also be described as isolated; isolated meaning separated from any cellular constituents.P153160PC00

[0061] Tenascin targeting sequence:

[0062] The first targeting sequence may bind to a tenascin protein. That is, it may specifically bind tenascin proteins with substantially no binding to other proteins. That is, there is preferential binding to tenascin proteins. There are various tenascin isoforms. The targeting sequence may bind to any of these isoforms or proteins.

[0063] For example, the targeting sequence may bind to Tenascin-C (TNC) and / or Tenascin-W (TNW). TNC and TNW are homologues. The targeting peptide may bind to any of the TNC or TNW isoforms (e.g. splice variants). For example, the targeting peptide may bind to the TNC isoform NCBI Reference Sequence: NP_002151.2 (SEQ ID NO. 9) or a tenascin sequence with at least with at least 70%, 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO. 9.

[0064] The TNC targeting sequence may comprise (or consist of) FHKHKSPALSPV (SEQ ID NO.

[0065] 1) or a sequence with at least 80% sequence identity to SEQ ID NO. 1 which binds to TNC. The TNC targeting peptide may comprise (or consist of) SEQ ID NO. 1 in reverse which also binds TNC, i.e. VPSLAPSKHKHF (SEQ ID NO. 2) or a sequence with at least 80% sequence identity to SEQ ID NO. 2 which binds to TNC (for example, at least 85%, 90%, 95%, or 100% sequence identity).

[0066] Alternatively, the TNC targeting sequence may comprise (or consist of): PPRRGLIKLKTS (SEQ ID NO. 3); AGRGRLVR (SEQ ID NO. 4) or the reverse of these sequences, or a sequence with at least 80% sequence identity to either of SEQ ID NO.s 3 or 4 (or the reverse sequences) which binds to TNC (for example, at least 85%, 90%, 95%, or 100% sequence identity).

[0067] Nestin targeting peptide

[0068] The first targeting sequence may binds to a nestin protein. That is, it may specifically bind nestin with substantially no binding to other proteins. That is, there is preferential binding to nestin protein(s).

[0069] For example, the targeting sequence may bind to Genbank Reference sequence:

[0070] NP_006608 (SEQ ID NO. 20) or a nestin protein with at least 70%, 80%, 90% or 95% sequence identity to SEQ ID NO. 20. The targeting sequence may also bind to a nestin protein which is an isoform of SEQ ID NO. 20. SEQ ID NO. 20 is the canonical human protein of 1,621 amino acids in length, which displays a molecular weight of approximatelyP153160PC00

[0071] 200-240kDa on SDS-PAGE. There are isoforms of nestin which are expressed by cancer stem cells. The targeting peptide for nestin may specifically bind to one of these isoforms. Panning for such peptides which bind to such cancer specific isoforms may be carried out by methods known in the art, for example as described by using phage display (Beck etal, 2011, Biomaterials 32: 8518-8528).

[0072] The nestin targeting sequence may comprise (or consist) of: AQYLNPS (SEQ ID NO. 18) or SPNLYQA (SEQ ID NO. 19) or a peptide sequence with at least 80% sequence identity to these sequences. SEQ ID NO. 18 is hypothesised to bind to a 60KDa isoform of nestin as described in Beck et al (2011). This isoform is an N-terminal isoform (i.e. from the N-terminal of the 240kDa canonical sequence) which is found, at least in part, in the cellular membrane compartment of undifferentiated glioblastoma stem cells. The nestin protein targeted may therefore be a human N-terminal isoform of approximately 60Kda. The nestin protein may be any truncated form of nestin or the canonical form, or a protein with at least 70%, 80%, 90% or 95% sequence identity to the canonical form or a truncated isoform.

[0073] The composition may comprise or consist of two or more targeting peptides. For example a peptide which targets TNG and a peptide which targets nestin. Any of the peptides above targeting nestin or TNG may be part of a dual targeting composition. That is, a composition comprising separate targeting peptides, as an example only for illustration purposes: 50% of the total targeting peptides as tenascin targeting peptides and 50% as nestin targeting peptides, i.e. dual targeting may be described as where the composition comprises separate targeting peptides with different targets.

[0074] Extracellular tumour antigen

[0075] The targeting peptide may be any peptide capable of targeting an extracellular tumour antigen expressed by cancer stem cells. Nestin and tenascin are examples of such antigens. However, others are known in the art. By extracellular is meant on the cell surface or in the cellular (tumour) matrix.

[0076] Tenascin, nestin or any other target binding can be determined by any method known in the art, e.g. surface plasmon resonance from which a dissociation constant can be calculated which indicates binding affinity to tenascin, nestin or any other target protein.P153160PC00

[0077] The targeting sequence(s) are peptides. The targeting sequence may be at least 7 amino acids in length; for example, at least 8 amino acids in length. The targeting sequence may be 8-20 amino acids in length, for example 8-15, or 8-12 amino acids in length.

[0078] Nucleic acid binding sequence:

[0079] The nucleic acid binding peptide sequence is a sequence capable of binding nucleic acid in the composition. The peptide sequence may be a polycationic peptide sequence, for example a poly-arginine, poly-lysine or poly-histidine sequence. The peptide sequence may alternatively comprise a mixture of cationic amino acids (and optionally other non-cationic amino acids) to allow binding to nucleic acid. Therefore, the nucleic acid binding peptide may have an overall cationic charge to facilitate binding of the nucleic acid cargo.

[0080] The nucleic acid binding sequence may be at least 3 to 50 amino acids (AA) in length, for example 10-20 amino acids in length. For example, a poly-arginine, poly-lysine or polyhistidine sequence of 10-20 amino acids in length.

[0081] For example, a poly-arginine, poly-lysine or any non-canonical amino acid sequence of at least 10-20 amino acids in length that has a positive charge at physiological pH, i.e. has a pKa of over 7.4 (i.e. polycationic). That is, the nucleic acid binding amino acid sequence may consist or comprise, of arginineand / or lysine, i.e. the peptide has any one of these amino acids only (e.g. 100% arginines or 100% lysines). The poly-arg or poly-lys or poly-arg / lys sequence may comprise at least 9 AA, at least 10 AA, at least 11 AA, at least 12AA, at least 13AA, at least 14AA, at least 15AA or at least 16AA. The polycationic sequence may comprise at least 9 AA, at least 10 AA, at least 11 AA, at least 12AA, at least 13AA, at least 14AA, at least 15AA or at least 16AA, with each amino acid in the polycationic sequence having a positive charge at physiological pH, i.e. a pKa of over 7.4. The polycationic sequence may comprise lysine(s), arginine(s) and / or analogues of lysine or arginine. Analogues which have positive charges at physiological pH include: ornithine, homolysine, e-Trimethyllysine, canavanine, agmatine or homoarginine. The polycationic sequence may comprise a mixture of lysines and lysine analogues. The polycationic sequence may comprise a mixture of arginines and arginine analogues. The polycationic sequence may comprise a mixture of lysines and arginines; lysines and arginine analogues or arginines and lysine analogues.

[0082] Where the nucleic acid sequence is instead the broader nucleic acid-binding component, the component may be any composition capable of binding nucleic acid, including an amino acidP153160PC00

[0083] sequence, but also including, for example, other polymers, for example cationic polymers, (polymers with positively charged groups) capable of complexing with a nucleic acid.

[0084] Bipartite peptide sequence’.

[0085] The targeting amino acid sequence and the nucleic acid binding sequence may be part of the same peptide. That is, the composition comprises a lipid component and a peptide comprising both a targeting amino acid sequence and a nucleic acid binding amino acid sequence. By bipartite is meant comprising or consisting of 2 functional (i.e. binding) parts. The targeting amino acid sequence and nucleic acid binding amino acid sequence may be directly attached, e.g. H-R16FHKHKSPALSPV-OH, where the R16 is a 16 amino acid polyarginine nucleic acid binding sequence and FHKHKSPALSPV is a targeting sequence for TNC.

[0086] Alternatively, the targeting amino acid sequence and nucleic acid binding amino acid sequence (or component) may be indirectly attached, via a linker. The linker may be for example one or more amino acids. That is, a linker peptide. Alternatively, the linker may be a non-amino acid based linker. The linker may be a cleavable linker. For example, where the linker is an amino acid linker, the linker may be cleavable by furin. The furin-cleavable amino acid linker may be, for example, RVRR.

[0087] The bipartite peptide therefore has the structure of:

[0088] Nucleic acid binding tenascin / nestin protein

[0089] amino acid sequence - optional linker - targeting sequence

[0090] The bipartite structure may also be reversed, i.e. the peptide may have at the N-terminus the tenascin protein targeting sequence followed optionally by a peptide linker, and then the nucleic acid binding amino acid sequence at the C-terminus of the peptide.

[0091] Therefore, the bipartite peptide may have the structure (N to C terminus or vice versa) of:

[0092] N-optional linker peptide-T; wherein

[0093] N = nucleic acid binding peptide and T = tenascin protein targeting peptide (or another targeting peptide as described above).P153160PC00

[0094] The bipartite peptide sequence may be any of SEQ ID NO.s 5-8 or SEQ ID NO.s 10-15. or a sequence with at least 80% sequence identity to these sequences, wherein the bipartite peptide sequence binds to tenascin C and binds to a nucleic acid cargo. Alternatively, the bipartite sequence may have a sequence identity of at least 85%, at least 90%, at least 95%, at least 99% or 100% to any of SEQ ID NO.s 5-8 or 10-15.

[0095] The bipartite peptide sequence may be any of SEQ ID NO.s 16-17 or a peptide sequence with at least 80% sequence identity to these sequences, wherein the bipartite peptide sequence binds to nestin and binds to a nucleic acid cargo. Alternatively, the bipartite sequence may have a sequence identity of at least 85%, at least 90%, at least 95%, at least 99% or 100% to any of SEQ ID NO.s 16 or 17.

[0096] Sequence identity may also be 100% apart from conservative substitutions (for example 1-2 conservative substitutions over the SEQ ID NO.s for the targeting peptide). By conservative substitution is meant a substituted amino acid with a similar structure and / or charge (e.g. replacement of an alanine with a glycine).

[0097] The composition may also comprise one or more further targeting amino acid sequences. That is, as well as the composition comprising a first targeting amino acid sequence, the composition may also comprise one or more further targeting sequences, targeting the same or a different protein as the first targeting sequence. For example, the composition may comprise a first targeting peptide sequence which binds to tenascin and a second targeting peptide which binds to nestin. These may be in the form of a dual composition, i.e. the composition may comprise separate tenascin targeting peptides and a separate nestin targeting peptides.

[0098] Alternatively, the one or more further targeting amino acid sequences may be linked to the other peptide(s) in the composition. For example, the composition may comprise a tripartite peptide comprising a first targeting amino acid sequence which binds to a tenascin protein; followed by a second nucleic acid binding amino acid sequence, followed by a further targeting amino acid sequence which binds to tenascin or a different protein (e.g. nestin), i.e. the peptide comprises or consists of a tripartite structure. By tripartite is meant comprising or consisting of 3 functional (i.e. binding) parts. That is, the peptide has the following structure (From N-terminus on the left):P153160PC00

[0099] Tenascin protein Nucleic acid Further targeting amino targeting sequence - binding sequence - acid sequence (e.g. nestin)

[0100] This may also be reversed with the further targeting amino acid sequence at the N-terminus of the peptide. Optional linkers (cleavable or non-cleavable) may be used between any of the sequences. It can be envisaged that the two targeting sequences may reside on the surface of the particle (composition) with the nucleic acid binding sequence probing into the middle of the particle (composition) to stabilise the nucleic acid cargo.

[0101] By further targeting amino acid sequence is meant an amino acid sequence that specifically targets a protein, or group of proteins (family or isoforms) expressed by cancer cells, optionally cancer stem cells, for example, the further targeting amino acid sequence may target a protein other than tenascin expressed by cancer cells or cancer stem cells thus providing a further cancer targeting mechanism in addition to tenascin / nestin targeting.

[0102] The peptides, for example targeting peptide(s), may comprise D or L amino acids. For example, any of the targeting peptides in the bipartite peptides may comprise or consist of D amino acids or comprise of consist of L amino acids. Therefore, the first targeting amino acid sequence which binds to a tenascin protein may comprise or consist of D amino acids.

[0103]

[0104] Sequence identity may be at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100%. Sequence identity may also be 100% apart from conservative substitutions (for example 1-2 conservative substitutions over the SEQ ID NO.s for the targeting peptide). By conservative substitution is meant a substituted amino acid with a similar structure and / or charge (e.g. replacement of an alanine with a glycine).

[0105] Sequence identity can be calculated by using any suitable software such as BLAST (Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410.)

[0106]

[0107] The lipid component may comprise one type of lipid or more than one type of lipid. For example, the lipid component may comprise one or more cationic and / or ionisable lipid.P153160PC00

[0108] Suitable examples of cationic lipids include quaternary ammonium salt lipids. Suitable cationic lipids include: DOTMA (1,2-di- O-octadecenyl-3- trimethylammonium-propane); DOSPA (2,3- dioleyloxy-N-[2-(sperminecarboxamido) ethyl]- N,N- dimethyl-1- propanaminium trifluoroacetate), DOTAP (1,2- dioleoyl-3- trimethylammonium- propane) or DDAB (Dimethyldioctadecylammonium bromide). By cationic lipid is meant a lipid with a permanent positive charge.

[0109] Suitable ionisable lipids include: heptadecan-9- yl 8-((2- hydroxyethyl)(6- oxo-6-(undecyloxy)hexyl)amino) octanoate (referred to as Lipid H or SM-102); 4-hydroxybutyl)azanediyl)bis(hexane-6,1- diyl) bis(2- hexyldecanoate) (referred to as ALC-0315). By ionisable lipid is meant lipids which are protonated at low pH which makes them positively charged, but they remain neutral at physiological pH. By protonated at low pH is meant positively charged in aqueous solution at pH 6.5 or below. That is, they are ionised inside cells, e.g. in lysosomes, this being the mechanism for allowing nucleic acid escape into the cytosol. For example, ionisable lipid with a PKa within the range of 5.4-7. The lipid may be referred to as a ionizable cationic lipid. For this reason, cationic lipid in the claims (e.g. PCT claims 1 and 17) may be exchanged for ionizable cationic lipid.

[0110] The lipid component may comprise a phospholipid, for example DOPE (1,2- dioleoyl- sn-glycero-3- phosphoethanolamine); or phosphatidylcholine (e.g. DOPC: 1 ,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine). Other suitable lipids may be known and available to a person skilled in the art.

[0111] Instead of phospholipid, the lipid in addition to the cationic or ionisable lipids may be termed a “helper lipid”. By helper lipid is meant a lipid which aids transfection, for example by way of fusion of the bilayer with the membrane of the endosome. Helper lipids include zwitterionic lipids, phospholipids and cholesterol. Therefore, instead of a phospholipid (or in addition to), this may be exchanged for cholesterol or a zwitterionic lipid which performs the same function.

[0112] Where the lipid component comprises a mixture of lipids, the mixture may comprise: a cationic lipid and a phospholipid. For example, the lipid component may comprise a cationic lipid such as DOTMA or DOTAP and a phospholipid such as DOPE, including analogues and homologs of DOTMA and DOPE. The cationic lipid and phospholipid may be in a ratio of 10:1 to 3:1, for example 5:1 cationic lipid:helper lipid. DOPE may be replaced or inP153160PC00

[0113] addition to DOPG (Dioleoylphosphatidylglycerol) or DOPS (Dioleoylphosphatidylserine) which have similar small headgroups and therefore have similar properties. In addition to a cationic lipid and DOPE (or DOPG / DOPS), the composition can additionally include cholesterol as a further helper lipid.

[0114] Example compositions

[0115] Example compositions include as follows:

[0116] • Lipid composition comprising DOTMA / DOTAP with DOPE; Bipartite peptide comprising or consisting of SEQ ID NO. 5; further comprising a nucleic acid encoding a suicide gene. The suicide gene may be thymidine kinase, optionally under the control of the survivin promoter.

[0117] • Lipid composition comprising DOTMA / DOTAP with DOPE; Bipartite peptide sequence comprising or consisting of SEQ ID NO. 17; further comprising a nucleic acid encoding a suicide gene. The suicide gene may be thymidine kinase, optionally under the control of the survivin promoter.

[0118] • Dual targeting lipid composition comprising DOTMA / DOTAP with DOPE; Bipartite peptide comprising or consisting of SEQ ID NO. 5; Bipartite peptide sequence comprising or consisting of SEQ ID NO. 17; further comprising a nucleic acid encoding a suicide gene. The suicide gene may be thymidine kinase, optionally under the control of the survivin promoter.

[0119] Nucleic acid

[0120] The composition may further comprise a nucleic acid. This may also be referred to as a cargo, as this is delivered to the interior of a cell as a result of transfection. The nucleic acid may be DNA or RNA or a chemically modified nucleic acid. It may, for example, code for a protein that has a utility in the target cell. Alternatively, it may be an anti-sense nucleic acid or an RNAi nucleic acid. The RNA may be a chemically modified nucleic acid, e.g. comprise a 5’cap and / or 3’ cap and / or non-naturally occurring nucleotides. The DNA may be in an expression vector (e.g. plasmid), comprising an expression cassette comprising an open reading frame encoding a protein.

[0121] Other cargos instead of the nucleic acid comprised in the composition may be a protein, peptide or drug (pharmaceutically active ingredient).

[0122] The nucleic acid may be described as isolated and / or non-naturally occurring.

[0123] D-amino acid targeting peptide compositionP153160PC00

[0124] More generally, there is provided a composition for non-viral transfection into a cell, the composition comprising:

[0125] a) a lipid component;

[0126] b) a first targeting amino acid sequence wherein the amino acids comprise or consist of D amino acids.

[0127] The lipid component may be as described above.

[0128] By targeting is meant the amino acid sequence is designed to bind to a specific protein, e.g. on the surface of a cell. For example, a cancer cell. The specific protein may alternatively or additionally be in the tumour matrix.

[0129] The composition may additionally comprise a second nucleic acid binding amino acid sequence. The nucleic acid binding amino acid sequence may be as described above. The first and second amino acid sequences may be comprised in one bipartite peptide sequence. The composition may comprise a further targeting amino acid sequence also as described above, which may also comprise or consist of D-amino acids.

[0130] Pharmaceutical composition

[0131] The composition may be a pharmaceutical composition, comprising one or more pharmaceutical excipients.

[0132] The pharmaceutical excipient may for example be one or more immune adjuvants to stimulate the immune system to the cancer antigens released by destruction of the tumour. Immune adjuvants may include MPLA (monophosphoryl Lipid A), or an analogue thereof, e.g. PHAD®, QS-21 (a plant extract) or any other immune adjuvant to elicit a strong immune response.

[0133] Medical uses

[0134] The composition may be for use as a medicament. For example, the composition may be for use in a method of treating cancer. For example, a composition for treating solid tumours. The cancer may be described as a tenascin / nestin positive cancer (i.e. a cancer which expresses tenascin / nestin). The cancer may be of the brain, e.g. the cancer may be a glioma. The cancer may be glioblastoma, breast, ovarian, colon, pancreatic, bladder, prostate, lung cancer, mesothelioma or other solid tumours.

[0135] The subject for treatment may be a human or the subject may be an animal, i.e. the treatment is a veterinary treatment.P153160PC00

[0136] The composition or pharmaceutical composition may comprise a nucleic acid cargo for a suicide gene. For example, herpes simplex thymidine kinase. The method may further comprise administering a compound which is converted by the suicide gene into a toxic product which causes cell death. For example, where the encoded suicide gene is for herpes simplex thymidine kinase, the compound may be gancicloviror an oral analogue. Other suicide gene therapeutic strategies may be used other than enzyme-prodrug suicide genes such as TK with ganciclovir. For example, an apoptosis-inducing gene may instead be encoded by the nucleic acid cargo. For example, IL24, I L12 or IL36 gamma.

[0137] The composition or pharmaceutical composition may also be a vaccine, for example a T-cell vaccine. That is, the composition comprises, for example, a nucleic acid which when transfected into cells induces a T-cell response to the cells. The T-cell vaccine may be used to treat cancer - to induce a T-cell response to cancer cells. Therefore, as well as directly killing cancer cells, e.g. via suicide gene therapy as explained above, the composition may also be used to indirectly kill cancer cells by way of activating the immune system. The vaccine may be for prophylactic immunisation.

[0138] The nucleic acid when for use in transfecting cancer cells, or for treating cancer may comprise a cancer-specific promoter, optionally a survivin promoter.

[0139] The compositions described (as shown in the examples) transfect and kill cancer cells and / or cancer stem cells. Therefore, the composition is for use in treating cancer (a tumour), for example by transfecting and killing cancer stem cells. These are a subset of cells, for example in a tumour, which drive tumour drug resistance due to their ability to enter a quiescent state, activate DNA repair mechanisms, reactivate drug efflux system and protect against reactive oxygen species, ultimately being responsible for disease relapse. The compositions described herein can penetrate deep within solid tumours where such cancer stem cells are found. Furthermore, after reaching these deep penetration depths, the compositions can transfect and kill the cancer stem cells. The composition is therefore for treating cancer stem cells, for example in solid tumours. That is, alternatively the composition may described as being for use in a method for treating a cancer associated with cancer stem cells or a cancer stem cell-positive cancer. The composition may be administered in an amount effective to inhibit proliferation and / or reduce survival of the cancer stem cells. The cancer may be a therapeutically-resistant cancer (for example refractory to chemotherapy or radiotherapy). The treatment may reduce (or prevent) the risk of cancer relapse in the subject. As a result, the method may be for treating cancer relapse.P153160PC00

[0140] The treatment may be for inhibiting a cancer stem cell to treat cancer in the subject. The method targets cancer stem cells in a subject.

[0141] In vitro use

[0142] The composition may also be used in vitro for transfecting cells, for example tenascin expressing cells. These may be cancer cells or cancer stem cells. That is, the composition may be used as a research tool to aid delivery into cells expressing a tenascin / nestin protein, e.g. spheroids.

[0143] The composition may be in the form of a kit of parts comprising the separate components of the composition (i.e. lipid component(s) and amino acid sequence(s)). The composition may then be mixed in its component parts with a nucleic acid or other component to be transfected.

[0144] Also therefore disclosed is an in vitro method of transfecting cells with a nucleic acid, the method comprising: incubating one or more cells with the composition described in the claims comprising a nucleic acid cargo and delivering this cargo into the interior of the cell to obtain a transfected cell.

[0145] Kit

[0146] By kit is meant a number of components of the transfection compositions sold together, those components being ready to be combined to form the transfection composition or complex.

[0147] Sequence listing

[0148]

[0149] P153160PC00

[0150]

[0151] P153160PC00

[0152]

[0153] The following clauses also describe the invention and may be combined with any of the disclosure above:

[0154] A1. A composition for non-viral transfection into a cell, the composition comprising:

[0155] a) a lipid component;

[0156] b) i) a first targeting amino acid sequence which binds to a tenascin protein; and ii) a second nucleic acid binding amino acid sequence.

[0157] A2. The composition of clause A1 , wherein the lipid component comprises: a) one or more cationic lipid(s) optionally a quaternary ammonium salt lipid(s); or one or more ionisable lipid(s); and / or

[0158] b) one or more phospholipid(s).P153160PC00

[0159] A3. A composition for non-viral transfection into a cell, the composition comprising:

[0160] a) a lipid component, wherein the lipid component comprises one or more cationic or ionisable lipid(s); and one or more phospholipid(s); and

[0161] b) i) a first targeting amino acid sequence which binds to a tenascin protein.

[0162] A4. The composition of clause A3, further comprising: b) ii) a second nucleic acid binding amino acid sequence.

[0163] A5. The composition of any of clauses A1 , A2 or A4, wherein the first and second amino acid sequences are comprised in one bipartite peptide sequence.

[0164] A6. The composition of any of the preceding clauses, wherein the lipid component comprises a cationic lipid, optionally DOTMA; and a phospholipid, optionally DOPE.

[0165] A7. The composition of any of the preceding clauses:

[0166] a) wherein the tenascin protein is tenascin-C, optionally wherein the targeting amino acid sequence comprises: any of SEQ ID NO.s 1-4, or an amino acid sequence with 80% sequence identity with SEQ ID NO.s 1-4; or

[0167] b) wherein the first targeting amino acid sequence is comprised in a bipartite peptide sequence with a second nucleic acid binding sequence wherein the bipartite peptide sequence comprises: any of SEQ ID NO.s 5-8 or 10-15 or 80% sequence identity with SEQ ID NO.s 5-8 or 10-15; or

[0168] c) wherein the tenascin protein is tenascin-W; and / or

[0169] d) comprising a further targeting amino acid sequence.

[0170] A8. The composition of any one of clauses A1 , A2, A4-6, A7a) or A7c), wherein the nucleic acid binding amino acid sequence comprises or consists of a plurality of positively charged amino acids, optionally wherein the positively charged amino acids are arginine, lysine and / or histidine.

[0171] A9. The composition of any of the preceding clauses, further comprising a nucleic acid.P153160PC00

[0172] A10. A pharmaceutical composition comprising the composition of any of clauses A1-9 and one or more pharmaceutical excipients, optionally wherein one or more pharmaceutical excipient(s) is an immune adjuvant.

[0173] A11. The composition of clause A9 or pharmaceutical composition of clause A10 for use as a medicament or a vaccine, optionally wherein the vaccine is a cancer vaccine.

[0174] A12. The composition of clause A9 or pharmaceutical composition of clause A10 for use in a method of treating cancer, optionally comprising a nucleic acid encoding a suicide gene, optionally wherein the method further comprises administering a compound capable of being converted by the suicide gene into a toxic product causing cell death.

[0175] A13. In vitro use of the composition of clauses A1-9 for transfecting cells, optionally cancer cells.

[0176] A14. An in vitro method for transfecting a cell with a nucleic acid, the method comprising contacting the cell with the composition of clause 9 to obtain a transfected cell.

[0177] A15. A kit comprising:

[0178] a) a lipid component;

[0179] b) a first targeting amino acid sequence which binds to a tenascin protein; and c) a second nucleic acid binding amino acid sequence;

[0180] d) or

[0181] e) b) a lipid component, wherein the lipid component comprises one or more cationic or ionisable lipid (s) and one or more phospholipid(s); and

[0182] f) a first targeting amino acid sequence which binds to a tenascin protein.

[0183] A16. A composition for non-viral transfection into a cell, the composition comprising:

[0184] a) a lipid component; and

[0185] b) a first targeting amino acid sequence wherein the amino acids comprise or consist of D amino acids.P153160PC00

[0186] A17. A composition for non-viral transfection into a cell, the composition comprising:

[0187] a) a lipid component, wherein the lipid component comprises a cationic or ionisable lipid, optionally further comprising a phospholipid; and

[0188] b) i) a first targeting amino acid sequence which binds to tenascin.

[0189] A18. A composition for non-viral transfection into a cell, the composition comprising:

[0190] a) a lipid component; and

[0191] b) i) a targeting amino acid sequence which binds to tenascin; and

[0192] ii) a nucleic acid binding component, optionally wherein the second nucleic acid binding component is an amino acid sequence.

[0193] A19. A composition for non-viral transfection into a cell, the composition comprising:

[0194] a) a cationic lipid and a helper lipid, wherein the helper lipid is DOPE, DOPG or DOPS;

[0195] b) i) a first targeting amino acid sequence which binds to a protein overexpressed in cancer stem cells; and

[0196] ii) a second polycationic nucleic acid binding amino acid sequence, wherein the amino acids in the first targeting amino acid sequence comprise or consist of D amino acids.

[0197] Throughout the specification, unless the context demands otherwise, the terms ‘comprise’ or ‘include’, or variations such as ‘comprises’ or ‘comprising’, ‘includes’ or ‘including’ will be understood to imply the method or kit includes a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0198] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in the text is not repeated in this text is merely for reasons of conciseness. Reference to cited material or information contained in the text should not beP153160PC00

[0199] understood as a concession that the material or information was part of the common general knowledge or was known in any country.

[0200] Description of the Figures

[0201] Figure 1 shows the pSELECT HSVTK-GFP plasmid map. Displayed is the structure and size of the pSELECT HSVTK-GFP plasmid created inhouse. This plasmid contains both the GFP gene to enable identification of plasmid expression in cells, and the HSVTK gene which will be utilised for gene therapy.

[0202] Figure 2 shows formulated LAT 4 stability.

[0203] Figure 3 shows LAT 7 stability (a-c); and polydispersity index of all other batches (d).

[0204] Figure 4 shows successful transfection of cells with LAT 7.

[0205] Figure 5 shows GCV exposure with and without LAT 7.

[0206] Figure 6 shows cells after the addition of GCV (48 hours).

[0207] Figure 7 shows distribution of LAT 7 throughout the U87 spheroids.

[0208] Figure 8 shows addition of HSV-TK-GFP (LAT 7) to 3D U87 spheroids.

[0209] Figure 9 shows Ganciclovir Concentration Curve on Normal, Parental U87.

[0210] Figure 10 shows Exposure of the U87 cells to increasing concentrations of Ganciclovir (GCV).

[0211] Figure 11 shows removal of GCV after a certain period of time.

[0212] Figure 12 shows comparison of GCV exposed control (bottom) with GCV exposed spheroids containing the LAT particles (top)

[0213] Figure 13 shows comparison of GCV exposed controls with spheroids containing LAT particles at different concentrations of GCV.

[0214] Figure 14 shows the same comparison as Figure 13 but with higher concentrations of GCV. Figure 15 shows the use of GCV on mosaic spheroids with a)-e) showing various percentages of cells that had the HSVTKGFP plasmid.

[0215] Figure 16 shows a comparison of transfection efficiency of various tenascin targeting peptides.P153160PC00

[0216] Figure 17 shows the size and zeta potential of the compositions incorporating the various tenascin targeting peptides.

[0217] Figure 18 shows the changes in tumour volume in mice over the 10 day treatment period for Groups 1, 2, 3, 4, 6, and 8.

[0218] Figure 19 shows the % survival in all control groups verses all treatment groups as described in Example 7. Survival was determined by the size of the tumour with mice being sacrificed when the tumour size exceeded 16mm in any 1 dimension.

[0219] Figure 20 shows SEM images of LAT 7 particles. This is a form of microscopy that uses a beam of electrons to create an image of a samples surface. SEM take high resolution images and can provide information about a samples composition and texture. This image shows the presence of spherical particles (lipid + targeting peptide) and confirms these are nanoparticles.

[0220] Figure 21 shows U87 cells transfected using various targeting peptides. The percentage of transfected cells was calculated from the number of GFP positive cells over the total number of cells 24 hours post transfection. Data presented is an average of 3 independent experiments ±standard deviation.

[0221] Figure 22 shows U87 spheroids transfected using compositions with various targeting peptides 24 hours post transfection.

[0222] Figure 23 shows U87 spheroids transfected using dual targeting compositions (TNG and nestin, i.e. SEQ ID NO.s 5 and 17).

[0223] Figure 24 shows SEQ ID NO. 5 with polycationic amino acids (lysine) in U87 (a) and LN229 spheroids (b).

[0224] Figure 25 shows U87 Spheroid transfected with TRJ10C (Lysine Peptide - SEQ ID NO. 5) on the confocal microscope; a) = middle section of spheroid x63 magnification; b) = whole section of spheroid x20 magnification Red =HSVTK, Blue = nuclei, Green = GFP (from plasmid)

[0225] Figure 26 shows U87 Spheroid transfected with TRJ10CN (Nestin Peptide - SEQ ID NO. 16) on the confocal microscope ; a) = middle section of spheroid x63 magnification; b) = whole section of spheroid x20 magnification Red =HSVTK, Blue = nuclei, Green = GFP (from plasmid)P153160PC00

[0226] Figure 27 shows LN229 Spheroid transfected with TRJ10CN (Nestin Peptide) on the confocal microscope . a) = middle section of spheroid x63 magnification; b) = whole section of spheroid x20 magnification Red =HSVTK, Blue = nuclei, Green = GFP (from plasmid) Figure 28 shows LN229 Spheroid transfected with PL1 peptide (SEQ ID NO. 11) on the confocal microscope : middle section of spheroid x63 magnification. Red =HSVTK, Blue = nuclei, Green = GFP (from plasmid)

[0227] All previous figures shown are particles comprising DOTMA and DOPE.

[0228] Figure 29 shows U87 cells transfected with alternative lipid formulations to assess transfection efficiency 24 hours post transfection. Data presented is an average of 3 independent experiments ± standard deviation.

[0229] Figure 30 shows 3D spheroid cell transfection with the various lipid combinations in Figure 29: a) and b) are DOTMA plus DOPE in U87 spheroids; c) and d) are DOTAP plus DOPE in U87 spheroids.

[0230] Figure 31 shows 3D spheroid cell transfection with further lipid combinations in LN229 spheroids namely: a) DOTMA plus DOPE; b) DOTAP plus DOPE; and c) DOTMA plus DSPC.

[0231] Figure 32 shows the stability of the particles after the addition of pegylated lipid and / or cholesterol.

[0232] Figure 33 shows the 2D transfection efficiency after the addition of pegylated lipid and / or cholesterol 24 hours post transfection.

[0233] Figures 34 and 35 show the 3D spheroid transfection efficiency after the addition of pegylated lipid and / or cholesterol in U87 human glioma spheroids and LN229 human glioma spheroids respectively.

[0234] Figure 36 shows U87 Tumour (control no treatment) probed with nestin antibody and imaged on an oil immersion confocal microscope: a) = control (no antibody; b) = tumour section x10; c) = X63 oil immersion. Red = nestin; Blue = nuclei.

[0235] Figure 37 shows U87 Tumour (control no treatment) probed with Sox2 antibody and imaged on an oil immersion confocal microscope: a) = control (no antibody); b) = x63 oil immersion. Red = Sox2; Blue = nuclei; and c) U87 Tumour (control no treatment) probed with CD133 antibody and imaged on an oil immersion confocal microscope: c) = control (no antibody); d) = x63 oil immersion; Red = CD133; Blue = nuclei.P153160PC00

[0236] Figure 38 shows U87 Tumour (control no treatment) probed with HIF-1a antibody and imaged on an oil immersion confocal microscope: a) = control (no antibody); b) = zoomed in 6x from x63; c) = x63 oil immersion. Red = Hif- 1 a; Blue = nuclei.

[0237] Figure 39 shows immunofluorescence in tumour sections from mice showing transfection of TRJIOc deep into the mouse tumours by probing for HsvTK.

[0238] Figure 40 shows Z-stack images of U87 spheroid transfected with Lysine peptide formulation (SEQ ID NO. 5): a) 3D render of particle penetration into the spheroid, which demonstrates particles are 180.6818 pm inside the spheroid, b) shows superimposed z-stack images which demonstrates particles are 437.5pm inside the spheroid.

[0239] Figure 41 shows Human Glioblastoma stem cells transfected with various formulations, showing formulations can transfect stem cells: a) Dual targeting peptide mixture (SEQ ID NO. 5 and SEQ ID NO. 17), b) Nestin 2 peptide (SEQ ID NO. 17) c) Survivin plasmid formulation utilising Lysine peptide (SEQ ID NO. 5) & d) Lysine peptide (SEQ ID NO.

[0240] 5). Figure 42 (a) shows a representative image of glioblastoma stem cells stained with trypan blue following the addition of GCV alone, with blue cells indicating cell death. Figure 42 (b -e) displays representative images of glioblastoma stem cells stained with trypan blue following transfection with various formulations and the subsequent addition of GCV, with blue cells indicating cell death. Figure 42 (f) displays the percentage kill of glioblastoma stem cells for each condition tested.

[0241] Figure 43 shows the commercially available IL24 containing plasmid map (Origene®, rg216502). Displayed is the structure and size of the IL24 plasmid. This plasmid contains both the GFP gene to enable identification of plasmid expression in cells, and the IL24 gene which will be utilised for gene therapy.

[0242] Figure 44 shows cell transfection 24 hours post addition of the IL24 particle to LN229 cells. Cells that fluoresce green indicate the presence of the IL24 plasmid within the cell, which indicates successful transfection.

[0243] Figure 45 shows the concentration of IL24 present in LN229 cell supernatant 72 hours post transfection with the IL24 particle. The IL24 particle was administered at different volumes to determine if the amount of IL24 produced was proportionate to the volume of particle administered. IL24 concentration was determined via IL24 specific ELISA assay.

[0244] Examples

[0245] Aspects of the present invention will now be illustrated by way of example only and with reference to the following experimentation.P153160PC00

[0246] Additional methods: Testing forTenascin binding

[0247] Testing for Tenascin binding in vivo can be carried out using immunohistochemistry, for example using the method from McCabe et al, 2023, Analyst, Issue 14. Briefly, compositions comprising a tenascin binding peptide (produced in accordance with the below examples) may be incubated with a tenascin expressing cell culture, for example:

[0248] 1) LI87-MG glioblastoma cell line may be used as they overexpress the tenascin-C targeting protein. LI87-MG glioblastoma cancer cells were cultured in Eagle's minimum essential medium (EMEM) containing 0.02 mM phenol red and 2 mM L- glutamine supplemented with 10% heat-inactivated foetal bovine serum (FBS), 100 pg mL-1penicillin / streptomycin and 2 pg mL-1amphotericin B. Cells were incubated at 37 °C and 5% CO2 in a humidified incubator. The cells were grown in T75 cell culture flasks and those with a confluence of ca. 70-80% were detached using 0.05% trypsin-EDTA and re-suspended in media and counted using a haemocytometer before reseeding or using in experiments. LI87-MG cells were harvested and counted, and 2.5 x 1 o6cells were added to a Techne spinner flask with 75 mL of complete (with supplements) EMEM medium, including 10% heat- inactivated foetal bovine serum (FBS), 100 pg mL-1penicillin / streptomycin and 2 pg mL-1amphotericin B. The flasks were gassed with CO2 and left to stir in a Techne stirrer flask for 4 days in an incubator set to 37 °C, with regular media changes and addition of CO2 every two days.

[0249] In vitro tenascin binding can be carried out according to Kim et al, 2011. Mol Cells: Selection and characterization of Tenascin C targeting peptide. Briefly, to find further tenascin binding peptides, a phage display library may be panned for tenascin binding, for example using the tenascin C protein of SEQ ID NO. 9 coated onto 96 well plates. After incubation of the phage with the protein-coated plates, unbound phage may be removed by rinsing with buffer. The bound phage may then be eluted and used to infect E. coli. A detailed protocol is set out below:

[0250] A phage-display peptide library may be panned in 96-well plates coated with tenascin protein in 0.1 M NaHCOs (pH 8.6) overnight at 4°C. The wells were blocked with buffer comprised of 0.1 M NaHCOs (pH 8.6), 0.5% bovine serum albumin (BSA) and 0.02 % NaNs for 1 h at4°C, and 2 x 1011plaque forming units / ml were added to the tenascin-coated plates in blocking buffer. After incubation for 30 min at 25°C, unbound or weakly bound phages were removed by rinsing ten times with Tris buffered saline-Tween 20, and bound phages were eluted by incubation for 8 min in 0.1 ml of an elution buffer comprised of 0.2 M glycine-HCI (pH 2.2)P153160PC00

[0251] and 0.1% BSA. The recovered phages were used to infect E. coli, amplified and were purified by precipitation with 1 / 6 volume of a mixture containing 20% (w / w) polyethylene glycol 8000 and 2.5 M NaCI (PEG / NaCI). Individual phage clones were purified by precipitation with PEG / NaCI. The phage pellets were suspended in iodide buffer comprised of 10 mM Tris-HCI (pH 8.0), 1 mM EDTA and 4 M Nal, and single-stranded phage DNA was precipitated with ethanol and sequenced. The corresponding peptides may then be synthesized.

[0252] Binding of any putative tenascin binding peptide may be determined accurately using Surface Plasmon Resonance (SPR). For example, to attach the protein to the sensor chip, the surface of the chip was pre-equilibrated with HEPES and activated with 0.05 M N-hydroxysuccinimide (NHS) and 0.2 M N-ethyl-N' -(dimethylaminopropyl) carbodiimide (EDC) to modify the carboxymethyl groups of dextran. The TNC was injected into one of the cells whereas binding buffer was injected into the other. When the baselines were stabilized, various concentrations of the peptide were injected. Following each experiment, the sensor chip was regenerated with 10 mM NaOH.

[0253] Example 1 : Creation of the pSELECT HSVTK (Herpes Simplex virus thymidine kinase) -GFP Plasmid to be used as cargo

[0254] Materials

[0255]

[0256] P153160PC00

[0257]

[0258] The pSELECT-GFPzeo-mcs plasmid was purchased from InvivoGen (Catalogue number: psetgz-mcs) and resuspended according to the manufacturer’s specifications - 20 pL of sterile DNase free water was added for a final concentration of 1 pg / pL.

[0259] The pSELECT-HSVItk plasmid was purchased from InvivoGen (Catalogue number: psetz-hsvltk) and resuspended according to the manufacturer’s specifications - 20 pL of sterile DNase free water was added for a final concentration of 1 pg / pL. This plasmid provides the HSVTK gene (Herpes simplex virus thymidine kinase gene) for the creation of the pSELECTHSVTK-GFP plasmid in which the HSVTK gene will be inserted into the pSELECT-GFPzeo-mcs backbone.

[0260] To prepare the plasmids for ligation, both plasmids were cut using Kasl (New England Biolabs) and Nhel-HF (New England Biolabs) in the following reaction: 5 pL plasmid DNA (5 pg), 5 pLrCut smart buffer (New England Biolabs), 1 pL Kasl, 1 pL Nhel-HF, and 38 pL DNase free water. The reactions were then incubated at 37°C for one hour to allow the plasmid DNA to be digested. Following incubation, the samples were then incubated at 80°C to heat inactivate the restriction enzymes.P153160PC00

[0261] Following restriction digest, the samples were run on a 1.5% agarose gel (TAE buffer) at 100 volts to visualize DNA bands. For pSELECT-HSVItk, the smaller 1,156 bp band containing the HSVTK gene insert was cut out of the gel using a sterile scalpel and stored in a bijou container. For pSELECT-GFPzeo-mcs, the larger band of 4,194 bp was cut out of the gel using a sterile scalpel and stored in a bijou container.

[0262] The DNA from the excised gel bands was then extracted using the Pure Link Quick Gel Extraction kit (Invitrogen) according to the manufacturer’s specifications. Briefly, excised gel bands were weighed and gel solubilisation buffer (L3) added based on the weight of the gel in a ratio of 3:1 (buffer to gel). The gel / buffer mixture was then incubated at 50°C until the gel fragment had fully dissolved. Once dissolved one gel volume of isopropanol (VWR) was added to the gel mixture before loading into the Quick Gel Extraction Column, with one column used per 400 mg of agarose gel. Columns were centrifuged at >12,000 * g to bind plasmid DNA to the column, then washed with buffer W1. Plasmid DNA was then eluted by adding 50 pL of DNase free deionised water directly to then membrane and incubated at room temperature for one minute prior to centrifugation at >12,000 * g to collect the extracted plasmid DNA in a recovery tube (Invitrogen).

[0263] Now that the desired plasmid DNA had been recovered, the extracts were run on a 1.5% agarose gel (TAE buffer) with high and low DNA mass ladders (Invitrogen) to enable the quantity of DNA to be determined prior to ligation. From this gel the pSELECT-GFPzeo-mcs was calculated to have a concentration of 12 ng / pL, and the HSVTK insert calculated to be 1.5 ng / pL.

[0264] To ligate the pSELECT-GFPzeo-mcs with the HSVTK insert, the Anza™ T4 DNA Ligase Master Mix kit was used according to the manufacturer’s specifications. Briefly, 20 ng of the pSELECT-GFPzeo-mcs and 20 ng of HSVTK insert were mixed with 5 pL Anza™ T4 DNA Ligase Master Mix, then incubated at room temperature at for 15 minutes.

[0265] Following ligation, the plasmid mixture was transformed into DH5a cells (Thermo Scientific) according to the manufacturer’s specifications. Briefly, 5 pL of plasmid DNA was gently mixed with 50 pL of DH5a cells and incubated on ice for 30 minutes, then incubated at 42°C for 30 seconds, before returning the samples to the ice for 2 minutes. Following incubation, 250 pL of S.O.C. medium was added and the samples incubated at 37°C at 225 rpm. Following incubation, 20 pL of cell suspension was spread on a Luria Broth (LB) zeocin agar plate (25 pg / mL zeocin) and incubated at 37°C overnight.

[0266] From the transformed bacterial plates, individual bacterial colonies were picked and added to 5 mL LB zeocin broth (25 pg / mL zeocin) individually. These cultures were then incubated atP153160PC00

[0267] 37°C at 160 rpm overnight. Following incubation, cultures were spun at 4,000 * g to create a bacterial cell pellet for each sample. The PureLink™ Quick Plasmid Miniprep Kit was then used to isolate the plasmid DNA from the bacterial pellets as per the manufacturer’s instructions. Briefly, bacterial cell pellets were resuspended with 250 pL of Resuspension Buffer (R3). Next, bacterial cells were lysed by adding 250 pL of Lysis Buffer (L7). Following lysis, 350 pL of Precipitation Buffer (N4) was added and the samples loaded into individual spin columns and centrifuged at 12,000 x g for 1 minute. Next, columns were washed with 700 pL of Wash Buffer (W9) and the plasmid DNA eluted in 75 pL DNase free deionised water into a collection tube.

[0268] To screen isolated plasmids for HSVTK insert, samples were digested with Kpnl-HF (New England Biolabs) and Nhel-HF (New England Biolabs) in the following reaction: 5 pL plasmid DNA, 5 pLrCut smart buffer (New England Biolabs), 1 pL Kpnl-HF, 1 pL Nhel-HF, and 38 pL DNase free water. The reactions were then incubated at 37°C for one hour to allow the plasmid DNA to be digested. Following incubation, the samples were then incubated at 80°C to heat inactivate the restriction enzymes. Following digestion, samples were run on a 1.5% agarose gel (TAE buffer) at 100 volts to visualise samples and identify those with the HSVTK insert, as samples without the insert would generate a singular DNA band, whereas samples with the insert would produce two bands of 4,751 bp and 607 bp in size.

[0269] The pSELECT HSVTK-GFP was then transformed into DH5a cells as previously described, and individual colonies grown in 5 L of LB zeocin (25 pg / mL zeocin) broth in preparation for isolation of large quantities of plasmid to be used in formulations of LAT-7. Isolation was carried out using the PureLink™ Expi Endotoxin- Free Giga Plasmid Purification Kit (Invitrogen) according to the manufacturer’s specifications. Briefly, bacterial cells were pelleted at 4,000 x g, and resuspended with 125 mL Buffer R3. Next bacterial cells were lysed with 125 mL Buffer L7 and 125 mL Buffer N3 added. The resulting mixture was passed through a lysate filtration cartridge attached to a receiver flask under vacuum. Towash the lysate, 50 mL of Wash Buffer (W8) was added, and the vacuum applied. Next 30 mL of Endotoxin Removal Buffer (ER) was added to remove endotoxins. To equilibrate a DNA binding cartridge to receive the plasmid DNA, 200 mL Equilibration Buffer (EQ1) was added, and a vacuum applied. The clarified lysate was then added to the DNA binding cartridge and a vacuum applied. The DNA cartridge was then washed with 275 mL W8 buffer twice. To elute plasmid DNA 100 mL of Elution Buffer (E4) was passed through the DNA binding cartridge under vacuum. The eluted DNA was then precipitated by adding 70 mL of isopropanol and centrifuged at 12,000 x g for 30 minutes. The resulting supernatant was discarded, and the pellet washed with 5 mL of 70% ethanol before centrifuging at 12,000 x g for 10 minutes to create a plasmid DNA pellet. The ethanolP153160PC00

[0270] supernatant was then discarded and the plasmid DNA pellet air dried before resuspension in 2 mL of DNase free deionised water.

[0271] Results:

[0272] The resulting plasmid is shown in Figure 1.

[0273] Example 2: Manufacture of the delivery particles (LAT-4 / 5 / 7 / 8 / 9 / 10)

[0274] Materials & Equipment:

[0275] Table 1: Materials & Equipment Used

[0276]

[0277]

[0278] P153160PC00

[0279]

[0280] Procedure:

[0281] Lipid Solutions Preparation

[0282] 1. Filter ~50 mL of PW into a sterile, RNAse-free 50 mL tube.

[0283] 2. Remove the lipids, 50 mg / mL peptide stock solution, and the desired GFP peptide from storage at -20°C and equilibrate to room temperature.

[0284] 3. Prepare the individual lipid stock solutions as per Tables below.

[0285] Table 2A: Preparation of DOTMA stock solution

[0286]

[0287] Table 2B: Preparation of DOPE stock solution

[0288]

[0289] Note: The DOTMA and DOPE will be prepared in a solution containing 95% ethanol and 5% filtered PW. The water is to help facilitate the charged lipids dissolving in ethanol.

[0290] Peptide Solution

[0291] Table 3.

[0292]

[0293] P153160PC00

[0294]

[0295] Take 50 mg out of the desired Tenascin binding peptide and dissolve in 1 mL of PW.

[0296] Organic Solution (Peptide + Lipids) Preparation

[0297] To be prepare before pGFP-L / VP Preparation

[0298] 4. Rapidly add (all at once, not dropwise) the peptide solution to the combined lipid working solution (in the 15 mL tube) according to Table 4 and mix well by vortexing.

[0299] Table 4: Preparing Organic (Peptide + Lipid) Solution

[0300]

[0301] P153160PC00

[0302] Stocks required: DOTMA 5 mg / ml in 95% ethanol

[0303] DOPE 1 mg / ml in 95% ethanol

[0304] Dilute stocks as follows:

[0305]

[0306] Table 5.

[0307] Neonano: Flow Rate = 9:3 ml / min (aqueous:organic)

[0308] Table 6: Flow Rates and Volumes of Aqueous and Organic Solutions

[0309]

[0310]

[0311] The organic phase in Table 6 is the following: DOPE, DOTMA, Ethanol and the peptide (Tenascin-C). The aqueous phase is the plasmid in water. These two phases are then mixed together in the microfluidic device.P153160PC00

[0312] Stock solutions prepared:

[0313] DOTMA 10 mg / mL in 95% ethanol and 5% filtered PW

[0314] DOPE 10 mg / mL in 95% ethanol and 5% filtered PW Tenascin-C 50 mg / mL in PW

[0315] Desirable GFP 1 mg / mL in PW

[0316] Size and Zeta Potential measurements

[0317] Dynamic light scattering (DLS) and zeta measurements were obtained using a Malvern Zetasizer, Nano ZS system (Malvern, UK). Before sample analysis, a 40 nm polystyrene latex bead standard was run to calibrate the instrument. To measure the size, the NPs were placed into a 1 cm PMMA cuvette. The samples were measured in triplicate and the average mean and standard deviation was recorded. The zeta potential of the samples was measured using a dip cell placed into the 1 cm PMMA cuvette.

[0318] Results:

[0319] The various delivery vehicles manufactured are described below in Table 7.

[0320] Table ?.

[0321]

[0322] P153160PC00

[0323] Formulated LAT delivery vehicles thus far. LAT 9 and 10 can be differentiated by the GFP gene being constantly switched on (LAT 7) and being switched on and off in the presence only of cancerous cells (LAT 9 and 10). It is envisaged that different peptides may be used with different plasmids, and the above combinations are provided as examples only.

[0324] Figures 2-3 show the stability of the formulated compositions.

[0325] Figure 20 shows a SEM image of LAT7 particles, and shows the presence of spherical particles (lipid + targeting peptide) and confirms these are nanoparticles.

[0326] Example 3: The compositions show superior transfection into cells

[0327] Materials and methods (for examples 3-5):

[0328] All materials used were purchased from Sigma-Aldrich Ltd, UK, unless stated otherwise. The U87-MG glioblastoma cancer cell line was purchased from Caliper Life Sciences (American Type Culture Collection (ATCC, US). Histoplast, pelletised Paraffin Wax and DPX mounting medium were purchased from Thermo Scientific (Leicestershire, UK). Histoclear clearing agent was purchased from National Diagnostics (Hull, UK). Rabbit monoclonal antibody against tenascin-C antibody (ab108930), goat anti-rabbit IgG (ab6702), goat anti-rabbit IgG (Alexa 488) (ab150077), goat anti-rabbit horse radish peroxidase (HRP) conjugated antibody (ab6721), mounting media with DAPI (ab104139) and DAB substrate kit (ab64238) were purchased from Abeam (Cambridge, UK).

[0329] pGFP Solution Preparation

[0330] Prepare the 0.333 mg / mL pGFP desired working solution as per Table 8 in a sterile 50 mL tube.

[0331] Table 8: Preparing 0.333 mq / mL pGFP Working Solution

[0332]

[0333] P153160PC00

[0334] Cell culture

[0335] LI87-MG and 11251 glioblastoma cancer cells were cultured in Eagle’s minimum essential medium (MEM) and LN229 cultured in Dulbecco's Modified Eagle Medium (DMEM) with 2 mM L-glutamine supplemented with 10% heat-inactivated foetal bovine serum (FBS), 100 mg / mL penicillin / streptomycin and 2 pg / mL amphotericin B. Cells were incubated at 37 °C and 5% CO2 in a humidified incubator. The cells were grown in T75 cell culture flasks and those with a confluence of 70-80% were detached using 0.05% trypsin-EDTA and re-suspended in media and counted using a haemocytometer before reseeding or using in experiments. Glioblastoma stem cells were cultured in human glioblastoma cancer stem cell complete serum free media (Celprogen, USA). The cells were grown in cancer stem cell extracellular matrix coated T75 cell culture flasks (Celprogen, USA) and those with a confluence of 70% were detached using Accutase solution and re-suspended in media and counted using a haemocytometer before reseeding or using in experiments.

[0336] Multicellular Tumour Spheroid (MTS) Culture

[0337] For the formation of the MTS, U87-MG cells were harvested and counted, and 2.5 x 1 o6cells were added to a Clino-Reactor with 10 mL of complete (all supplements included) MEM medium. The reactor was the placed into the Clinostar for 5-6 days at 37 °C with 5% CO2. For the first 2 days the reactor was set to rotate at 5 RPM. This was then increased to 10 RPM for the remaining duration of the spheroid growth.

[0338] Transfection

[0339] The cells were transfected by either the addition of the LAT-Xorvia Effectine. For transfections, the cells were plated at 1.0 x 106cells per well of a 6-well dish with 3 mL of complete MEM media and left for 24 hours to adhere to the plates at 37 °C with 5% CO2. For transfection of the LAT-X, the complete MEM media was removed and 3 mL of optiMEM was added for 1 hour to the cells in an incubator set to 37 °C with 5% CO2. After 1 hour, 30 pL of LAT-X was added to the cells for 4 hours and then they were topped up with 3-5 mL of complete MEM media and left in an incubator set to 37 °C with 5% CO2. The cells were then checked after 24 hours and if they had begun to stick then 5 pL of zeosin was added in 5 mL of complete MEM media.

[0340] For transfection via Effectine transfection reagent, the cells were plated as above and left for 24 hours. Once they were stuck to the 6-well plates, the Effectine protocol was used. This involved added 1 pg, 2 pg and 3 pg of the desired plasmid to 150 pL of EC buffer. Then 8 pL of enhancer was then added and the samples were mixed using a vortex. Following this, 25P153160PC00

[0341] pL of Effectine was added and the Eppendorf tube was micro-centrifuged and incubated at room temperature for 10 minutes. To this, 1 mL was complete MEM media was then added, and the media was removed from the cells. The above solution was then added straight to the cells dropwise.

[0342] Spheroid incubation with LAT-X

[0343] Once the spheroids had grown to the desired size (> 250 pm), the delivery vehicles were then added to the spheroids (denoted as LAT - X), depending on the various plasmids that have been added (Table 7). This was carried out by adding the spheroids to 3 mL of optiMEM (this is cell culture medium that does not contain serum proteins) thus improving the uptake of the LAT into the spheroids, in a tissue culture bijou. This was added to a rolling platform at 37 °C for 1 hour. After 1 hour, 30 pL of the appropriate LAT formulation was added and placed back onto the roller for an additional 4 hours. The roller is used to prevent the spheroids from sticking to each other. After 4 hours, an additional 3 - 5 mL of complete MEM media was added to allow the spheroids to have the appropriate nutrients for growth. To the bijou’s, 5% CO2 was added, and they were left on the roller at 37 °C overnight. These spheroids were then added to individual wells of 24-well plates in complete MEM media that was dosed with different concentrations of GCV (5, 10, 25, 50 and 100 pg / mL). The spheroids were kept in an incubator set to 37 °C with 5% CO2 and refreshed with GCV dosed complete MEM media every 7 days for constant exposure. For experiments where there was not constant exposure to GCV, this GCV dosed media was removed, and fresh complete MEM media was added. MTS sectioning - microtome

[0344] The MTS were sectioned using a microtome (LEICA, RM2125RTF). Firstly, the spheroids with and without addition of the various formulations were fixed in 4% paraformaldehyde (PFA) for 1 hour at RT. They were then washed in phosphate buffered saline (PBS) and then added to a bio-wrap (Leica Biosystems Richmond, USA) and placed in a plastic cassette. The cassette was then placed into increasing concentrations (70%, 90% and 100%) of ethanol for 1 hour each, then into Histoclear for 1 hour. The fixed spheroids were then placed into paraffin wax at 59 °C overnight to allow the wax to penetrate the spheroids. The next day, the spheroids in wax were placed into a mould and allowed to cool to RT and placed at -20 °C for at least 1 hour before being sectioned. The sections were added to adhesion glass slides and baked at 60 °C for 2 hours for drying.

[0345] MTS sectioning - Cryostat + immunofluorescenceP153160PC00

[0346] The MTS were sectioned using a cryostat (LEICA, CM 1950). Spheroid samples were prepped by fixing in 4% PFA (Thermo Fisher J 19943. K2) at room temperature. PFA was removed and spheroids were washed in PBS (Gibco, 10010-015). Parafilm was taken and a small circle of OCT (CellPath, 30226281) placed on top. Spheroids were then placed in media and pipetted into the liquid OCT. Using tweezers spheroids were evenly distributed among the OCT. Cryomoulds were taken and a layer of OCT placed on the bottom and left to freeze on dry ice. Parafilm with the OCT and spheroids were also placed on dry ice to set. Once set, the spheroids in OTC were placed vertically into the cryo-mould and OCT added to hold the samples in place. The mould was then placed on dry ice again to set, labelled and stored at -80°C until ready to be sectioned. Once ready to be sectioned, Cryo-moulds were taken and placed at -16°C in the cryostat. Frozen samples were removed from the moulds and placed on top of fresh liquid OCT on a “puck” and left to set. Slides (Superfrost+, epredia, J1820AMNZ) were labelled appropriately and numbered. Moulds were trimmed until the media colour could be observed within the mould and then 10-micron sections were taken. 4-5 sections of spheroids were placed on each slide and then placed in the -80°C freezer until ready to process. Once spheroids were sectioned, slides were placed in cold 100% acetone (Sigma-Aldrich 270725) for 20 minutes. Slides were washed in cold PBS and placed in 0.1% Triton X-100 (Sigma-Aldrich 1003339793) for 5 minutes. Slides were then washed in PBS and placed in blocking buffer for 1 hour. Slides were then washed in PBS and placed in a humidifier chamber. The Primary Antibody for TK (Anti-thymadine Kinase HHV1 antibody AB128880) was diluted 1:1000 in fresh blocking buffer and added onto spheroid sections on the slides. The humidifying chamber was placed overnight at 4°C. The next day, secondary Goat AntiRabbit IgG H&L antibody Alexa fluro 647 (AB150079) was diluted 1:1000 (red) in fresh blocking buffer, slides were washed 3 x in PBS and then secondary antibody added to the sections. The humidifying chamber with the slides and secondary antibody was incubated at room temperature for 60 mins. After slides were washed again in PBS, mounting media containing Dapi (AB104139) was added to the slides with cover slips. Slides were left to set at 4°C. Slides were imaged on the confocal microscope on a x63 oil immersion lens using Dapi and Alexa 647 lasers. Images taken on the confocal were then processed using Image J software.

[0347] Staining

[0348] Haematoxylin and Eosin (H&E)

[0349] The spheroid sections were added to histoclear to remove the paraffin wax, then rehydrated in decreasing concentrations of ethanol (100%, 90% and 70%) for 2 minutes each. They wereP153160PC00

[0350] then fixed in ice cold acetone: ethanol (30:70) for 15 minutes. Next, they were placed into trisbuffered saline (TBS) for 10 minutes for washing. They were then placed into Haematoxylin for 10 minutes, washed in running tap water, dipped in Scott’s tap water then placed into Eosin stain for 2 dips and washed in running water until the desired colour was observed. The sections were then rehydrated again in increasing concentrations of ethanol (70%, 90% and 100%) before being mounted to a glass coverslip using DPX mounting medium. The sections were imaged using an EVOS FL Auto system (Life technologies, UK).

[0351] Immunohistochemistry

[0352] The spheroid sections were added to histoclear to remove the paraffin wax then rehydrated in decreasing concentrations of ethanol (100%, 90% and 70%) for 2 minutes each. For antigen retrieval, the sections were placed into retrieval buffer (10 mM sodium citrate, 0.05% Tween20 at pH 6) and placed into a plastic pressure cooker and heated in the microwave for 10 minutes at the highest power then left to cool for 20 minutes until reaching RT. They were then washed in TBS-T (tris-buffered saline with Tween20 (1%)) on a rocker for 10 minutes. The slides were then added to 5% bovine serum albumin (BSA) in TBS-T for 30 minutes for blocking and washed again in TBS-T for 10 minutes on a rocker. Rabbit monoclonal antibody to tenascin-C (primary antibody) was added (diluted 1:200 in TBS-T) and left in a humidified chamber at 4 °C overnight. The sections were washed with TBS-T on a rocker for 10 minutes and then a goat anti-rabbit HRP conjugated secondary antibody (ab6721) was added for 1 hour in a humidified chamber at RT. Sections were developed using DAB substrate kit (ab64238) for 10 minutes at RT, sections were washed to stop the reaction with the DAB and counterstained in haematoxylin before being mounted to a glass coverslip using DPX mounting medium. The sections were imaged using an EVOS FL Auto system (Life technologies, UK).

[0353] Immunofluorescence:

[0354] The spheroid sections were added to histoclear to remove the paraffin wax then rehydrated in decreasing concentrations of ethanol (100%, 90% and 70%) for 2 minutes each. For antigen retrieval, the sections were placed into retrieval buffer (10 mM sodium citrate, 0.05% Tween20 at pH 6) and placed into a plastic pressure cooker and heat in the microwave for 10 minutes at the highest power then left to cool for 20 minutes until reaching RT. They were washed in TBS-T (tris-buffered saline with Tween20 (1%)) on a rocker for 10 minutes. They were then added to 5% BSA in TBS-T for 30 minutes for blocking and washed again in TBS-T for 10 minutes on a rocker. Rabbit monoclonal antibody to tenascin-C (primary antibody) was added (diluted 1 :200 in TBS-T) and left in a humidified chamber at 4 °C overnight. The sections were washed with TBS-T on a rocker for 10 minutes and then a goat anti-rabbit IgG (Alexa 488)P153160PC00

[0355] (ab150077) secondary antibody was added for 1 hour in a humidified chamber. After, the sections were washed in TBS-T on a rocker for 15 minutes. For nuclei stain, DAPI- mounting medium was added to the slides. Coverslips were added on top and sealed. The sections were imaged using an EVOS FL Auto system (Life technologies, UK) for both DAPI (405 nm) and Alexa488 (488 nm).

[0356] Clonogenic Assay

[0357] Clonogenic assays were carried out on U87 or U251 glioblastoma cells to calculate the clonogenic capacity of these cells, representing cell survival after exposure to LAT-X and / or GCV. The cells were plated at 1.0 x 106cells per well of a 6-well dish with 3 mL of complete MEM media and left for 24 hours to adhere to the plates at 37 °C with 5% CO2. After 24 hours, the LAT 7 was added (30 pL) and left for 24 hours in an incubator set to 37 °C with 5% CO2. After a further 24 hours, the ganciclovir (GCV) was added to the wells at different concentrations (5, 10, 25, 50 and 100 pg / mL) for either 24, 48 hours or continuously, in an incubator set to 37 °C with 5% CO2. After 12 days, the control (no LAT-X or GCV added) was checked and the colonies were ready to be fixed and stained to be counted. Thereafter, all the 6-well plates had the media removed and then were washed with PBS. After washing, reagent grade methanol (99.8%) was added to the cells for 10 minutes. After 10 minutes, the methanol was removed and then Giemsa stain was added to the colonies for 1 hour to allow them to be stained for counting. For the U87 cells, 4% PFD was used for 10 minutes after the wash and then crystal violet stain mixed (V5265) with water and 100% methanol. After 1 hour, the stain was removed, and the plates were allowed to dry prior to counting. The colonies were then counted, and all analysis was calculated as a percentage of the control.

[0358] Re-dosing

[0359] A re-dosing experiment was conducted on the U87 spheroids to determine whether there would be a higher level of kill on the spheroids. The spheroids were set up exactly as described above. For the re-dosing with the LAT7, one 24 well plate of the spheroids was left exactly as above, one 24 well plate of spheroids was left for 7 days and then on day 7, the LAT7 was added to each individual well (10 pL into 1 mL). Finally, a plate had LAT7 added to the 24-well plate as above on both day 7 and day 14.

[0360] Re-dosing experiment for the clonogenic was also carried out for this where the LAT-X was added again after 7 days (1 re-dose) and every 7 days, for 14 days (2 re-dose) similarly to above.

[0361] RESULTS:P153160PC00

[0362] The above materials and methods were used for an experiment in which a monolayer of U87 cells (2D) were transfected with LAT 7 (HSV-TK-GFP). This is done by addition of LAT 7 and then cells were incubated with i the LAT 7 for both 24 and 48 hours BEFORE ganciclovir addition. Transfection with LAT 7 is shown in Figure 4a. Qualitatively 50-60% of cells are transfected.

[0363] Figure 4b then compares transfection efficiency with Effectine. This figure shows that transfection using LAT 7 is superior compared to the Effectine protocol typically used for commercial transfection. Specifically, there is more LAT 7 plasmid added to the cells compared to when Effectine is used (30 pg vs. 1 pg) respectively.

[0364] It was also noted that Effectine induces a lower level of transfection in spheroids (U87). Furthermore, it is not possible to add 30 pg of plasmid using the Effectine protocol because the concentration of the Effectine would need to be increased if there was an increase in the plasmid, and therefore this would be extremely toxic to the cells.

[0365] Therefore, overall, LAT 7 transfection shows higher plasmid transfection than Effectine AND this can also be increased further due to the low toxicity of the LAT particles to cells.

[0366] Example 4: The compositions can deliver their nucleic acid load to cells to facilitate kill

[0367] Ganciclovir (GOV) concentrations of both 50 pg / mL and 100 pg / mL were added for BOTH time points. The concentrations were chosen based on a ganciclovir curve added to NON transfected cells. What is needed for the experiment is a concentration that causes cell death only in transfected cells. This concentration range was also based on the literature, and we see similar death rates. 50 pg / mL roughly provided 30% death.

[0368] To obtain quantitative results, a clonogenic assay was also used to determine the ultimate fate of cells after administration of LAT7 and Ganciclovir (see details above).

[0369] RESULTS:

[0370] The results are shown in Figures 5 and 6.

[0371] Figure 5 demonstrates that with no treatment (controls) 100% of cells survived. On administration of both the plasmid plus Ganciclovir at 100pg / ml, all glioblastoma tumour cells were effectively killed (fifth and sixth bars). This effect was dose dependent with more kill observed with the higher dose of ganciclovir administered (100pg / ml) versus the lower dose (10pg / ml). There is limited cell kill with either agent alone.P153160PC00

[0372] Figure 6 shows images of the cells 48 hours after the addition of ganciclovir. Ganciclovir is converted by the thymidine kinase gene in LAT 7 to a toxic product and kills cells. Figure 6 shows the ability of LAT 7 to transfect the cells, delivering the killer TK gene as after GCV is added, the cells are beginning to die and with higher concentrations the number of GFP cells diminishes as the transfected cells succumb.

[0373] Example 4: The compositions are able to deeply penetrate the tumour mass

[0374] The penetration capabilities of the LAT particles within spheroids was then investigated. This was done by optical sectioning and physical sectioning. Spheroids (three-dimensional cell culture models) are an in vitro model of solid tumours. They are able to closely mimic the main features of human solid tumours, including their structural organization, cellular layered assembling, hypoxia, and nutrient gradients. These properties imprint in the spheroids an anticancer therapeutics resistance profile, which is similar to that displayed by human solid tumours.

[0375] RESULTS:

[0376] The results are shown in Figures 7 and 8.

[0377] To determine whether the LAT 7 plasmid was transfecting cells within the 3D spheroid models, the LAT 7 was added to the spheroids and then these were subject to paraffin sectioning (see methods above). These results are shown in Figure 7.

[0378] Figure 7a shows different images of the same spheroid using two different filters in the microscope lenses. The top image is noted to be a ‘TRANS’ image and this is taken on brightfield. This means that the image is taken with a white light camera. Underneath this we have the same image using a GFP illumination (fluorescent image). The bright areas (green colour) is what denotes the protein expressed by the plasmid as it has the gene encoding GFP within the same plasmid which contains the HSVTK gene. Finally, at the bottom, this is an overlay of the two images to prove that where there is GFP, this matches with the brightfield image to ensure that it is true and in the cells.

[0379] Figure 7b shows Haematoxylin (purple, darker spots) and Eosin (pink, lighter spots) Staining. H&E staining is carried out to prove the presence of cells. The haematoxylin (purple, darker spots) stains for cell nucleus. The eosin (pink, lighter spots) stains for the cell cytoplasm. These sections show that once the U87 are incubated with LAT 7, the LAT 7 is distributed throughout the entire spheroid. Different sections were taken randomly at different areas of the spheroids.P153160PC00

[0380] Figure 8 shows the addition of HSV-TK-GFP (LAT 7) to 3D U87 spheroids. Figure 8a shows an image of the cells 24 hours after the LAT 7 was added. Figure 8b shows sections of U87 spheroids transfected with LAT 6 also (as part of the development process, we also created a human TK plasmid to act as a negative control to guide the future design). This is LAT6. As for LAT 7, GFP gene expression was observed throughout the entire spheroid.

[0381] Example 5: Deep penetration by the LAT particles allows better kill of spheroids To assess whether this enhanced penetration observed in Example 4 leads to enhanced tumour cell killing, GOV was added.

[0382] Again, as for Example 2, a GOV concentration curve on normal parental U87 cells was carried out to ensure that the concentration of the Ganciclovir (GOV) added to the cells was NOT causing any cell death to those that did not have the plasmid (LAT 7: which is the delivery vehicle resulting from the plasmid pSELECT-HSVTK-GFP).

[0383] All spheroid experiments had 24 spheroids per treatment group to ensure there were sufficient biological replicates. The results of this experiment are shown in Figure 9 to 14. Mosaic Spheroids

[0384] Mosaic spheroids were created to determine if there was a different level of kill to the spheroids when there was a different percentage of the HSV-TK expressing cells with the plasmid (i.e. , 100% transfected). Comparisons were carried out for 100% HSVTKGFP, 50% HSVTKGFP:50% U87, 5% HSVTKGFP:95% 2% HSVTGFP and 98% U87. Spheroids are formed by adding different proportions of stably transfected glioma cells (with the LAT7 HSVTK plasmid and GFP) and non-transfected glioma cells to the spheroid forming vessel. In this experiment only for example 1 and 2% of the total number of cells in the spheroid are expressing the transgene and GFP. The spheroids are then treated with Ganciclovir and growth measured over 12 days. The spheroids were measured over a time course and the area converted to a volume. The volume at day x is then divided by the volume at day 0 (V / V0) and the results plotted verses control spheroids which were not administered with ganciclovir.

[0385] RESULTS:

[0386] The results of exposing the spheroids to the GCV concentrations obtained from Figure 9 are shown in Figures 10-15.

[0387] Figure 10 shows exposure of the U87 cells to increasing concentrations of GCV. The U87 cells were stably transfected with HSVTK-GFP plasmid and then used make spheroids.P153160PC00

[0388] Once the spheroids were formed, the GCV at increasing concentrations was added and their change in volume (V / Vo) was measured. As can be seen from this figure, even lower concentrations of GCV led to cell death.

[0389] To determine whether the GCV needed to be present at all times (continuous) to achieve the significant cell death as shown in Figure 10, GCV was removed after 24 and 48 hours. This would determine whether the cells were still being killed even after the GCV had been removed. This was done simply by removing the GCV media and replacing it with fresh media without GCV. Spheroids were then continually imaged and their V / Vo was determined. The results of this experiment are shown in Figure 11.

[0390] To ensure that the cell death was solely due to the presence of the LAT composition, the control spheroids were also exposed to the same concentrations of GCV. The results of this experiment are shown in Figure 12. In Figure 12a spheroids that were incubated with LAT were incubated with the increasing concentrations of GCV. In Figure 12b the U87 spheroids were also incubated with the same concentrations of the GCV, except they were not incubated with LAT prior to addition to GCV. They were added as controls to ensure that any cell death we observe is purely due to the combination of the LAT with the GCV. As can be seen from Figure 12, we started this experiment using a range of concentrations of GCV with both the control and the LAT 7 treated spheroids so that we could then determine the point at which the cells that had no plasmid added were being killed by the GCV and therefore causing non-targeted killing. Therefore, Figure 12 shows that at 25, 50 and 100 ug / mL the GCV induced kill in the spheroids without the plasmid. Whereas, for the lower concentrations of 5 and 10 ug / mL, we only observe cell death when the plasmid is present. Figure 13 shows the comparison between control spheroids and LAT 7 containing spheroids at different concentrations of GCV. As can be seen from this figure, the LAT 7 containing spheroids showed higher cell death. The GCV is therefore not responsible for the superior kill but rather the presence of the LAT particle. This can also be seen in Figure 14.

[0391] Although at higher concentrations GCV does show toxicity towards the controls, the LAT containing particles still induce superior killing. This could allow low concentrations of toxic drugs like GCV to be used which in combination with the LAT particles have the potency to kill tumours but importantly to do not kill normal non-malignant cells surrounding the tumour meaning there is less chance of side effects.

[0392] In the next experiment, mosaic spheroids were formed to determine if there was a variable level of death with the addition of ganciclovir (GCV), depending on the percentage of cells that had the HSVTK-GFP plasmid or not. This was done by mixing stably transfected U87P153160PC00

[0393] cells with HSVTK-GFP and normal parental U87 cells at various percentages. The results are shown in Figure 15.

[0394] Figure 15a and b show that when the U87 spheroids are parental (100% U87) or stably transfected with the plasmid (LAT7 in this case) labelled as HSVTKGFP. This shows that cells should only die when the plasmid is present as these lower concentrations. These graphs show this for the administration of 5 and 10 ug / mL Ganciclovir. For those that are 100% transfected, all concentrations induced cell kill.

[0395] To then investigate the degree of cell death achievable if there was not going to be 100% transfection (which is likely clinically) when the LAT is added, different percentages of the 2 cell lines were mixed. Figure 15 c demonstrates that there is still complete eradication of the cells within the spheroid when only 50% of them have the plasmid and thus express the TK gene at the concentrations tried. This has also been tested in spheroids with only 2% transfected cells to 98% non-transfected cells and this again shows that there is complete eradication of the spheroid even with a very low percentage of transfected cells. Therefore, we have shown that when there is only 2% of the spheroid containing HSV-TK transfected cells, the concentrations of GOV are still causing complete eradication of the spheroids (as shown in Figure 15d; Fig 15e provided shows the result with 5% transfected cells).

[0396] Despite a high transfection efficiency, these experiments highlight that with in an in vivo context, a lower transfection rate would not equate to treatment failure since it highlights that kill is not related to the transfection efficiency. This additional cell kill is result of the well documented bystander effect observed with suicide genes such as HSVTK, where the conversion of the prodrug to the toxic compound results in drug being excreted out of the transfected cells to interact with non-transfected cells in their vicinity. This phenomenon demonstrates that in a clinical setting we do not need to achieve 100% transfection of all tumour cells.

[0397] Example 6: Various tenascin binding peptides were tested

[0398] U87 cells were seeded in 6-well plates at a density of 100,000 cells per well in a total volume of 5 mL of complete MEM media per well. Cells were incubated for 24 hours at 37 °C with 5% CO2 until approximately 70% confluent. To begin the transfection, all MEM media was removed and 3 mL OptiMEM (Gibco 11058-021) was added, and the cells incubated for 1 hour at 37 °C with 5% CO2. Following incubation, 30 pL of each unique LAT was added to individual wells dropwise and incubated for 4 hours at 37 °C with 5% CO2. Following incubation, 5 mL of complete MEM media was added per well and the cells returned to the incubator at 37 °C with 5% CO2.P153160PC00

[0399] Cells were then harvested 24 hours and 48 hours post transfection by removing all media from individual wells, and 0.5 mL 1X 0.05% trypsin-EDTA solution added. Cells were then incubated at 37 °C with 5% CO2 for 10 minutes to detach cells, then 0.5 mL complete MEM media was added to create a cell suspension.

[0400] To determine the total number of cells vs the number of transfected cells, the cell suspensions were then counted using the Logos LUNA-FX7 automated cell counter.

[0401] Transfected cells were identified by the presence of GFP. Transfection efficiency was calculated as follows:

[0402] Transfection efficiency: (number of transfected cells / total number of cells) x 100

[0403] The peptides tested were as follows:

[0404]

[0405] Table 9.

[0406] Lipids used in the composition were DOTMA and DOPE as described in Example 2.

[0407] RESULTS: The results in terms of the percentage of transfected cells observed at 24 hours and 48 hours post-transfection for each of the peptides is shown in Figure 16. Stability of the resulting particles with these peptides is shown in Figure 17.

[0408] Example 7 Animal models show deep penetration translates to effective tumour kill Protocol:P153160PC00

[0409] Germ free authenticated athymic nude mice approximately 8 weeks old were purchased from Charles River UK. All procedures were carried out under Home Office Project Licence PP2953508 following Ethical Approval.

[0410] On arrival, the mice were separated into sterile cages and fed sterile feed and water for 1 week to allow animals to settle.

[0411] During this time, animals were handled daily to make them accustomed to being handled and reduce stress for handler and mice- making experimentation easier and less stressful for the animals

[0412] On day 8 mice were earmarked by ear clipping in various conformations to enable identification of individual animals.

[0413] On day 10 mice were injected on the right flank (with the mice facing away from the handler) with 2 million mycoplasma free U87 glioma cells. The cells were seeded earlier and collected when approximately 70-80% confluent. Cells were detached using trypsin which was subsequently neutralised by addition of equal volume of complete MEM medium. Cells were centrifuged and all liquid removed from the pellet before resuspension in 100 pL of Matrigel (Corning 354230) that had been thawed briefly and then maintained on ice to ensure it remained in liquid form.

[0414] The 100 pL cell mixture was then injected subcutaneously using a 0.3mm (30G) insulin syringe.

[0415] The mice were weighed before injection and approximately every 2 days thereafter.

[0416] Mice were monitored daily for signs of tumour growth and to confirm wellbeing and once tumours appeared the tumours were measured using callipers at least every 2 days.

[0417] Approximately 10 days later, the mice were randomised into cages of 4 mice / cage with a range of sizes of tumours in each cage then randomly assigned a treatment group as follows:

[0418] Group 1: LAT 7 intratumourally once plus GCV intraperitoneally every 72 hours

[0419] Group 3: LAT 7 intratumourally day 1 then day 4 plus GCV intraperitoneally every 48 hours and GCV intratumourally every 72 hours

[0420] Cage 8: LAT 7 intratumourally plus GCV intraperintoneally every 48 hours then LAT 7 Intratumourally day 4

[0421] Group 2: LAT 7 alone intratumourally day 0 then day 4 intratumourallyP153160PC00

[0422] Group 4: Ganciclovir intraperitoneally every 48 hours

[0423] Group 6 control- no treatment

[0424] Each treatment group had 8 mice.

[0425] Treatments were initiated and mice weighed and tumours measured at least every 2 days. Mice with tumours with a measurement of 1 axis reaching 16mm were sacrificed due to this being the designated end point in the animal licence.

[0426] In treatment groups, mice with tumours that had ulcerated due to tumour shrinkage were also euthanised due to animal welfare issues and the final tumour size recorded for the remainder of the experiments.

[0427] Tumour volume was measured then converted to a volume using the equation: 0.5 x longest measurement x shortest measurement squared. Volumes were plotted on a graph using Graph pad prism software (also used to undertake statistical analysis) as volume at day x / volume at day 0 (V / Vo) against time. Statistical analysis was undertaken after determination of normality via a Shapiro Wilkes test via Kruskal-Wallis test with significance determined via a with Dunn’s Post Hoc test. Error bars represent the mean with SEM and each group consists of 8 animals.

[0428] Results:

[0429] The results are shown in Figures 18-19

[0430] As shown by Figure 18 and Table 10 below, there was a statistically significant reduction in tumour growth in all treatment groups verses the control untreated tumour.

[0431] Dunn's multiple comparisons test i Mean rank diff. Significant? Summary Adjusted P Value >

[0432]

[0433] < Table 10.

[0434] There was no statistically significant difference between any of the control groups suggesting LAT7 alone nor ganciclovir alone had any effect on tumour growth.

[0435] There was no statistically significant difference in tumour volume between groups 3 and 1 or 1 and 8.P153160PC00

[0436] However, there was a statistically significant change in tumour volume between groups 3 and 8. This suggests that there was no advantage to multiple intra-tumour (IT) injections of Ganciclovir.

[0437] Out of the treatment group of animals: 4 out of 24 tumours completely disappeared and have not reformed even after cessation of treatment 62 days after treatment inception. The starting sizes of these tumours were 6x8mm, 5x5 mm, 5x4 mm and 5.5x7 mm and came from groups 1 and 8 the treatment rather than the control groups.

[0438] Weights did not reduce across the experimental time course in any groups, in fact animals steadily increased in weights indicating no toxicity and healthy mice. No other toxic effects were observed; animals were lively and displaying all normal behaviours.

[0439] These results collectively suggest that the treatment of LAT7 plus ganciclovir delivered either IP or IT significantly reduced tumour growth in nude mice.

[0440] These results also suggest that any combinations of IP or IT or frequency has little bearing on the reduction in tumour size.

[0441] Figure 19 shows that the treatment had a significant effect on mouse survival, shown up to 15 days post treatment. From the graph we can clearly see that mice which had a combination of LAT7 and ganciclovir (all double treatment groups pooled) had a significant survival advantage compared to all control groups pooled (Ganciclovir alone, LAT7 alone or no treatment). Each group contained 24 mice.

[0442] Example 8: The same deep penetration into spheroids is seen for particles with further targeting peptides overexpressed in tumours, for example Nestin targeting peptides

[0443] To prove that the tumour kill effect is not limited to tenascin targeting peptides, we tested the same compositions (in terms of lipid and DNA load) but changed the TNG targeting peptide to a Nestin targeting peptide. Nestin is an intracellular protein with some protein excreted into the surrounding matrix.

[0444] Firstly, we checked the transfection efficiency of Nestin alongside various TNG peptides in 2D culture. Methodology is as described in example 6, except cells were only imaged 24 hours post transfection.

[0445] We then looked for further confirmation that Nestin targeting could produce the same deep penetration as the various TNG targeting peptides tested above. This was done by adding the spheroids to 3 mL of optiMEM (this is cell culture medium that does not contain serumP153160PC00

[0446] proteins) thus improving the uptake of the particle into the spheroids, in a tissue culture bijou. This was added to a rolling platform at 37 °C for 1 hour. After 1 hour, 30 pL of the appropriate LAT formulation was added and placed back onto the roller for an additional 4 hours. The roller is used to prevent the spheroids from sticking to each other. After 4 hours, an additional 3 - 5 mL of complete MEM media was added to allow the spheroids to have the appropriate nutrients for growth. To the bijou’s, 5% CO2 was added, and they were left on the roller at 37 °C overnight. These spheroids were then individually added to single wells of a 24-well plate in complete MEM media and imaged to check for transfection.

[0447] The same experiments were applied to a dual targeting peptide composition. For this experiment a mixture of targeting peptides was added to the lipid composition (50% nestin targeting and 50% tenascin targeting). .

[0448] Results:

[0449] The results are shown in Figures 21-24.

[0450] Figure 21 shows 2D transfection using various TNG and Nestin targeting peptides (with lipid composition = DOTMA and DOPE).

[0451] The control contains untreated cells that have not been transfected with any particles.

[0452] “Lysine” in the figure refers to SEQ ID NO. 5. Arginine = SEQ ID NO. 6; D-Amino = SEQ ID NO. 15; Reverse = SEQ ID NO. 10; PL-1 = SEQ ID NO. 11; PL3 = SEQ ID NO. 12; PL3R = SEQ ID NO. 13; N1 = SEQ ID NO. 16; N2 = SEQ ID NO. 17; Dual Lysine+N2 = composition containing SEQ ID NO. 5 and SEQ ID NO. 17.

[0453] Figure 22 shows the transfection results in 3D U87 spheroids. A representative image is shown. However, each experiment was carried out in triplicate and with a different cell line (LN229) to verify the penetration. Figure 23 shows the dual peptide targeting compositions penetrating both U87 (a) and LN229 spheroids (b).

[0454] Figure 24 a) and b) shows deep penetration in the 2 spheroid models with SEQ ID NO. 5. These results show that deep penetration into the spheroid core is generally applicable to peptides which target tumours in combination with the lipid combination of cationic lipid plus DOPE.

[0455] Example 9: Spheroid penetration is supported by spheroid section magnification In addition to the 3D cell transfection in Example 3, we also confirmed spheroid penetration using confocal microscopy. Spheroids were fixed sectioned and stained following methodology of example 3. Immunofluorescence was performed on sections of MTS probingP153160PC00

[0456] for TK. The MTS were sectioned using a cryostat (LEICA, CM 1950). Spheroid samples were prepped by fixing in 4% PFA at room temperature. PFA was removed and spheroids were washed in PBS. Parafilm was taken and a small circle of OTC placed on top. Spheroids were then placed in media and pipetted into the liquid OTC. Using tweezers spheroids were evenly distributed among the OTC. Cryo-moulds were taken and a layer of OTC placed on the bottom and left to freeze on dry ice. Parafilm with the OTC and spheroids was also placed on dry ice to set. Once set, the spheroids in OTC were placed vertically into the cryo-mould and OTC added to hold the samples in place. The mould was then placed on dry ice again to set, labelled and stored at -80°C until ready to be sectioned. Once ready to be sectioned, Cryo-moulds were taken and placed at -16°C in the cryostat. Frozen samples were removed from the moulds and placed on top of fresh liquid OTC on a “puck” and left to set. Slides were labelled appropriately and numbered. Moulds were trimmed until the media colour could be seen within the mould and then 10 micron sections were taken. 4-5 sections of spheroids were placed on each slide and then placed in the -80°C freezer until ready to process. Once spheroids were sectioned, slides were placed in cold 100% acetone for 20 minutes. Slides were washed in cold PBS and placed in 0.1% Triton X-100 for 5 minutes Slides were then washed in PBS and placed in blocking buffer for 1 hour. Slides were then washed in PBS and placed in a humidifier chamber. Primary Antibody for TK was diluted 1:1000 in fresh blocking buffer and dropped onto spheroid sections on the slides. The humidifying chamber was placed overnight at 4°C. The next day, secondary Antibody Alexa fluro 647 was diluted 1:1000 (red) in fresh blocking buffer, slides were washed 3 x in PBS and then secondary antibody added to the sections. The humidifying chamber with the slides and secondary antibody was left at room temperature for 60 mins. After slides were washed again in PBS, and then mounting media containing DAPI was added to the slides with cover slips. Slides were left to set at 4°C. Slides were imaged on the confocal microscope on a x63 oil immersion lens using DAPI and Alexa 647 lasers. Images taken on the confocal were then processed using Image J software.

[0457] Results:

[0458] The results are shown in Figures 25-28.

[0459] Figure 25 shows a U87 spheroid transfected with TRJ10C (SEQ ID NO.5; DOTMA+DOPE, these lipids also used for the other particles in this example) on the confocal microscope. Figure 25a is the middle section of a spheroid at x63 magnification. Figure 25b is the whole section of a spheroid atx20 magnification. Red =HSVTK, Blue = nuclei, Green = GFP (from plasmid).P153160PC00

[0460] As you can see from Figure 25, the composition has penetrated deep into the core of the spheroid. The same deep penetration into the spheroid is shown in Figure 26 which instead of a Tenascin C targeting peptide has a Nestin 2 targeting peptide (SEQ ID NO. 17). Again, Figure 26a shows middle section of spheroid at x63 magnification. Figure 26b shows the whole section of a spheroid at x20 magnification. Red =HSVTK, Blue = nuclei, Green = GFP (from plasmid).

[0461] The Nestin targeting composition was also tested in an alternative Human glioma cell line LN229 spheroid cell line as shown in Figure 27a and b, which also confirmed deep penetration of the Nestin targeting composition into these spheroids.

[0462] In the same LN229 spheroids, Figure 28 shows penetration into the spheroid core using a different TNG targeting peptide (SEQ ID NO. 11).

[0463] Example 10: Alternative Manufacture of the delivery particles (LAT-4 / 5 / 7 / 8 / 9 / 10) Materials & Equipment: as described in example 2, with exception of a NovaTM Benchtop UM LNP Manufacturing System (Helix Biotech, USA) with a size 1 impinged jet mixer was used as the microfluidic device.

[0464] Procedure: as described in example 2, with exception of the flow rates, which were as displayed in table 11.

[0465] Table 11: Flow Rates and Volumes of Aqueous and Organic Solutions

[0466]

[0467] The organic phase in Table 11 is the following: DOPE, DOTMA, Ethanol and desired peptide. The aqueous phase is the plasmid in water. These two phases are then mixed together in the microfluidic device at the displayed flow rates. The volumes can be scaled accordingly to the required batch side.

[0468] Particles are collected in filtered PW in an appropriate volume to achieve a 1 in 5 dilution, i.e. if 10 mL of particles produced, they would be collected in 40 mL of filtered PW.

[0469] Results:P153160PC00

[0470] This manufacturing method can be used to make any of the previously mentioned formulations, with characteristics similar to the other manufacturing methodology described in example 2.

[0471] Example 11: Different Lipid compositions allow deep penetration

[0472] For lipid penetration, methodology following MTS transfection, fixing and microtome sectioning was followed. Staining processes also followed example 3.

[0473] U87 and LN229 spheroids were grown and imaged as described above. Compositions used were as in the table below. Manufacture of the compositions was in accordance with Example 2 above.

[0474] DOTMA was replaced with DOTAP (Avanti, catalogue number: 890890P) on a molar percentage basis. Additionally, DOPE was replaced with DOPC (Avanti, catalogue number: 850375P) or DSPC (Avanti, catalogue number: 850365P) on a molar percentage basis. When substituting lipids a ratio of 1:1 of DOTMA / DOTAP:DOPE / DOPC / DSPC was always maintained and the same working concentrations as previously described used. The size, PDI, and zeta potential of particles manufactured with alterative lipids is displayed below in table 12.

[0475] Table 12: Properties of alternative lipid particles

[0476]

[0477] Pegylated lipids and cholesterol in the cationic lipid plus phospholipid mix were also investigated. The PEGylated lipid and / or cholesterol was substituted into the formulation on a % molar basis whilst maintaining the 1:1 ratio of DOTMA / DOTAP:DOPE.Both cholesterol and PEGylated lipid were prepared at a stock concentration of 5 mg / mL dissolved in 100% ethanol.

[0478] The PEGylated lipid selected for this was DMG-PEG 2000 (Avanti, catalogue number: 880151 P) . The PEGylated lipid was at a concentration of 1.5%.

[0479] The Cholesterol (Sigma Aldrich, catalogue number: C8667) was at a concentration of 30%.P153160PC00

[0480] We have made the following combinations:

[0481] • DOTMA + DOPE + PEGylated lipid

[0482] • DOTMA + DOPE + Cholesterol

[0483] • DOTMA + DOPE + PEGylated lipid + Cholesterol

[0484] • DOTAP + DOPE + PEGylated lipid

[0485] • DOTAP + DOPE + Cholesterol

[0486] • DOTAP + DOPE + PEGylated lipid + Cholesterol

[0487] Also tested were ionisable lipids Dlin-MC3-DMA (TargetMol, catalogue number: T5823), SM-102 (TargetMol, catalogue number: T9410), and ALC-0315 (Avanti, catalogue number: 8909000). lonisable lipids were prepared to 5 mg / mL working solution in 100% ethanol, lonisable lipids were substituted into the formulation on a molar percentage basis in place of DOTMA, to maintain a 1:1 ratio with DOPE.

[0488] Results:

[0489] The results are shown in Figures 29-35.

[0490] 2D transfection efficiency for the cationic lipids and ionisable cationic lipids is shown in Figure 29.

[0491] Exemplar cationic lipids DOTMA and DOTAP were then taken forward and showed deep penetration into the spheroid core following significant transfection in the 2D experiment. Additionally, formulations with DOPE and DSPC with either cationic lipid had exceptional penetration into the spheroid core (Figure 30: which shows deep penetration with DOTMA / DOTAP and DOPE; and Figure 31: which shows DOTMA and DOTAP in a different cell line with DOPE or DSPC).

[0492] It was also investigated if the addition of cholesterol or pegylated lipids supported the deep penetration of the composition. The addition did not affect the stability of the particles as shown in Figure 32. Furthermore, the addition of a PEGylated lipid and / or Cholesterol did not impact transfection efficiency in 2D cells in either cell line as shown in Figure 33.

[0493] The addition of a pegylated lipid or cholesterol also did not affect the deep penetration of the particles as shown in Figures 34 and 35. Figure 34 shows 3D transfection efficiency in U87 spheroids. Figure 35 shows 3D transfection efficiency in LN229 spheroids. In summary, the particles still transfect even with the addition of a PEGylated lipid and / or Cholesterol.P153160PC00

[0494] Example 12: The deep penetration of the particles allows stem cell targeting and kill In Example 7 we showed the particles conferred a significant survival advantage compared to all control groups pooled in an in vivo mouse tumour model. We then performed further experiments to support that the compositions not only deeply penetrated tumours but also were able to kill cancer stem cells within the hypoxic core at these tumour depths.

[0495] Immunofluorescence was used to confirm stem cell markers in xenograft tissue.

[0496] Xenografts formed on nude mice were harvested following euthanisation and removal of the tumours from mice. Tumours were washed in PBS before being placed in cryomoulds and surrounded by OCT to prevent tissue damage. Samples once in OCT were stored at -80°C until ready to be sectioned.

[0497] Tissue was sectioned on the cryostat (LEICA, CM 1950). Once ready to be sectioned, Cryomoulds were taken and placed at -16°C in the cryostat. Frozen samples were removed from the moulds and placed on top of fresh liquid OCT on a “puck” and left to set. Slides were labelled appropriately and numbered. Moulds were trimmed until slices were well within the tissue sample. 10 micron sections were then taken. Six sections of each tissue sample were taken with a trim of 100 microns between sections to allow full depth of the tissue sample to be procured. 3 to 4 slices were placed on each slide and then placed in the -80°C freezer until ready to process. Once samples were sectioned, slides were placed in cold 100% acetone for 20 minutes. Slides were washed in cold PBS and placed in 0.1% Triton X-100 for 5 minutes. Slides were then washed in PBS and placed in blocking buffer for 1 hour. Slides were then washed in PBS and placed in a humidifier chamber. Primary Antibody for each stem cell marker was diluted as per the manufacturer guidelines (Hif 1 -a AB51608, Nestin AB316018, Sox2 AB137385, CD34 AB198395, CD133 AB284389) in fresh blocking buffer and dropped onto sections on the slides, with 1 control section.

[0498] The humidifying chamber was placed overnight at 4°C. The next day, secondary Antibody Alexa fluro 647 was diluted 1:1000 (red) in fresh blocking buffer, slides were washed 3 x in PBS and then secondary antibody added to the sections. The humidifying chamber with the slides and secondary antibody was left at room temperature for 60 mins. After slides were washed again in PBS, mounting media containing DAPI was added to the slides with cover slips. Slides were left to set at 4°C. Slides were imaged on the confocal microscope on a x63 oil immersion lens using DAPI and Alexa 647 lasers. Images taken on the confocal were then processed using Image J software.

[0499] Results:P153160PC00

[0500] The results are shown in Figures 36-40.

[0501] Figure 36 shows a U87 tumour tissue from nude mice (control no treatment) probed with Nestin antibody and imaged on an oil immersion confocal microscope: a)= control (no antibody; b) = tumour section x10; c) = X63 oil immersion. Red = Nestin; Blue = nuclei. Figures 37 and 38 show probing of the U87 xenograft tumour with further stem cell markers (SOX2, CD133 and HIF). This shows the U87 spheroids contain stem cells.

[0502] Following on from this, we used immunofluorescence to show penetration of the formulation (DOTMA + DOPE, Lysine Ten C pep, non survivin HSVTK plasmid (i.e. LAT7 / TRJ10C + survivin with DOTMA and DOPE) throughout tumour tissue taken from mice instead of a spheroid model, i.e. to the stem cell containing areas.

[0503] Figure 39 shows the results from this experiment. Tumours were taken from mice at the end of the experiment. These were sliced and probed with HSVTK antibody to see penetration of our product into solid tumours. As our particles already have Fluorescent green GFP, we used a red colour to detect the HSVTK. As is evident from Figure 39, penetration is deep within the mouse tumour in agreement with the spheroid model. Overlap of the green GFP with the red HSVTK shows delivery of the TK by the particles deep within the tumour mass. Figure 40 shows Z-stack images also to support the depth of penetration into the tumour mass.

[0504] As Figure 40 demonstrates that the particles penetrate over 180 pm into the spheroid when measured as a 3D image. Image 40 (A) shows the 3D side view of a U87 spheroid which was 1000um in size TK was probed for with our secondary red alexa fluro stain 647. Using Image J we were able to determine a penetration of 180pM and from Figure 40 (B) an overlay of each 3D image flattened shows our red fluorescent TK marker penetrating >400pm in depth.

[0505] Spheroids were grown to ~1000pM in size and transfected with TRJ10c overnight following a similar methodology to Example 3. Spheroids were imaged on the evos to determine is transfection was successful with the GFP laser. Spheroids were then washed with PBS, fixed in 4% PFA for 1 hour at room temp and then washed again in PBS. Once washed spheroids were permeabilised in 1% Triton x-100 for 48h, washed in PBS and then left in blocking buffer for a further 48h. After 48h spheroids were washed in PBS and left in Primary TK antibody diluted 1:1000 for 48h. Spheroids were then washed again in PBS and left in secondary Antibody Alexa Fluro 647 diluted 1:1000 (red) in fresh blocking buffer for a further 24h, After 24h, the spheroids were washed in PBS and then placed in DAPI solution for 1P153160PC00

[0506] hour (ThermoFisher D1306). Spheroids were then washed again in PBS and imaged immediately.

[0507] Example 13: Further evidence that the composition kills stem cells after deep penetration of tumour mass

[0508] The ability of the particles to transfect and kill stem cells once the stem cells are reached via the deep penetration into the tumour core was investigated further by human glioblastoma stem cell transfection.

[0509] Glioblastoma stem cells were cultured in extracellular matrix coated 6-well dishes in human glioblastoma cancer stem cell complete serum free media, then 150 pL of formulation added to relevant wells. To determine stem cell kill, GBM stem cells were transfected with particles, then 48 hours later 5 pg / mL GOV was added. Following 24 hours of incubation with 5 pg / mL GOV, the treatment was removed and cells stained with Trypan Blue (a dye that is selectively taken up by dead cells, turning them blue). Following staining, stem cells were counted to determine the total number of cells versus the number of blue (dead) cells as a means of calculating the percentage kill.

[0510] Results:

[0511] The results are shown in Figures 41 - 42. Figure 41 demonstrates that the following formulations; Dual peptide (a), Nestin 2 (b), Survivin (c), and Lysine (d) successfully transfect the glioblastoma stem cells. DOTMA and DOPE were the lipids used.

[0512] Figure 42 (a - e) displays representative images of glioblastoma stem cells stained with trypan blue following transfection with various formulations and the addition of GCV, with blue cells indicating cell death, and in (f) the percentage kill of each formulation is displayed. This demonstrates that not only did the particles transfect the stem cells, but we were able to achieve cell kill.

[0513] Example 14: Alternative suicide genes

[0514] As an alternative to TK, IL24 was also used. IL24 is a cytokine which can induce apoptosis. The plasmid used to express the alternative suicide gene and encapsulated in the particles is shown in Figure 43. Methodology for transfection was as described above (DOTMA + DOPE, Lysine peptide and IL24 plasmid), except the formulation was made utilising the IL24 plasmid.

[0515] To test for IL24 expression a Enzyme-Linked Immunosorbent Assay (ELISA) designed to quantify the level of Human IL-24 in cell culture supernatants was used (Thermo Fisher,P153160PC00

[0516] catalogue number: EH269RB). Briefly, cell supernatants were harvested 72 hours post transfection in LN229 cells and incubated in an ELISA plate precoated with antibodies specific to IL24, meaning any IL24 antigen present in the cell supernatant will bind to the antibody. A biotin conjugate in conjunction with streptavidin-HRP is then used to visualise IL24 levels via colorimetric reaction which is quantified by reading the absorbance of the wells using a plate reader.

[0517] Results:

[0518] The results are shown in Figures 44-45.

[0519] Figure 44 shows very few cells were GFP positive - indicating presence of IL24. In this example in LN229 cells, the cells looked very unhealthy just 24 hours after the addition of the IL24 particle and there were very few GFP positive cells - this suggests IL24 is being produced.

[0520] Figure 45 shows the amount of IL24 produced was proportional to the amount of IL24 particle added, and considerably higher than the untreated control at the highest volume of particle administered. This confirms that IL24 was being produced and is responsible for the cell death seen previously.

Claims

P153160PC00CLAIMS1. A composition for non-viral transfection into a cell, the composition comprising:a) a lipid component comprising a cationic lipid and DOPE;b) i) a first targeting peptide sequence which binds to a tenascin protein and / or a nestin protein; andii) a second polycationic nucleic acid-binding peptide sequence.

2. The composition of claim 1 wherein the first and second peptide sequences are comprised in one bipartite peptide sequence.

3. The composition of any of the preceding claims, wherein the cationic lipid is DOTMA or DOTAP.

4. The composition of any of the preceding claims:a) wherein the tenascin protein is tenascin-C (TNG) and the TNG targeting peptide sequence binds to SEQ ID NO. 9 or a sequence with 80% sequence identity to SEQ ID NO. 9; and / orb) wherein the tenascin protein is tenascin-C (TNG), optionally wherein the TNC targeting peptide sequence comprises: any of SEQ ID NO.s 1-4, or a peptide sequence with 80% sequence identity with SEQ ID NO.s 1-4; orc) wherein the tenascin targeting peptide sequence is comprised in a bipartite peptide sequence with the polycationic nucleic acid-binding peptide sequence, wherein the bipartite peptide sequence optionally comprises: any of SEQ ID NO.s 5-8 or 10-15 or a peptide sequence with 80% sequence identity with SEQ ID NO.s 5-8 or 10-15; ord) wherein the tenascin protein is tenascin- W; and / ore) comprising a further targeting peptide sequence.

5. The composition of any of claims 1-3:a) wherein the nestin targeting peptide sequence binds to SEQ ID NO. 20 or a sequence with 80% sequence identity to SEQ ID NO. 20; and / orP153160PC00b) wherein the nestin targeting peptide sequence comprises: any of SEQ ID NO.s 18- 19 or a peptide sequence with 80% identity with SEQ ID NO.s 18-19;c) wherein the nestin targeting peptide sequence is comprised in a bipartite peptide sequence with the polycationic nucleic acid-binding peptide sequence, wherein the bipartite sequence comprises: any of SEQ ID NO.s 16-17 or a peptide sequence with 80% sequence identity with SEQ ID NO.s 16-17; and / ord) comprising a further targeting peptide sequence.

6. The composition of any of the preceding claims, wherein the composition comprises a first targeting peptide sequence which binds to a tenascin protein and a further targeting peptide which binds to nestin.

7. The composition of any of the preceding claims wherein the polycationic nucleic acid-binding peptide sequence comprises or consists of a plurality of lysine or lysine homologues and / or arginine or arginine homologues.

8. The composition of any of the preceding claims, further comprising a nucleic acid.

9. A pharmaceutical composition comprising the composition of any of claims 1-8 and one or more pharmaceutical excipients, optionally wherein one or more pharmaceutical excipient(s) is an immune adjuvant.

10. The composition of any one of claim 8 or pharmaceutical composition of claim 9 for use as a medicament or a vaccine, optionally wherein the vaccine is a cancer vaccine.

11. The composition of claim 8 or pharmaceutical composition of claim 9 for use in a method of treating cancer, optionally comprising a nucleic acid encoding a suicide gene, optionally wherein the method further comprises administering a compound capable of being converted by the suicide gene into a toxic product causing cell death.P153160PC0012. The composition or pharmaceutical composition for use of claim 11, wherein the nucleic acid encoding the suicide gene comprises a cancer-specific promoter, optionally wherein the cancer-specific promoter is a survivin promoter.

13. The composition or pharmaceutical composition for use of any of claims 8-12, wherein the composition is for use as a cancer vaccine and / or for use in a method of treating cancer and wherein the composition transfects and kills cancer stem cells.

14. In vitro use of the composition of claims 1-8 for transfecting cells, optionally cancer cells, optionally cancer stem cells.

15. An in vitro method for transfecting a cell with a nucleic acid, the method comprising contacting the cell with the composition of claim 8 to obtain a transfected cell, optionally wherein the cell is a cancer cell, optionally wherein the cell is a cancer stem cell.

16. A kit comprising:a) a lipid component comprising a cationic lipid and DOPE;b) i) a first targeting peptide sequence which binds to a tenascinprotein and / or a nestin protein; andii) a second polycationic nucleic acid-binding peptide sequence.

17. A composition comprising:a) a lipid component comprising a cationic lipid and DOPE;b) i) a first targeting peptide sequence which binds to an extracellular tumour antigen expressed by cancer stem cells; andii) a second polycationic nucleic acid-binding peptide sequence wherein the composition further comprises a nucleic acid, for use in a method of treating cancer in a subject, wherein the composition transfects and kills one or more cancer stem cells.P153160PC0018. In vitro use of a composition for transfecting cancer stem cells, the composition comprising:a) a lipid component comprising a cationic lipid and DOPE;b) i) a first targeting peptide sequence which binds to an extracellular tumour antigen expressed by cancer stem cells; andii) a second polycationic nucleic acid-binding peptide sequence.

19. An in vitro method for transfecting a cancer stem cell with a nucleic acid to obtain a transfected cancer stem cell, the method comprising contacting the cancer stem cell with a composition comprising:a) a lipid component comprising a cationic lipid and DOPE;b) i) a first targeting peptide sequence which binds to an extracellular tumour antigen expressed by cancer stem cells; andii) a second polycationic nucleic acid-binding peptide sequence, wherein the composition further comprises the nucleic acid.

20. The composition for use, in vitro use or in vitro method of claims 17-19, wherein the first and second peptide sequences are comprised in one bipartite peptide sequence.

21. The composition for use, in vitro use or in vitro method of claims 17-20, wherein the cationic lipid is DOTMA or DOTAP.

22. The composition for use, in vitro use or in vitro method of claims 17-21, wherein the polycationic nucleic acid binding amino acid sequence comprises or consists of a plurality of lysine or lysine homologues and / or arginine or arginine homologues.

23. The composition for use of claims 17-22, wherein the composition is a pharmaceutical composition additionally comprising one or more pharmaceuticalP153160PC00excipients, optionally wherein one or more pharmaceutical excipient(s) is an immune adjuvant.

24. The composition for use of claims 17-23, wherein the composition is a cancer vaccine.

25. The composition for use of claims 17-24, wherein the composition additionally comprises a nucleic acid encoding a suicide gene, optionally wherein the method further comprises administering a compound capable of being converted by the suicide gene into a toxic product causing cell death.

26. The composition for use of claim 25, wherein the nucleic acid encoding the suicide gene comprises a cancer-specific promoter, optionally wherein the cancer-specific promoter is a survivin promoter.

Images