Transduction

Abstract

This invention relates to transduction of cargo molecules into living cells, such as protein transduction, in particular a delivery molecule for transduction of a cargo into a cell comprising: a cargo-binding molecule and/or a cargo; a glycosaminoglycan (GAG) binding element, which is capable of binding to GAG on the surface of the cell; and a protein transduction domain. Methods of transduction, methods of producing or modifying cargo for transduction, delivery molecules for transduction and methods of treatment using transduction, or using transduced cells are also provided.

Claims

1. A delivery molecule for transduction of a cargo into a cell comprising: a cargo, or a cargo-binding molecule for binding to a cargo, and optionally wherein the cargo is bound to the cargo-binding molecule; a glycosaminoglycan (GAG) binding element comprising: P21 of Heparin-binding EGF-like growth factor (HB-EGF); or a GAG binding antibody, which is capable of binding to GAG on the surface of the cell; and a protein transduction domain (PTD) wherein the PTD is not a heparin binding domain.

2. The delivery molecule according to claim 1, wherein the GAG binding element comprises P21 of HB-EGF.

3. The delivery molecule according to claim 1, wherein the GAG binding element comprises a GAG binding antibody.

4. The delivery molecule according to claim 3, wherein the GAG binding antibody comprises a single-domain antibody.

5. The delivery molecule according to claim 1, wherein the protein transduction domain is hydrophilic or amphiphilic.

6. The delivery molecule according to claim 1, wherein the protein transduction domain comprises a majority of hydrophilic amino acid residues.

7. The delivery molecule according to claim 1, wherein the protein transduction domain comprises a majority of arginine and/or lysine amino acid residues.

8. The delivery molecule according to claim 1, wherein the protein transduction domain is selected from any of the group comprising: TABLE-US-00008 Penetratin or Antenapedia PTD (SEQ ID NO: 37) RQIKWFQNRRMKWKK; HIV transactivator protein (TAT) (SEQ ID NO: 38) YGRKKRRQRRR; Synembryn B (SynB)1  (SEQ ID NO: 39) RGGRLSYSRRRFSTSTGR; SynB3 (SEQ ID NO: 40) RRLSYSRRRF; PTD-4 (SEQ ID NO: 41) PIRRRKKLRRLK; PTD-5 (SEQ ID NO: 42) RRQRRTSKLMKR; Flock house virus (FHV) Coat-(35-49) (SEQ ID NO: 43) RRRRNRTRRNRRRVR; Brome mosaic virus (BMV) Gag-(7-25) (SEQ ID NO: 44) KMTRAQRRAAARRNRWTAR; Human T-cell lymphotrophic virus (HTLV)-II Rex- (4-16) (SEQ ID NO: 45) TRRQRTRRARRNR; D-Tat (SEQ ID NO: 46) GRKKRRQRRRPPQ; R9-Tat (SEQ ID NO: 47) GRRRRRRRRRPPQ; Transportan (SEQ ID NO: 48) GWTLNSAGYLLGKINLKALAALAKKIL chimera; Microtubule-associated protein (MAP) (SEQ ID NO: 49) KLALKLALKLALALKLA; Streptavidin-binding Peptide (SBP) (SEQ ID NO: 50) MGLGLHLLVLAAALQGAWSQPKKKRKV; Folate-binding protein (FBP) (SEQ ID NO: 51) GALFLGWLGAAGSTMGAWSQPKKKRKV; Human 3-methyladenine-DNA glycosylase (MPG) (SEQ ID NO: 52) ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya; MPG-nuclear localisation sequence (NLS) (SEQ ID NO: 53) ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-cya; Pep-1 (SEQ ID NO: 54) ac-KETWWETWWTEWSQPKKKRKV-cya; and Pep-2 (SEQ ID NO: 55) ac-KETWFETWFTEWSQPKKKRKV-cya; or polyarginines, polylysines, (RAca)6R, (RAbu)6R, (RG)6R, (RM)6R, (RT)6R. (RS)6R, R10, (RA)6R, R7, and R8.

9. The delivery molecule according to claim 1, wherein the protein transduction domain comprises about 8 arginine residues.

10. The delivery molecule according to claim 1, wherein the cargo is selected from any of the group comprising a peptide, a protein, a nucleic acid, and a nanoparticle.

11. The delivery molecule according to claim 1, wherein the cargo is selected from any of the group comprising a therapeutic molecule; a drug; and a pro-drug; a functional protein or peptide.

12. The delivery molecule according to claim 1, wherein the cargo is between about 20 and about 30,000 amino acids in length.

13. The delivery molecule according to claim 1, wherein the cargo-binding molecule comprises any of a peptide, a protein, streptavidin, a nucleic acid-binding molecule, an antibody, or fragment thereof, an antibody mimetic, a chemical linker molecule, an affinity tag, or an affinity tagged molecule.

14. A cell comprising or encoding the delivery molecule in accordance with claim 1.

15. A nucleic acid encoding the delivery molecule according to claim 1.

Description

(1) Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.

(2) FIG. 1 Inefficient Protein Delivery in Pluripotent Cells. (a) Schematic of the proteins created to determine the efficiency of protein delivery. mR is mRFP only as a poorly transducing control protein representing background transduction. mR-8R is mRFP with a C-terminal fusion of eight Arginine residues (8R) to promote transduction. (b) mR-8R transduces efficiently into NIH3t3 cells. Fluorescence microscopy images of NIH3t3 cells treated with mR or mR-8R (100 μg/ml) for twelve hours in standard media conditions. Scale bar, 100 μm. (c) mR-8R transduces inefficiently into human and mouse embryonic stem cells (HUES7 and CGR-8, respectively), human induced pluripotent stem cells (IPS2) and mouse cardiomyocyte cell line HL1. Fluorescence microscopy images of multiple cell lines treated with mR-8R (100 μg/ml) for twelve hours in cell-type specific media conditions. Scale bar, 100 μm. (d) Flow cytometry analyses of the multiple cell lines treated with different dosages of mR-8R (0, 1, 5, 10, 20, 50 and 100 μg/ml) for twenty-four hours. (e) Flow cytometry analyses of the 100 μg/ml dose for twenty-four hours. Graphs show relative fluorescence units (R.F.U.). Error bars indicate s.d.

(3) FIG. 2 P21 improves PTD-mediated transduction. (a) Schematic of the proteins created after screening domains which improve efficiency of protein delivery to cells. mR and mR-8R are described in FIG. 1. P21-mR is mRFP with an N-terminal fusion of the P21 domain of heparin-binding EGF (HB-EGF). P21-mR-8R is mRFP with N-terminal fusion of P21 and C-terminal fusion of 8R. (b) Fusion of P21 to mR-8R significantly improves transduction into NIH3t3 cells. Fluorescence microscopy images of NIH3t3 cells treated with proteins (20 μg/ml) for twelve hours in standard media conditions. Scale bar, 100 μm. (c) P21-mR-8R transduces efficiently into human and mouse embryonic stem cells (HUES7 and CGR-8, respectively) and human induced pluripotent stem cells (IPS2) and mouse cardiomyocyte cell line HL1. Flow cytometry analyses of the mR-8R-inefficiently transduced cell lines treated with proteins mR-8R (20 μg/ml) for twelve hours. (d) P21-mR-8R initially strongly interacts with cell membranes and progressively transduces be localised peri-nuclearly. Fluorescence (top) and confocal laser scanning microscopy (bottom) images of NIH3t3 cells treated with P21-mR-8R (20 μg/ml) for either 1 hour, 1 hour with washes and a further 5 hours incubation (in serum-free media) or 6 hours treatment. Cells were pre-incubated for 1 hour in serum-free media, transduced for the desired time in serum-free media. Scale bars, 50 μm (top) and 10 μm (bottom). (e) Enhancement of transduction mediated by P21 and 8R are affected by Trypsin proteolysis. Flow cytometry analyses NIH3t3 cells treated with proteins (20 μg/ml) for 1 hour and a further 5 hour incubation (in serum-free media), with or without 10 min pre-digestion with Trypsin or treatment with non-proteolytic cell dissociation solution (CDS). Cells were pre-incubated for 1 hour in serum-free media, treated with Trypsin and transduced for 1 hour in serum-free media. (f) Cell surface interaction of P21-containing proteins is disrupted by Tritonx100 treatment. Flow cytometry analyses NIH3t3 cells treated with proteins (20 g/ml) for 1 hour and a further 5 hour incubation (in serum-free media) with 10 min pre-treatment of PBS or PBS containing 0.1% (v/v) Tritonx100 (Tx100). Cells were pre-incubated for 1 hour in serum-free media, treated with PBS or PBS with Tx100 and transduced for 1 hour in serum-free media. Error bars indicate s.d.

(4) FIG. 3 P21 binds directly to Heparin and cell surface HS-GAG. (a) Soluble Heparin in media during transduction inhibits cell membrane interaction and transduction of P21-containing proteins. Fluorescence microscopy images of CGR-8 cells treated with P21-mR-8R (20 μg/ml) for 6 hours in serum-free media containing 0 or 50 μg/ml Heparin. Scale bar, 100 μm. (b) Flow cytometry analyses NIH3t3 cells treated with P21-mR-8R (20 μg/ml) for 6 hours with or without a variety of GAGs (50 μg/ml) in serum-free medium. CS is Chondroitin sulphate. Cells were pre-incubated for 1 hour in serum-free media and transduced for 6 hours in serum-free media with or without GAGs. (c-d) Only high-doses of Heparin inhibit 8R activity whereas P21 activity is inhibited dose-dependently by Heparin in (c) NIH3t3 cells and (d) CGR-8 cells. (e) and (f) Cell surface Heparan sulphate is required for efficient P21-mediated protein delivery. Heparan sulphates/Heparin-containing FCS inhibits P21-mediated transduction but can also replace cell surface GAGs and mediate P21-transduction in cells deficient for Heparan sulphate. Fluorescence microscopy images of CGR-8 cells and EXT1−/− mESCs treated with P21-mR-8R (20 μg/ml) for 6 hours in media containing 0 or 20% FCS. Scale bar, 100 m. Error bars indicate s.d.

(5) FIG. 4 GET/HETD-mediated nuclear delivery of Cre Recombinase. (a) Schematic of the construct created to mark Cre activity in cells. Cre-mediated excision of a transcriptional STOP region flanked by loxP sites induces the constitutive expression of eGFP. Pr, promoter; βGal, β-galactosidase; Neo, Neomycin phosphotransferase. The NIH3t3 LSL-eGFP cell line was created by transfection and selection of NIH3t3 cells. (b) eGFP expression in untreated NIH3t3 LSP-eGFP cells or those transduced with SIN Cre lentivirus. Left shows fluorescence microscopy and right shows flow cytometry histogram of eGFP expression. Scale bar, 50 μm (c) Scheme of testing transduction of Cre activity in NIH3t3 LSL-eGFP cells. Cells were transduced with Cre proteins for 1 hour, washed and cultured for 2 days before analyses. (d-e) P21-mR-Cre-8R is efficiently transduced and recombines DNA. (d) Fluorescence microscopy images Cre-transduced NIH3t3 LSL-eGFP with the variety of dosages. Scale bar, 50 μm. (e) Flow cytometry analyses of NIH3t3 LSL-eGFP cells transduced for 1 hour with mR-Cre, mR-Cre-8R and P21-mR-Cre-8R at a variety of dosages (0, 1, 10, 100 and 500 μg/ml), washed and cultured for 2 days. Graph shows % recombination (i.e. % of eGFP+ve from total cell population). Error bars indicate s.d.

(6) FIG. 5 GET/HETD-mediated delivery of NEO promotes antibiotic-resistance in mouse embryonic fibroblasts. (a) Scheme of testing the antibiotic-resistance activity of transduced NEO (Neomycin Phosphotransferase) in mouse embryonic fibroblast (MEF) cells. MEF cells (300,000) were cultured in DMEM containing 10% (v/v) FCS and P21-mR-NEO-8R (0, 10 or 100 μg/ml) for 3 days feeding with fresh media daily. G-418 was then added to media for a further 3 days with daily feeding of fresh media. (b-e) P21-mR-NEO-8R rescues MEF and NIH3t3 cells from G-418 selection by conferring antibiotic-resistance. (b-c) P21-mR-NEO-8R maintains living cultures of MEFs under G-418 selection. (b) MEF cell numbers when supplemented with SIN NEO (to overexpress NEO) or transduced with P21-mR-NEO-8R and selected with G-418 (0, 75, 150 or 300 μg/ml). (c) MEF cell numbers for the 150 μg/ml dose of G-418. Error bars indicate s.d. (d-e) P21-mR-NEO-8R promotes viability in G-418 selected MEF and NIH3t3 cells. (d) Quantitation of the percentage of viable MEF cells using Trypan Blue staining. (e) Fluorescence microscopy of NIH3t3 cells selected with 150 μg/ml G-418 assessed for viability using LIVE/DEAD staining. Scale bar, 100 μm. Error bars indicate s.d.

(7) FIG. 6 GET/HETD-mediated delivery of NANOG promotes the Self-renewal of mouse Embryonic Stem cells. (a) Scheme of testing activity of transduced NANOG in CGR-8 cells. Cells were transduced with P21-mR-NANOG-8R proteins (0, 1, 10 and 50 μg/ml) for three consecutive days (1 passage, 1:3 split), passaged 1:3 and plated into growth media with P21-mR-NANOG-8R but lacking LIF (−LIF). Cells were fed daily with −LIF media containing P21-mR-NANOG-8R and passaged 1:3 every 3 days for 2 passages (a total of 3 passages −LIF) (b) P21-mR-NANOG-8R rescues self-renewal of mESCs lacking LIF dose dependently. Alkaline phosphatase (AP) staining of CGR-8 cells treated with P21-mR-NANOG-8R proteins and LIF withdrawal. AP activity and colony morphology is retained in CGR-8 cells cultured in LIF or without LIF but supplemented with SIN NANOG (to overexpress NANOG) or transduced with P21-mR-NANOG-8R. Scale bar, 100 m. (c) P21-mR-NANOG-8R maintains the proliferation of mESCs lacking LIF dose dependently. Percentage of the number of CGR-8 cells cultured without LIF verses those with LIF (% −LIF/+LIF) at passaging. In LIF-deficient CGR-8 cultures proliferation is promoted when supplemented with SIN NANOG (to overexpress NANOG) or transduced with P21-mR-NANOG-8R. Error bars indicate s.d. (d) NANOG-dependent rescue in LIF-deficient cultures generates a more epiblast-like gene expression profile. Relative gene expression analyses of LIF-deficient CGR-8 cultures using quantitative PCR (QPCR). Cultures supplemented with SIN NANOG (to overexpress NANOG) or transduced with P21-mR-NANOG-8R have increased Fgf5 expression, reduced Rex1 expression and retain Oct4 expression. Error bars indicate s.e.

(8) FIG. 7 GET/HETD-mediated delivery of MYOD promotes Myogenic differentiation of Human Embryonic Stem cells. (a) Scheme of testing the differentiation activity of transduced MYOD in HUES7 cells. HUES7 cells were plated onto gelatinised plastic and cultured in DMEM containing 10% (v/v) FCS. Cells were fed daily with DMEM containing 10% (v/v) FCS and P21-mR-MYOD-8R (0, 1, 5, 10 or 50 μg/ml) for 7 days. Media was then changed to DMEM containing 2% (v/v) Horse serum (HS), Human recombinant Insulin and P21-mR-MYOD-8R and fed daily for 3 days. (b-f) P21-mR-MYOD-8R drives myogenic differentiation of HUES7 cells to multinucleated Myotubes. (b) Light microscopy of HUES7 cells cultured under the myogenic regime supplemented with SIN MYOD (to overexpress MYOD) or transduced with P21-mR-MYOD-8R. Elongated fused Myotubes and single myocytes are generated with SIN-MYOD or high doses of P21-mR-MYOD-8R. Scale bar, 100 μm. (c) MYOD-dependent myogenic differentiation of human embryonic stem cells. Relative gene expression analyses of HUES7 cultures using quantitative PCR (QPCR). Cultures supplemented with SIN MYOD (to overexpress MYOD) or transduced with P21-mR-MYOD-8R have increased endogenous MYOD expression and skeletal muscle-specific ACTA1 expression. Error bars indicate s.e. (d-e) P21-mR-MYOD-8R differentiated cells are multinucleated. (d) Quantitation of mean nuclei number per cell using PI staining. Error bars indicate s.d. (e) Fluorescence microscopy images of HUES7 cells differentiated with P21-mR-MYOD-8R (50 μg/ml) and stained with nuclear dye DAPI. Scale bar, 50 μm. (f) P21-mR-MYOD-8R differentiated cells are MYOGENIN positive. Quantitation of the percentage MYOGENIN positive cells, using immunofluorescent-labelling. Error bars indicate s.d.

(9) FIG. 8 PTD-proteins bind to Pluripotent cells but transduce poorly. Fluorometry of media to determine the remaining fluorescent protein remaining after incubation with cells. mR-8R was diluted to 20 μg/ml in serum-free media and 1 ml/well incubated for either 1 hour (a) or 12 hours (b) with confluent NIH3t3, MEF, CGR-8, HUES7 or IPS2 cells in 6 well plates. Fluorescence pre-incubation was assigned as 100% of units and background of serum-free media subtracted. Error bars indicate s.d.

(10) FIG. 9 P21 binds directly to Heparin. Fluorometry to determine the fluorescent protein remaining after incubation with Heparin-sepharose beads. Recombinant proteins were diluted (20 μg/ml) in serum-free media and 1 ml/tube incubated for 1 hour at 37° C. with 50 μl Heparin-sepharose with rotation. Fluorescence pre-incubation was assigned as 100% of units and background of serum-free media subtracted. Error bars indicate s.d.

(11) FIG. 10 GET/HETD-mediated delivery of domain position protein variants. (a) Schematic of the proteins created to test the effect of domain position on protein delivery to cells. (b) Fusion of P21 and 8R to mR in any orientation significantly improves transduction. Flow cytometry analyses of NIH3t3 and HUES7 cells incubated with the protein variants (20 g/ml) for twelve hours. Error bars indicate s.d.

(12) FIG. 11 GET/HETD-mediated delivery of PTD protein variants. (a) Schematic of the proteins created to test the enhancing effect of P21 on other PTDs for protein delivery to cells. 8R is RRRRRRRR (SEQ ID NO: 19), TAT is HIV-1 TAT protein RKKRRQRRR (SEQ ID NO: 20), 8K is KKKKKKKK (SEQ ID NO: 56), 8RQ is RQRQRQRQ (SEQ ID NO: 57) (b) Fusion of P21 and any PTD to mR significantly improves transduction. Flow cytometry analyses of NIH3t3 and HUES7 cells incubated with the protein variants (20 μg/ml) for twelve hours. Error bars indicate s.d.

(13) FIG. 12 PTD-mediated delivery is inhibited by free Heparin and Chondroitin sulfate. Flow cytometry analyses NIH3t3 cells treated with mR-8R (20 μg/ml) for 6 hours with or without a variety of GAGs (50 μg/ml) in serum-free medium. CS is Chondroitin sulphate. Cells were pre-incubated for 1 hour in serum-free media and transduced for 6 hours in serum-free media with or without GAGs. Error bars indicate s.d.

(14) FIG. 13 GET/HETD-Protein delivery is inhibited by GAG-containing FCS, Heparinase treatment and competing soluble Heparin. (a-b) Flow cytometry of transduced cells pre-treated with Heparinase. (a) NIH3t3 cells were pre-incubated in serum-free media for 1 hour with different amounts of Heparinase III (0, 0.01, 0.1 or 1 U/ml) and transduced with mR or P21-mR-8R (20 μg/ml in serum-free media) containing Heparinase III for 12 hours. (b) NIH3t3 cells were pre-incubated in serum-free media for 1 hour with different amounts of Heparinase III and transduced with P21-mR-8R (20 μg/ml) containing Heparinase III and different amounts of FCS (0, 1 or 10% (v/v)) for 12 hours. (c) Flow cytometry of NIH3t3 cells transduced in different concentrations of FCS or FCS which has been depleted for P21-binding material. NIH3t3 cells were pre-incubated in serum-free media for 1 hour and transduced with P21-mR-8R (20 μg/ml) containing different amounts of FCS (0, 0.1, 0.5, 1, 2, 5, 10 or 20 (v/v)) for 6 hours. (d) Flow cytometry of NIH3t3 cells transduced in 10% FCS or 10% P21 binder-depleted FCS with the addition of soluble Heparin. NIH3t3 cells were pre-incubated in serum-free media for 1 hour and transduced with P21-mR-8R (20 g/ml) containing either type of FCS and different amounts of soluble Heparin (0, 0.1, 0.5, 1, 2, 5 or 50 μg/ml) for 6 hours. Error bars indicate s.d.

(15) FIG. 14 GET/HETD-mediated delivery is inhibited by HS-GAG synthesis inhibitor Sodium Chlorate. Flow cytometry analyses NIH3t3 cells treated with P21-mR-8R (20 μg/ml) for 6 hours with or without Sodium Chlorate (SC, 20 mM) in serum-free medium. Cells were pre-incubated with or without SC for 1 hour in serum-free media and transduced for 6 hours in serum-free media with or without SC. Error bars indicate s.d.

(16) FIG. 15 GET/HETDs can achieve higher intracellular levels of cargo delivery than transgenic systems. (a) Fluorometry of soluble extracts generated from NIH3t3 mR (transgenic NIH3t3 cells transduced with SIN mR) compared with those from NIH3t3 cells transduced for 6 hours with different doses of mR-8R or P21-mR-8R (0, 10, 20, 50, 100 or 200 μg/ml in serum-free media) (b) Flow cytometry of NIH3t3 mR (transgenic NIH3t3 cells transduced with SIN mR) compared with those from NIH3t3 cells transduced for 6 hours with different doses of mR-8R or P21-mR-8R (0, 10, 20, 50, 100 or 200 μg/ml in serum-free media). Fluorescence is normalised to untreated NIH3t3 cells. Error bars indicate s.d.

(17) FIG. 16 GET/HETD-tagged proteins are rapidly depleted from culture media by efficient transduction. (a-b) Fluorometry of media to determine the remaining fluorescent protein remaining after incubation with cells. Recombinant proteins were diluted to 20 μg/ml in serum-free media and 1 ml/well incubated for 12 hours with confluent NIH3t3s in 6 well plates. Fluorescence pre-incubation was assigned as 100% of units and background of serum-free media subtracted. (a) HUES7-mediated depletion of recombinant proteins from culture media in 12 hours. (b) NIH3t3-mediated depletion of recombinant proteins from culture media in 12 hours. (c) The Tl/2 depletion was calculated by incubating NIH3t3 cells with recombinant proteins (20 μg/ml) for different times. Bar chart shows the time (hours) required to deplete the recombinant protein to half the starting concentration in the described system. Error bars indicate s.d.

(18) FIG. 17 HERD-mediated Cre Recombinase nuclear activity is promoted by vesicle escape but repressed by inhibitors of macropinocytosis or cholesterol depletion. NIH3t3; LSL-eGFP cells were pre-incubated in serum-free media (with or without drugs), transduced with Cre proteins (mR-Cre: 100 g/ml or P21-mR-8R: 10 μg/ml) for 1 hour in serum-free media (with or without drugs), washed and cultured for 12 hours in full growth media (with or without drugs) and a further 36 hours in full growth media before analyses. (a) Methyl-β-cyclodextrin (used to deplete cholesterol) inhibits Cre transduction and recombination. (Methyl-β-cyclodextrin doses were 0, 1 2 and 5 mM). (b) Nystatin (a drug which sequesters cholesterol) inhibits Cre transduction and recombination. (Nystatin doses were 0, 10, 20 and 50 μg/ml). (c) Amiloride (a specific inhibitor of Na.sup.+/H.sup.+ exchanged required for macropinocytosis) inhibits Cre transduction and recombination. (Amiloride doses were 0, 1, 5 or 10 mM). (d) Cytochalasin D (an F-actin elongation inhibitor) inhibits Cre transduction and recombination. (Cytochalasin D doses were 0, 1, 5 or 10 μM). (e) Chloroquine promotes the release of Cre from endosomal vesicles and increases recombination (Chloroquine doses were 0, 10 and 100 μM). (f) Picogram per millilitre amounts is required to induce recombination with enhanced vesicle escape. The dose of transduced P21-mR-Cre-8R was varied in ten-fold dilutions (0-100 μg/ml) with 1 hour incubation in the presence of Chloroquine. All data is presented as % of the maximal recombination. Error bars indicate s.d.

(19) FIG. 18 GET/HETD-mediated transduction increases general cellular macropinocytosis. Flow cytometry of cells incubated in 70 kDa FITC-Dextran and transduced with recombinant proteins. NIH3t3 cells were pre-incubated in serum-free media for 1 hour and transduced with mR, P21-mR, mR-8R or P21-mR-8R (20 μg/ml in serum-free media) containing 70 kDa FITC-Dextran (neutral) for 1 hour. Error bars indicate s.d.

(20) FIG. 19 GET of non-protein Cargoes. (a) GET of biotinylated cargoes using monomeric streptavidin (mSA2). (i) Schematic of the mSA2 proteins engineered to bind to and transduce biotinylated cargoes. P21-8R was used as a non-interacting control, mSA2 as a non-transducing control, and P21-mSA2-8R as the test protein. (ii) Schematic of the antibody (Ab) complexes of a biotinylated primary (1°) antibody (Goat anti-rabbit; GtαRb) bound to an FITC-conjugated secondary (2°) antibody (Rabbit anti-mouse; Rb αMu) used to test activity. (iii) GET-delivery of Ab complexes were visible by fluorescence microscopy (scale bar, 50 μm). With co-incubation of P21-mSA2-8R (10 μg/ml, bottom image), Ab complexes were efficiency delivered to cells (iv) Flow cytometry demonstrating that 1′/2 Ab complexes (l g/ml) are taken into NIH3t3 cells poorly by direct incubation or when co-incubated with mSA2 only. (b) GET of nucleic acids by employing LK15 peptide. (i) Schematic of the LK15 proteins engineered to bind to and transduce nucleic acids. (ii) Transfection of human mesenchymal stem cells (iHMSCs) using GET-LK15. Initially we assessed binding capacity of LK15 peptides for plasmid (p)DNA (SIN GFP, to express GFP on transfection), modified synthetic messenger RNA (modRNA) (Miltenvi Biotech; to express GFP on transfection) and small-inhibitory (si)RNAs (labelled with FAM fluorophore to detect delivery). After optimising ratios we transfected iHMSCs with P21-LK15-8R and pDNA (10 μg), modRNA (10 μg) or siRNA (1 μg) and visualised transfection by fluorescence microscopy (scale bar, 100 μm). (iii) Quantification of GET-LK15 transfection of iHMSCs by flow cytometry (% transfection efficiency or relative fluorescence for siRNA) compared to lipofectamine (LIPO)2000 as a commercial standard. Error bars indicate s.d. (c) GET of Magnetic Nanoparticles. (i) Schematic of the P21-8R peptide synthesised and test magnetic nanoparticles (MNPs). We tested 250 nm Nanomag-D dextran shell/iron oxide core MNPs and conjugated P21-8R peptide to surface COOH groups. (ii) MNPs are taken into NIH3t3 cells most efficiently in serum-free media (SFM; left panel). Light microscopy images of Prussian blue iron stained NIH3t3 cells treated with MNPs (50 μg/ml) for twelve hours in standard media conditions (10% FCS) or SFM. Conjugation of P21-8R to MNPs significantly increases cellular uptake in both 10% FCS and SFM conditions (circular image is of entire well, scale bar, 100 m).

(21) FIG. 20 Ligand auto-labelling of intracellular and extracellular membrane-anchored HALO proteins. (a) Schematic of the HALO (intracellular) and LAMP2b-HALO (extracellular membrane-anchored) transgenic SIN lentivirus constructs. In LAMP2b-HALO the expressed protein is localised to the cell membrane by the signal peptide (SIG) which is cleaved and presented on the extracellular side of the cell membrane (b) NIH3t3 cells transgenic for intracellular HALO protein (NIH3t3-HALO) are only efficiently labelled by cell permeant ligands (HALO.sup.TAG Oregon Green). NIH3t3 cells transgenic for membrane-anchored extracellular HALO protein (NIH3t3-LAMP2b) is efficiently labelled by both cell permeant and cell impermeant ligands (HALO.sup.TAG Alexafluor.sup.488). Data shows flow cytometry of the NIH3t3 cell-lines incubated in ligand (1 μM) for 15 mins, followed by 3 media washes and a 15 mins incubation to remove unbound ligand. Error bars indicate s.d. This provides an assay to assess intra-verses extracellular localisation of HALO proteins.

(22) FIG. 21 Ligand labelling of GET-HALO proteins demonstrates rapid cell binding and transduction. (a) Schematic of HALO proteins created (as described for mRFP in FIG. 1). (b) P21-HALO-8R and P21-HALO efficiently bind NIH3t3 cells but do not significantly internalise with a 1 hour incubation. (c) Bound P21-HALO-8R efficiently transduces into NIH3t3 cells with further incubation (1 h-5 h). Bound P21-HALO does not as efficiently enter cells and remains bound to cell membrane with further incubation. Data shows flow cytometry analyses of NIH3t3 cells treated with proteins (20 μg/ml) for 1 hour followed direct ligand labelling (1 h) or further incubation of 5 hours (1 h-5 h). Error bars indicate s.d.

(23) FIG. 22 pH-sensitivity of GET-mNectarine (mNect) proteins demonstrates rapid cell binding and transduction. (a) Schematic of HALO proteins created (as described for mRFP in FIG. 1). (b-c) GET-mNect or GET-mR proteins (20 μg/ml) were transduced into NIH3t3 cells for 1 h (to demonstrate membrane binding activity), 1 h followed by a further 5 h incubation without protein (1 h-5 h) (to demonstrate transduction activity) or 6 h (to demonstrate sustained delivery). Flow cytometry was used to compare intensities of mNect and mR GET-proteins. Fluorescence signal from transducing mNect proteins (unlike mR versions) is rapidly lost after internalisation new to endosomal acidification and protein unfolding. Error bars indicate s.d. (d-f) GET-mNect proteins (20 μg/ml) were transduced into NIH3t3 cells for the same regimes but washed in DMEM at pH7.5 or pH5.5 before cytometry. Membrane localised mNect protein fluorescence is extinguished by pH5.5 but is retained at pH7.5 indicating at 1 h incubations leave P21-mNect-8R external to cells, bound to membranes and not protected from pH-mediated unfolding. 1 h-5 h incubations demonstrate that P21-mNect-8R localisation is shifted and protected from pH-mediated unfolding demonstrating internalisation of the protein and protection by the cell membrane. Error bars indicate s.d.

(24) FIG. 23 GET protein must be delivered intracellularly to allow successful re-transduction (a) Scheme of testing the effect of re-transduction of GET proteins in NIH3t3 cells. Cells were pre-incubated in fresh media for 1 hour and transduced with P21-mR-8R (20 μg/ml) for 1 hour. Cells were then either analysed for fluorescence or re-transduced with P21-mR-8R (20 μg/ml) for a further 1 hour. This re-transduction was either immediate (0 h) or with a 1-6 hour incubation between re-transduction before fluorescence analyses by flow cytometry. Immediate re-transduction is inhibited whereas >1 hour incubation between transductions allows the most efficient re-transduction of GET-protein (b). Error bars indicate s.d. * p<0.05.

(25) FIG. 24 GET compared to CPP-only transduction of NEO. (a) Schematic of the mR-NEO-8R (CPP-only protein) and P21-mR-NEO-8R (GET protein). (b) P21-mR-NEO-8R maintains living cultures of MEFs under G-418 selection whereas mR-NEO-8R has poor rescue activity. P21-mR-NEO-8R activity is comparable to cells genetically supplemented with SIN NEO (to overexpress NEO). Data shows MEF cell numbers for the 150 μg/ml dose of G-418. Error bars indicate s.d.

(26) FIG. 25 GET compared to CPP-only transduction of NANOG. (a) Schematic of the mR-NANOG-8R (CPP-only protein) and P21-mR-NANOG-8R (GET protein). (b) P21-mR-NANOG-8R maintains the proliferation of mESCs lacking LIF dose dependently whereas mR-NANOG-8R has poor self-renewal activity. Data shows the percentage of the number of CGR-8 cells cultured without LIF verses those with LIF (% −LIF/+LIF) at passaging. In LIF-deficient CGR-8 cultures proliferation is promoted when supplemented with SIN NANOG (to overexpress NANOG) or transduced with P21-mR-NANOG-8R but poorly by mR-NANOG-8R. Error bars indicate s.d.

(27) FIG. 26 GET compared to CPP-only transduction of MYOD. (a) Schematic of the mR-MYOD-8R (CPP-only protein) and P21-mR-MYOD-8R (GET protein). (b) P21-mR-MYOD-8R drives myogenic differentiation of HUES7 cells to MYOGENIN positive multinucleated Myotubes whereas mR-MYOD-8R has poor myogenic activity. P21-mR-MYOD-8R activity is comparable to cells genetically supplemented with SIN MYOD (to overexpress MYOD). Data shows the quantitation of the percentage MYOGENIN positive cells using immunolabelling. Error bars indicate s.d.

(28) FIG. 27 Delivery of a tissue-extracted pro-apoptotic protein by GET-mSA2. (a) Schematic of bovine heart cytochrome-C(Cyt-C) and biotinylated Cyt-C (BIO-Cyt-C) and Cyt-C complexes with mSA2. (b) Co-incubation of Cyt-C (as a non-interacting control) or BIO-Cyt-C (10 μg/ml) with GET-mSA2 proteins in the presence of Chloroquine (100 μM) to induce apoptosis of NIH3t3s. As assessed by trypan blue exclusion after a 12 hour incubation only the fully interacting and transducing complex (with Chloroquine) mediated loss of cell viability. Error bars indicate s.d. (c) BIO-Cyt-C transduced with P21-mSA2-8R in the presence of chloroquine caused complete loss of cell viability demonstrated by light microscopy. (scale bar, 50 μm).

(29) FIG. 28 GET of Antibodies using SpA B domain. (a) Schematic of the SpAB and P21-SpAB-8R proteins engineered to bind to and transduce IgG antibodies. We tested the delivery of an FITC-conjugated Rabbit anti-mouse IgG (Rb IgG-FITC). (b) IgG (1 μg/ml) was taken into NIH3t3 cells poorly by direct incubation or when co-incubated with SpAB (left panel). With co-incubation of P21-SpAB-8R (10 g/ml, right panel), IgG was efficiency delivered to cells visible by fluorescence microscopy (scale bar, 50 μm). (c) Flow cytometry of delivered cells confirmed an increase in IgG delivery of over 2-orders of magnitude. Error bars indicate s.d.

(30) FIG. 29 P21 and CPP addition to cargoes improves delivery by >two-orders of magnitude. Cell lines (described in FIG. 1) including rat aortic smooth muscle cells (rSMC) and neonatal cardiomyocytes (rCMs) were transduced with mRFP fused-variants of cargo proteins (mR, mR-Cre, mR-NEO, mR-NANOG or mR-MYOD) or with cargoes with N-terminally fused P21 and C-terminally fused 8R (CPP) sequences (GET proteins; P21-mR-8R, P21-mR-Cre-8R, P21-mR-NEO-8R, P21-mR-NANOG-8R or P21-mR-MYOD-8R). Transductions were at 20 μg/ml protein for 24 hours in the cell-line standard media. Data shows the fold increase of transduction with addition of GET sequences (P21 and 8R) to the cargoes as assessed by flow cytometry. Error bars indicate s.d.

(31) FIG. 30 High level or constant GET-transduction does not affect cell viability (a) Assessment of cell viability by trypan blue exclusion of NIH3t3s transduced for 12 hours with 0-200 μg/ml P21-mR-8R protein either directly after transduction (0 h; left) or 24 hours-post transduction (24 h; right). (b) Assessment of cell proliferation after transduction by passaging and cell counts of NIH3t3s transduced for 24 hours with 0-200 μg/ml P21-mR-8R protein. (c) Assessment of cell proliferation during constant transduction by passaging and cell counts of NIH3t3s transduced with 0-200 μg/ml P21-mR-8R protein (protein refreshed at each passage). Cells were passaged daily and re-plated at 100,000 cells per 12 well. Error bars indicate s.d.

(32) FIG. 31 GET is biocompatible in multiple clinically relevant cell types. Cell lines were transduced with P21-mR-8R at 20 or 200 μg/ml over 24 hours and assessed by trypan blue for cell viability (cell lines were those described in FIG. 1 including rat aortic smooth muscle cells (rSMC) and neonatal cardiomyocytes (rCMs)). Viability remained high in all cell types for both concentrations tested. Error bars indicate s.d.

(33) FIG. 32 A: Schematic diagram of a dextran coated nanoparticle, B: Prussian blue staining of 50 μg Nanomag-D nanoparticles labelled with P21mR and particles and protein added separately, C: Fluorescence of Nanomag-D particles labelled with red fluorescent protein, D: Fluorescence of pre and post labelling solutions.

(34) FIG. 33 A: Prussian blue staining of Nanomag particles incubated with 3t3 cells for 24 hours, B: Iron assay results for the amount of iron per cell after the 24 hour incubation.

(35) FIG. 34 A: Prussian blue staining of nanomag-D particles incubated in 3t3 cells for varying times, B: The iron assay quantification results.

(36) FIG. 35 A: 3t3 cells incubated with 50 μg nanoparticles, 1 μM of P218R and various quantities of heparin analysed by iron assay. B: incubated with dextran. C: incubated with varying amounts of serum. D Fluorescence of 3t3 cells after incubation with Fitc-BSA

(37) FIG. 36 GET-mediated enhanced delivery of mRFP to cells via P21 mRFP 8R peptide. Fluorescence microscopy images of NIH3T3 cells treated with FmRFP, P21 mRFP, mRFP 8R and P21 mRFP 8R peptides (20 g/ml) for twelve hours. The images also include cells that weren't treated with any peptides, as a control. Scale bar, 100 μm.

(38) FIG. 37 Graph showing NIH3T3, CGR-8 and HUES7 cells treated with mRFP conjugated peptides (20 ug/ml) for 12 h. Flow cytometry analysis was used to quantify fluorescence (relative fluorescence units (RFU)) of cells. Error bars indicate s.d, n=3.

(39) FIG. 38 Graph showing the increase in transduction of mRFP into cells by modified CPPs (HS-GAG binding domain mRFP 8R) over an unmodified CPP (mRFP 8R). NIH3T3, CGR-8 and HUES7 cells were treated with mRFP conjugated peptides (20 ug/ml) for 12 h. Error bars indicate s.d, n=3.

(40) FIG. 39 Efficient delivery of mRFP to cells via modified peptides shown to promote GET. Fluorescence microscopy images of NIH3T3, CGR8 and HUES-7 cells treated with P21 mRFP 8R, FGF2B mRFP 8R, FGF7B mRFP 8R and PDGF mRFP 8R peptides (20 μg/ml) for twelve hours. Scale bar, 100 μm.

(41) FIG. 40 Optimization of (+/−) charge ratio of P21 LKL5 8R to pSIN GFP using YO-PRO-1 assay. Graph shows a decrease in % of fluorescence as P21 LK15 8R binds pSIN GFP. Error bars indicate s.d., n=3.

(42) FIG. 41 Examples of flow cytometry dot plots showing GFP expression of NIH3T3 cells following transfection with pSIN GFP for 6 h. Following transfection, cells were fixed at a 48 h time-point. Flow cytometry analysis was used to quantify % of GFP positive cells.

(43) FIG. 42 Transfection optimization of pSIN GFP into NIH3T3 cells by P21 LK15 8R peptide. Cells were treated with the optimum (+/−) charge ratio of P21 LK15 8R to pSIN GFP of 2:1, respectively. Optimization was carried out at varying transfection times (3, 6 and 24 h) in (A) 10% serum transfection media (B) serum free transfection media. Following transfection, cells were fixed at a 48 h time-point. Flow cytometry analysis was used to quantify % of GFP positive cells. Error bars indicate s.d., n=3.

(44) FIG. 43 Graph showing the optimization of the transfection of pSIN GFP into NIH3T3 cells by Lipofectamine2000, in serum free conditions. Flow cytometry analysis was used to quantify % of GFP positive cells. Error bars indicate s.d., n=3.

INTRODUCTION

(45) Human pluripotent stem cells (HPSCs) comprise HESCs derived from the inner cell mass of the pre-implantation embryo, and HiPSCs generated by epigenetic reprogramming of somatic cells {Robinton, 2012 #30}. The ability to control the behaviour and differentiation of these cells efficiently and reproducibly underpins current efforts in regenerative and personalized medicine. A transcription-factor driven transgenic method has been previously described to directly programming gene-regulatory networks in HPSCs to drive cardiac differentiation and create contractile Cardiomyocytes {Dixon, #11}. Many transgene-driven methods to control cell behaviours such as genome reprogramming {Takahashi, 2007 #82}, self-renewal {Chambers, 2003 #70}, differentiation {Dixon, #11}, apoptosis {Mohan, 2013 #83}, proliferation {Zhao, 2013 #85} or migration {Deboux, 2013 #84} have been described. These all involve the integration of DNA to allow exogenous gene expression usually using viral vectors or transient DNA transfection which is inefficient and may also lead to genome modification, Approaches such as RNA transfection {Warren, 2010 #4} or PTD-mediated protein delivery {Zhou, 2009 #72} are therefore an attractive alternative with controlled stoichiometry and have no possibility of genome integration {Gump, 2007 #39}. It was an aim to develop PTD-technology to allow robust delivery of bioactive proteins into pluripotent stem cells which could replace present technologies and be used to improve the adoption of HPSCs for regenerative medicine applications.

(46) Results

(47) Isolation of P21, a HBD that enhances PTD function through HS-GAG interaction A focus was improving the initial PTD interaction and lipid-bilayer transduction of cargo proteins rather than on endosomal escape. Initially, monomeric red fluorescent protein (mRFP1) was employed as a self-reporting cargo which is readily expressed and purified in Escherichia coli (FIG. 1a). Investigation led to the determination that TAT and 8R PTDs interact with the cell membranes of mESCs, HESCs and HiPSCs (FIG. 8a) but have very poor transduction of cargo (˜1-3 fold over control) (FIGS. 1 & 8b). This was in comparison to extensively tested cell-lines (such as NIH3t3, C2C12 and mouse embryonic fibroblasts) which showed efficient delivery (˜5-50-fold over control, p<0.01) (FIG. 1). Previous reports have demonstrated the functional delivery of PTD-tagged proteins in pluripotent stem cells with different degrees of robustness {Do Kwon, 2005 #74; Liang, 2013 #73}. The success achieved in these studies was hypothesized to be due to very minimal amounts of efficacious protein required, protein delivery into the differentiated derivatives rather than the starting population, or the use of vast amounts of protein to elicit low-level functionality.

(48) It was aimed to improve binding and ultimately transduction of cargo proteins into these target cell-types. Several short peptides were screened which have been reported to interact with molecules known to be present on mESC, HESC or HiPSC membranes including integrins, CD markers and GAGs. Peptides were fused N-terminally to mRFP1, expressed, affinity purified and incubated with the three cell-types. Screening of 12 variants yielded one which clearly increased localisation of mRFP1-fluorescence (mR) to cells and their membranes, termed P21 (KRKKKGKGLGKKRDPCLRKYK (SEQ ID NO: 1)) (FIG. 2). Interestingly this peptide also demonstrated transduction activity as evidenced by punctate intracellular fluorescence indicative of endosomal localisation (FIG. 2b,c). P21 was derived from HB-EGF, which belongs to the EGF family of cytokines. HB-EGF shows a strong affinity to heparin and binds to the same receptor as EGF and TGF-α {Sakuma, 1997 #76}. The interaction of HB-EGF with cell surface HS-GAG is essential for its optimal binding to EGFR and for promoting its growth/migratory activity toward vascular smooth muscle cells {Higashiyama, 1993 #90}. Mutagenesis and protease digestion of recombinant HB-EGF, coupled with analyses using synthetic peptides and heparin, revealed the P21 sequence in the amino-terminal region of the soluble HB-EGF is responsible for its binding to heparin so is considered a typical HBD {Thompson, 1994 #77}. In addition, P21 also interacts with cell surface HS-GAG but not the EGFR which is mediated by other sequences in HB-EGF. Therefore, a short 21-residue peptide, P21, was isolated, which enhances the association of a fluorescent reporter to both mouse and human pluripotent stem cells.

(49) To confirm direct binding of P21 to Heparin a binding assay was developed using Heparin-sepharose beads (FIG. 9). P21-tagged mR was efficiently sequestered by Heparin-sepharose unlike untagged mR (96.2±5.3%; p<0.01). This could be reversed by co-incubating with free Heparin in a dose dependent manner. The fact an HBD was isolated is interesting; HS-GAGs have long been considered important in the process of PTD-mediated transduction. However the exact role of HS-GAG in transduction is presently a point of contention with the most recent hypothesis being that HS-GAG is merely important in transduction by directly binding to PTDs and concentrating them on the membrane; this enhances lipid-bilayer translocation but is not necessary for its occurrence {Gump, 2010 #2}.

(50) Negatively charged sulphated proteoglycans and glycoproteins are present on all mammalian cells. However they are differently modified and specific variants are ubiquitous or present on very specific cell types {Lindahl, 1998 #56}. GPI-anchored proteoglycans and glycoproteins are present in lipid rafts suggesting that PTDs may have increased avidity for certain proteoglycans or perhaps that PTDs bind directly to cholesterol membrane constituents which trigger macropinocytosis. It is also possible that the P21-peptide may have specific avidity to certain HS-GAG forms and could recognise the same motifs at PTDs.

(51) P21-mediated binding of cell membranes was tested to determine if it could enhance PTD-mediated transduction of mR by combining both moieties in one molecule (FIG. 2a). P21-mR-8R was cloned, expressed and purified, and its activity compared to P21-only (termed P21-mR) or 8R-only (mR-8R) proteins (FIG. 2a). Data demonstrated that the inclusion of both P21- and 8R-moieties synergized in the same protein to significantly enhance the fluorescence of all cell-lines tested. (FIG. 2b). Importantly mouse and human pluripotent stem cells (CGR-8, HUES7 and IPS2) and cardiomyocytes (HL1) only transduced efficiently with the inclusion of P21-tag, and both tags synergized to produce the high-levels of transduction similar to that seen in other cell lines (FIG. 2c). These motifs were also placed in tandem at N- and C-terminal of mRFP (P21 or 8R first; P21-8R-mR, 8R-P21-mR or mR-P21-8R, mR-8R-P21) or switched their termini (i.e. 8R-mR-P21) with all variants demonstrating similar synergy and cell transducing behaviour (FIG. 10) as seen for P21-mR-8R. Furthermore 8R was swapped for alternative well characterised PTDs (TAT, 8K and 8RQ; {El-Andaloussi, 2005 #36}) and showed that these also synergized with P21 (FIG. 11).

(52) By incubating cells with protein for different timings and including a post-culture period it could be efficiently distinguished between fluorescence signal produced at the cell surface with that internalised (FIG. 2d). Cells demonstrated membrane localisation of fluorescence with short incubation times (1 hour), termed 1 h. With this short incubation and a subsequent culture period (1 hour with a 5 hour post-culture), termed 1 h-5 h we observed near exclusive intracellular fluorescence indicating transduction. Using longer incubation times (6 hours), termed 6 h, cells exhibited strong punctate peri-nuclear fluorescence indicative of endosomal-mediated transduction. This synergistic delivery mechanism is described as GAG-binding enhanced transduction (GET) or otherwise Heparan-sulfate enhanced transduction domain (HETD)-mediated delivery.

(53) GET Requires the Presence of Trypsin-Sensitive and Detergent-Soluble Cell Membrane Molecules

(54) To evaluate the mechanism of GET interaction and uptake by cells, a series of experiments were performed which were previously used to assess PTD. To assess which cell membrane components are required for both initial cell association and transduction by HETDs it was determined whether similar transduction would be obtained by enzymatic depletion of cell membrane before transduction. Cells were pre-treated with proteolytic enzyme trypsin and tested cell transduction using the 1 h-5 h regime protocol. Enzymatic removal of cell-surface proteins potently inhibited GET/HETD-mediated transduction (˜8.4-fold; p<0.05) (FIG. 2e). In contrast, non-enzymatic release of cells from the culture plastic using ionic cell dissociation solution (CDS) did not alter HETD-mediated uptake.

(55) Next it was tested if depletion of detergent-soluble cell-membrane molecules would also have a similar effect on transduction. Cells were pre-incubated in 0.1% (v/v) Triton X-100 and using the 1 h-5 h protocol a decrease (˜2.2-fold; p<0.05) was observed in GET (FIG. 2f) without a decrease in viability. Therefore, it was demonstrated that both protein and detergent soluble moieties on the cell membrane affect the efficacy of protein transduction through P21- and PTD-synergy in GET/HETD-transduction.

(56) GET Requires the Presence of Cell Membrane HS-GAG

(57) P21 is a HBD, and PTDs due to their cationic nature bind to negatively charged HS-GAGs. It was further aimed to confirm the mechanism of GET/HETD-mediated transduction by either enzymatic depletion of HS-GAG or competition with free GAGs (FIG. 3). Heparin prevented the cell-surface binding of HETD proteins and strongly inhibited transduction (99.8±2.1%; p<0.001) (FIG. 3a, b). Chondroitin sulphate (CS) A, B or C had little effect on either activity (FIG. 3b). CS-B and -C has previously been shown to affect the binding of PTD-proteins {Wadia, 2004 #25}. It was confirmed that Heparin, CS-B and -C have significant activities in inhibiting PTD-only protein transduction (8.1-, 2.4- and 4.1-fold, respectively; p<0.05) but that only Heparin affected GET/transduction mediated by HETDs (FIG. 3b and FIG. 12). P21-tagged proteins exhibit a dose-dependent inhibition of transduction with increasing concentrations of Heparin in NIH3t3 and CGR-8 cells (FIG. 3c & d, respectively). 8R-only protein is inhibited only with the highest doses of competing Heparin. In the context of the HETD protein (P21-mR-8R) it is therefore likely that the avidity of P21 for cell membrane HS-GAGs far outweighs that of CS-GAGs for PTD binding. Therefore, it can be concluded that Heparin and HIS-GAGs are the major target of P21 to enhance PTD activity.

(58) To further understand the requirement of cell-membrane HS-GAG for P21-activity cells were treated with HS-lyase enzyme, heparinase III, and tested cell transduction using the 6 h protocol (FIG. 13). Enzymatic removal of cell-surface HS-GAGs potently inhibited GET/HETD-mediated transduction (at 1 U/ml 97.2±3.4%; p<0.001). In contrast, neuraminidase treatment which depletes sialic acid GAGs (SA-GAGs) did not alter GET/HETD-mediated uptake. The analysis was further extended to define the effect of serum on transduction (FIG. 13b). It has previously been shown that serum has a negative effect on PTD-mediated delivery {Kaplan, 2005 #43}. By incubating serum with affinity purified-P21 peptide on sepharose its inhibitory effect on HETD-mediated recombination was capable of being removed (10% v/v, ˜7.3-fold increase with depletion; p<0.01) (FIG. 13c). This indicates that serum is likely to contain GAGs which act as competitive molecules for P21-membrane binding. These can be removed by pre-depletion using P21-bound sepharose. This inhibitory activity could be reconstituted by addition of soluble Heparin to depleted-serum further strengthening this hypothesis (FIG. 13d). In addition to enzymatic depletion, the HS-GAG requirement was confirmed by treating cells with Sodium Chlorate, a potent inhibitor of HS synthesis which also potently prevented HETD-mediated protein delivery (˜7.6-fold; p<0.05) (FIG. 14). Therefore, it was demonstrated that HS-GAG depletion specifically affects the efficacy of protein transduction and therefore endogenous HS-GAGs are important molecules for GET/HETD-mediated protein delivery. Also that exogenous HS-GAG are highly inhibitory to this process.

(59) HS-GAGs has a complex sugar structure, consisting of a backbone of repeating disaccharides of glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc), polymerized by a heteromeric complex of EXT1/EXT2 enzymes {Lawrence, 2008 #51}. To further investigate the role of HS-GAGs on GET/HETD-mediated transduction EXT1−/− mESCs were used, which lack endogenously synthesized cell membrane HS-GAG {Lin, 2000 #78}. When cultured in conventional mESC media (containing 20% w/v FCS) a significantly lower transduction in EXT1−/− mESC versus wild-type CGR-8 mESCs was observed (˜6.8-fold; p<0.01) (FIG. 3e,f). This was also apparent when cells were cultured in serum-free conditions likely to be due to interference from free GAGs in FCS (˜5.6-fold; p<0.01). Transduction/GET was not absent in EXT1−/− cells (˜9.7-fold over mR control levels) nor was the initially binding of P21-containing proteins. It was hypothesized that this may be due to incorporation of exogenous soluble serum-GAGs into the deficient EXT1−/− cell membrane. This is likely as subsequence Heparinase III treatment further inhibits binding and transduction in these cells.

(60) GET Generates Higher Intracellular Protein Levels than Lentiviral Transgenesis

(61) Several studies have concluded that PTD-mediated transduction is sufficiently refined to allow the transport of biologically active cargos for clinical studies. These now include trials of cancer therapies {Gump, 2007 #39}, siRNAs {Meade, 2007 #37} and in vivo imaging technologies {Bullok, 2006 #79}. As well as the benefits of avoiding genomic modification, if PTD-mediated transduction is to be preferential to gene-therapy approaches it must achieve the delivery of high-levels of molecule, be amenable to control of protein levels over short time-frames and also allow cell-type specific delivery. The levels achieved in cells by PTD- or GET/HETD-delivery were compared to those achieved by efficient lentiviral transduction {Dick, 2011 #10} and exogenous expression of mRFP1 (with stable EF1α-promoter driven) (FIG. 15). To achieve this soluble protein was extracted from transduced cells and measured amounts by fluorometry (FIG. 15a), or flow cytometry was used (FIG. 15b). Using 6 hour incubations, mR-8R levels were several times lower (˜3-fold; p<0.05) than that achieved by viral transgenesis even at the highest tested doses (200 g/ml). However P21-mR-8R levels under the same conditions were ˜16-fold higher (p<0.001) than transgenic cells. Importantly of the amount of P21-mR-8R protein incubated a significant proportion was recovered as soluble intracellular protein (˜46±3.5 μg/200 μg used; ˜2311.7% recovery). It is important to note that under these conditions transduced cells appear red/purple in colour under normal light, demonstrating the efficient enrichment of large quantities of HETD-tagged/GET proteins in cells.

(62) The rate at which these proteins were concentrated in cells was investigated by measuring the depletion of fluorescence in media over the incubation period (FIG. 16). Proteins were diluted (20 μg/ml) and incubated with cells for 12 hours in serum-free conditions. 8R-tagged proteins were depleted by ˜12% in NIH3t3- and ˜3.5% in HUES7-incubations. This precisely mirrors flow cytometric data with 8R-proteins poorly transduced into HUES7 cells but at moderate levels in NIH3t3 cells (FIG. 2). P21-tagged proteins were significantly depleted in both cell types (˜37% and ˜25% in NIH3t3 and HUES7 cells, respectively; p<0.05) with HETD-proteins depleted from media to the highest levels and majority of protein removed (˜72 and ˜66% in NIH3t3 and HUES7 cells, respectively; p<0.01).

(63) The time required to deplete half of the fluorescence (T½) was determined, with P21-mR-8R requiring only ˜9.4 hours, in comparison to mR-8R which required −62 hours and untagged protein never achieving half-depletion even after 7 days (FIG. 16). These data are corroborative with the cytometric data proving a rapid and efficient enrichment of exogenous GET-protein/HETD-protein into cells. Therefore, it was demonstrated that within a relatively short incubation period (6 hours) that a significant protein concentration can be achieved within cells. In less than a day, the majority of extracellular protein has been effectively internalised using GET delivery. This system will be amenable to precise regulation of protein stoichiometry, while avoiding the stochastic transgene expression variation and silencing of integrating vectors used in gene-therapy approaches.

(64) GET Enhances Cre-Mediated Genome Modification

(65) It was determined that HETDs bind rapidly to cell membranes through HIS-GAGs and transduce efficiently into cells, but it was yet to be confirmed if the mode of uptake was through macropinocytosis as for PTDs. Also, what proportion of this protein escaped endosomes and may be considered successfully delivered was not assessed. Previous studies have the avoided issues associated with direct measurement of fluorescent-tagged proteins (such as being unable to distinguish membrane, vesicle or functional cytosolic/nuclear protein) by assaying for the successful nuclear activity of Cre recombinase {Gump, 2010 #2}. This system was used to measure Cre-mediated recombination of a loxP-STOP-loxP (LSL) enhanced green fluorescent protein (eGFP) reporter gene in live NIH3t3 mouse fibroblast cells (NIH3t3: LSL-eGFP cells) as an indicator of cellular uptake (FIG. 4a). This system is rigorous as activation of green fluorescence requires exogenous Cre protein to enter the cell, undergo nuclear-translocation and excise the LSL fragment of the transgene. This must occur in live cells and be non-toxic for the subsequent expression of eGFP. However, this process requires only one functional Cre recombinase molecule to be delivered to activate eGFP so does not allow the determination of the precise amount of cargo delivered. To overcome this issue Cre proteins were delivered at limiting dilutions for a short exposure time (1 hour) and the minimum dose required to activate green fluorescence after 48 hours (FIG. 4c) was determined.

(66) Transduction of NIH3t3: LSL-eGFP cells with SIN Cre lentiviruses to overexpress Cre transgenically led to near complete (92±6%; p<0.001) activation of eGFP-expression in all cells confirming the utility of this system (FIG. 4b). The benefits of the fluorescence system and delivered fluorescent-versions of Cre-recombinase protein were retained by purifying proteins with mRFP1 cloned to the N-terminal of Cre cDNA. Treatment of NIH3t3: LSL-eGFP cells with mR-Cre (mRFP fused to Cre) resulted in recombination and eGFP activation (22.1±6.7%; p<0.05) at the highest doses (500 g/ml) (FIG. 4d). eGFP activation was inhibited at 4′C and negatively affected by serum concentration-dependently. mR-Cre-8R demonstrated that the 8R PTD enhanced functional delivery of Cre (˜22-fold; p<0.01).

(67) The GET-protein/HETD-protein, P21-mR-Cre-8R, required as little as one minute incubation with cells at a low dose (1 μg/ml) to elicit recombination (4.3±2.5%; p<0.05) confirming that binding and internalization is an efficient and rapid process. For a moderate dose (10 μg/ml) GET/HETD-transduction achieved a functional delivery ˜15-fold (p<0.01) above PTD only levels and completely recombined all NIH3t3: LSL-eGFP cells (FIG. 4d,e). Importantly this activity was ˜340-fold better than mR-Cre (p<0.001). Heparinase III, free-heparin and serum-free experiments were repeated using the Cre recombination system. It was confirmed that heparinase III pre-treatment reduced recombination to basal-levels and that media serum plays a role in replenishing cell membrane GAGs depleted by heparinase. Overall these data correlate well with the fluorescence delivery conclusions and show synergy between P21- and PTD-moieties to achieve significant increases in functional transduction of protein cargo.

(68) GET Protein Enters Cells by Lipid Raft Macropinocytosis

(69) Previously it has been shown that PTD-mediated internalization is via macropinocytosis rather than other endocytotic pathways {Wadia, 2004 #25}. It was next determined whether the cellular uptake of GET-proteins/HETD-proteins occurs through a specific endocytotic pathway employing the Cre assay system. Removal of cholesterol from the cell plasma membrane disrupts several lipid raft-mediated endocytotic pathways, including caveolae and macropinocytosis {Anderson, 1998 #29; Nichols, 2001 #30; Liu, 2002 #28}. NIH3t3: LSL-eGFP cells treated with methyl-p-cyclodextrin and nystatin were used to deplete or sequester cholesterol, respectively, then transduced HETD-tagged proteins. Both methyl-b-cyclodextrin (FIG. 17a) and nystatin (FIG. 17b) disruption of lipid rafts resulted in a dose-dependent inhibition of functional delivery. These data demonstrates that GET/HETD-mediated transduction specifically requires lipid raft-mediated endocytosis.

(70) Macropinocytosis is a rapid, lipid raft-dependent and receptor-independent form of endocytosis which requires actin membrane protrusions that envelope into vesicles termed macropinosomes {Nichols, 2001 #30; Liu, 2002 #28; Conner, 2003 #22}. To confirm macropinocytosis was indeed the endocytotic mechanism of HETD-mediated transduction cells were pre-treated with macropinocytosis-inhibiting compounds (FIG. 11a,b). Amiloride is a specific inhibitor of the Na.sup.+/H.sup.+ exchange required for macropinocytosis {West, 1989 #31}. Cytochalasin D is an inhibitor of F-actin elongation which is required for macropinosome-linked membrane protrusions {Sampath, 1991 #32}. Amiloride and Cytochalasin D did not disrupt cell binding of HETD-proteins but resulted in a dose-dependent reduction of functional transduction into cells (FIGS. 17c and 17d, respectively). These data confirm that P21 enhances the macropinocytotic pathway used by PTD to internalize cargo molecules.

(71) GET-Delivery Promotes General Macropinocytosis

(72) The effects of GET-binding/HETD-binding on the induction of macropinocytosis was investigated. PTD-mediated transduction has previously been shown to promote the uptake of other proteins by an increase in the overall level of macropinocytosis {Wadia, 2004 #25}. Cells were incubated with a fluorescent fluid-phase macropinocytotic maker, FITC-labeled 70 kDa neutral dextran, in combination with GET/HETD protein, P21-mR-8R (FIG. 18). Other studies {Oliver, 1984 #24; Araki, 1996 #23; Wadia, 2004 #25} have demonstrated that neutral dextrans are taken up by amiloride-sensitive macropinocytosis. P21-mR-8R induced a significant dose dependant increase in fluid-phase dextran uptake over steady-state control levels. PTD-tagged versus GET/HETD-tagged activity to stimulate this macropinocytosis was compared. P21-mR-8R enhanced FITC-dextran uptake ˜2.5-fold (p<0.05) over the stimulation achieved by the same concentration of mR-8R demonstrating that engagement with HS-GAG through P21 and its subsequent effect on PTD-mediated transduction stimulates macropinocytotic uptake.

(73) Significant Amounts of GET-Delivered Protein is Trapped in Endosomes which can be Efficiently Released with Chloroquine

(74) The majority of PTD-delivered molecules remain trapped in macropinosomes even after further incubation indicating that release from these vesicles is inefficient. If fine-tuned and graded amounts of delivery are to be controlled then it would be beneficial if the majority of internalized protein could be considered as functional. Cells were treated with chloroquine, an ion-transporting ATPase inhibitor that disrupts endosomes by preventing their acidification {Seglen, 1979 #33} (FIG. 17e). Similar doses have been demonstrated to significantly improve the functional delivery of PTD-delivered proteins {Wadia, 2004 #25}. Sub-cytotoxic doses of Chloroquine (100 μM) resulted in a marked increase (95.3±4.8-fold; p<0.001) in functional HETD-tagged/GET protein delivery at a sub-threshold dose (0.1 μg/ml) indicating that this is point in the pathway is still a major issue to resolve for GET/HETD medicinal application. Nevertheless the GET-delivery/HETD-delivery system was so efficient that with chloroquine treatment we achieved significant and measureable levels of recombination (4.8±2.9%; p<0.05) with short (1 hour) incubations of >10 μg/ml (FIG. 17f). Combination of GET/HETD-delivery efficiency with endosomal-escape technologies may therefore allow precise and temporally controlled amounts of cargo function in cells.

(75) GET-Mediated Internalisation is Efficient after Cell Membrane Association

(76) Even for incubations using low amounts of GET-protein functional quantities of protein activity were observed within cells. However, to categorically and stringently prove that most GET protein was indeed efficiently internalising a series of analyses was conducted using reporters that are responsive to their cellular or extracellular localisation. HALO (Halo.sup.Tag) was used, which is a self-labelling protein derived from DhaA.sup.29. HALO rapidly forms a covalent attachment to synthetic chloroalkane-based ligands; with cell permeant and impermeant ligands available. Intra-versus extracellular labelling of HALO was confirmed using transgenic over-expression of untagged HALO (for intracellular) and LAMP2b-HALO which is presented on the external cell membrane (for extracellular) and labeling with cell permeant (HALO.sup.TAG Oregon Green) or impermeant (HALO.sup.TAG Alexafluor.sup.488) ligands (FIG. 20). GET-HALO proteins were constructed and recombinantly expressed (FIG. 21a) and delivered them to cells testing the the internalisation by sensitivity to labelling with the cell impermeant ligand. One hour incubation demonstrated that GET-proteins remained mainly extracellularly localised and attached to the cell membrane (FIG. 21b). However, with further incubation (1 h exposure with 5 h further incubation: 1 h-5 h) GET-protein (P21-HALO-8R) is effectively internalised (remaining cell permeant ligand-sensitive but impermeant ligand-insensitive) (FIG. 21c). These experiments were repeated using the mNectarine (mNect) variant of RFP which is pH-sensitive and loses almost all fluorescence in environments <pH6.sup.30 (FIG. 22). In agreement with HALO transduction, mNect remained mostly membrane localised after 1 h and its fluorescence sensitive to acidic pH media incubation (pH5.5) (FIG. 22 d). After further incubation post-delivery (1 h-5 h) GET-mNect fluorescence was no longer sensitive to extracellular pH (FIG. 22c); however interestingly the absolute fluorescence levels were significantly decreased when compared to GET-mR, presumably due to the internal pH change the protein is experiencing during endosomal acidification.sup.1. These data demonstrates that GET protein membranes association is rapid and transduction is efficient post-cell binding. It was hypothesised that membrane clearance of GET-protein could be a rate-limiting step in the delivery process and this was tested by undertaking multiple transductions (1 h) of GET-mR varying the time between transductions (FIG. 23a). Indeed re-transduction directly after the initial transduction decreases the effectiveness of the second transduction however as little as I h between new transductions is required to obtain a maximal efficiency of re-transduction (FIG. 23b).

(77) GET-Delivery Promotes Survival by NEO-Conferred Resistance of Antibiotic Selection

(78) The GET/HETD system appeared to be able to deliver significant amounts of molecule several orders of magnitude better than untagged protein. Next it was sought to test if the system could deliver prolonged protein activity and a system was devised to attempt to provide resistance to cells under antibiotic selection (FIG. 5). This is believed to be a more reflective assay of the sustained delivery and function of GET-cargos/HETD-cargos that would be required for clinical translation. Unlike the Cre-based which requires a single enzyme to be delivered once into cells to be reported by eGFP as ‘success’, the NEO-based assay requires sustained delivery over several days and significant quantities to negate the activity of the antibiotic. G-418 sulfate (Geneticin), an aminoglycoside antibiotic, which blocks polypeptide synthesis by inhibiting chain elongation {Eustice, 1984 #69}.

(79) To test this, both MEF and NIH3t3 cells were subjected to a selection protocol in which cells were plated and subjected to three consecutive days of transduction with a GET/HETD NEO-cargo, P21-mR-NEO-8R. Cells were then re-plated and simultaneously selected with G-418 and ongoing GET/HETD NEO transduction for an additional three days (FIG. 5a). After selection these cultures were assessed for cell numbers remaining, viability using trypan blue exclusion, and stained using calcein AM/ethidium homodimer-1 to label LIVE/DEAD cells, respectively. These were compared to cells processed under the same regime but transduced with a NEO-expressing lentivirus, SIN NEO, representing the conventional transgenic approach to confer G-418 resistance.

(80) These data revealed a dose-dependent survival of selected cells with transduced P21-mR-NEO-8R (FIG. 5b). This survival was comparable to that conferred transgenically by SIN NEO integration (˜3.6-fold better survival; p<0.05) when P21-mR-NEO-8R was delivered at high doses with lower G-418 doses (˜3.3-fold better survival; p<0.05) (FIG. 5c-c). The highest-doses of G-418 could only be negated by SIN NEO transgenesis. Taken together, these experiments provide proof of principle that GET-delivered/HETD-delivered protein can provide sustained delivery of significant amounts of molecule and can promote the survival of mammalian cells under stringent antibiotic-selection.

(81) GET-Delivery of NANOG Promotes Self-Renewal of Pluripotency

(82) If this technology is to be adopted for clinical applications then it is important to demonstrate its use to alter cell-fate as well as control cell metabolism. A potentially important application of this technology would be in the driving of reprogramming, self-renewal and differentiation of stem cells. iPSC technology has been swiftly developed to allow genome non-integrating DNA {Yu, 2011 #91}, RNA {Warren, 2010 #4} and protein {Kim, 2009 #71; Zhou, 2009 #72} based technologies to supersede the original retroviral protocols {Takahashi, 2007 #82}. As PTD-delivery mediated reprogramming has already been demonstrated we sought to demonstrate that HETD-delivery was translatable to promote pluripotent cell fate {Kim, 2009 #71; Zhou, 2009 #72} (FIG. 6).

(83) To test this hypothesis, CGR-8 mESCs were employed to determine if GET/HETD-mediated delivery can sustain their pluripotent self-renewing phenotype with the withdrawal of Leukemia inhibitory factor (LIF). GET/HETD NANOG-cargo were delivered in an assay {Dixon, 2010 #16} similar to that used to initially isolate the role Nanog in mESCs {Chambers, 2003 #70}. CGR-8 cells were plated onto gelatinized plastic and subjected to three consecutive days of transduction with a GET/HETD NANOG-cargo. P21-mR-NANOG-8R, passaged and an additional three passages (three days per passage) continuing daily transduction in conditions lacking LIF (FIG. 6a). These cultures were assessed at each passage for cell number and after the third LIF-deficient passage, were assessed for morphological change, alkaline phosphatase (AP) activity and by QPCR for pluripotency- and differentiation-associated gene expression (Oct4, Rex1 and Fgf3, respectively). These were compared to cells transduced with NANOG-expressing lentivirus, SIN NANOG, which we have previously shown to rescue the self-renewal of CGR-8 cells in the same assay {Dixon, 2010 #16}.

(84) These data revealed that P21-mR-NANOG-8R rescued AP activity in significant numbers of CGR-8 even with relatively low doses (5-10 μg/ml) (FIG. 6b). AP activity in high-dose P21-mR-NANOG-8R samples was similar to that achieved by the SIN NANOG transgenic. Transgenics and high-dose P21-mR-NANOG-8R-transduced CGR-8 cells proliferated to a similar level in LIF-deficient cultures (˜87.6-fold more; p<0.001) (FIG. 6c) and also retained Oct4 expression to a similar level (albeit lower than LIF-containing cultures), indicative of retention of pluripotency (both p<0.05) (FIG. 6d). As observed previously, rescued cells by both DNA and protein methods up-regulated Igf5 and down-regulated Rex1 expression, which indicates an ICM-to-epiblast transition phenotype {Dixon, 2010 #16}. These experiments provide a demonstration that GET/HETD-delivered protein can prevent differentiation and retain the pluripotent phenotype which may be later applied to iPSC technology.

(85) GET-Delivery of MYOD Drives Myogenesis

(86) To realize the promise of iPSC technology for regenerative medicine or disease modeling, it is imperative that the multi-lineage differentiation potential of pluripotent cells is harnessed {Robinton, 2012 #30}. Although progress has been made in directing the differentiation of these cells to various lineages by modulating the extracellular cytokine milieu, such protocols remain relatively inefficient. Given the high efficiency of functional PTD-mediated cargo delivery by P21-enhancement, it was reasoned that GET/HETD technology might also be utilized to redirect pluripotent cells toward differentiated cell-fates beyond that already described for transduction of transcription-factors {Do Kwon, 2005 #74; Hidema, 2012 #75; Liang, 2013 #73}.

(87) For this the delivery of the efficacious MYOD myogenic factor {Bichsel, 2013 #92} was used to drive skeletal muscle specification (FIG. 7). To test this hypothesis, an in vitro differentiation protocol was devised in which HUES7 cells were plated onto gelatin and conventional media was withdrawn. Under these conditions, cells were then subjected to seven consecutive days of transduction with a GET/HETD MYOD-cargo, P21-mR-MYOD-8R, followed by an additional three days of culture in conditions with low concentrations of horse serum (FIG. 7a). These cultures were assessed for morphological change, immunostained for the myogenic marker MYOGENIN, assessed for myogenic-linked multinucleation by DAPI staining and by QPCR for myogenic gene expression (endogenous MYOD and ACTA1). These were compared to cells processed under the same regime but transduced with MYOD-expressing lentivirus, SIN MYOD.

(88) These data revealed a high percentage of large multinucleated MYOGENIN-positive myotubes (62.1±8.9%; p<0.01) (FIG. 7b-f) which had elevated MYOD and ACTA1 expression (p<0.01 and <0.05, respectively) (FIG. 7c). This was comparable to the differentiation observed in the SIN MYOD transgenic when P21-mR-MYOD-8R was delivered at high doses. Taken together, these experiments provide proof of principle that GET/HETD-delivered protein can direct the fate of HUES7 cells to a terminally differentiated somatic cell type.

(89) Discussion

(90) By combining a protein-transduction domain (PTD) with a cell membrane binding peptide, such as a HS-GAG binding (HBD)-peptide, to improve cell membrane targeting, a technology has been developed that enables highly efficient delivery of functionally relevant proteins to direct a variety of cell behaviours, even in hard to transduce cell-types. It was demonstrated that the GET/HETD system can be harnessed to promote survival, self-renewal or direct the differentiation of pluripotent cells toward a desired lineage. This system is not technically complex, as for modified RNA systems {Warren, 2010 #4}, and offers several key advantages over established techniques to deliver the exogenous function of a gene or protein. Furthermore by obviating the stringent biological containment required for viral gene-therapy approaches, GET/HETD-driven protein transduction technology should make such approaches more accessible.

(91) More fundamentally, because the technology is protein based, it completely eliminates the risk of genomic integration and insertional mutagenesis inherent to all DNA-based methodologies {Gump, 2007 #39}. Moreover if endosomal escape can be improved, our approach will allow protein stoichiometry to be tightly regulated within cultures. This will avoid stochastic variation in expression typical of integrating vectors, as well as the uncontrollable effects of viral silencing. GET/HETD technology may also be directly applied to reprogramming technologies. Given the stepwise character of the phenotypic changes observed during pluripotency induction {Chan, 2009 #63; Smith, 2009 #64} and for directed-differentiation protocols {Burridge, 2011 #81}, it seems likely that individual transcription factors play distinct, stage-specific roles. The unprecedented potential for temporal control over individual factor function afforded by GET/HETD technology should enable these variables to be tested to improve efficiency and kinetics of cell-fate control.

(92) The transient and non-mutagenic character of protein-based transduction could also deliver important clinical benefits outside those investigated here. Indeed, the use of protein transduction to express cancer or pathogen antigens for immunotherapy {Rabinovich, 2009 #67} may benefit from the non-immunogenic properties of protein transduction.

(93) The majority of delivered protein may be trapped in the inside of the endosome. The PTD field has focused on mechanistic aspects of TAT and other PTD interaction and escape from macropinocytomes with the goal of improving the release of cargo from vesicles {Heitz, 2009 #42}. The issue was approached from another angle with the aim to deliver proteins beyond that that may be achieved by PTDs and their avidity to the cell surface.

(94) Previous studies delivering TAT-Cre suggested that the rate-limiting step for effective PTD-mediated delivery of a functional cargo is macropinosome escape, however this data suggests that even though this escape process will ultimately control whether PTDs are successfully used to treat disease {Sugita, 2007 #80}, that the PTD-transduction process itself is not the most efficient it could physiologically be. GET/HETD-mediated transduction described here improves present systems by up to three orders of magnitude for hard-to-transduce cells. Marrying vesicle escape with the GET/HETD system will be a significant development. As proof-of-concept this was demonstrated by the use of general endosomal disruptor, Chloroquine. By decreasing macropinocytome-integrity during GET/HETD-transduction functional delivery of proteins using pg/ml concentrations was shown (FIG. 17f).

(95) The present understanding of PTD-mediated delivery consists of the hypothesis that a dense forest of GAGs provides the cell membrane with a ubiquitous negatively charge that PTDs bind to {Gump, 2007 #39}. Change in charge effect this binding but this is independent of the ability of PTDs to transduce cells or induce macropinocytotic uptake. GET/HETD has effectively created a more exaggerated version of this phenomenon with tagged-proteins more avidly binding cell membranes and promoting successful transduction mediated by a PTD. Alternative HBDs function in a similar manner to P21 with different efficacies and cell-type activities, depending on their source of isolation. Replacement of 8R has shown that other PTDs (TAT, 8K, 8RQ) can be employed in a successful GET/HETD (FIG. 11).

(96) It is known that GAGs, such as HS-GAGs, have diverse biological functions and are widely involved in many physiological and pathological processes such as blood coagulation and inflammatory responses {Lortat-Jacob, 2002 #53; Varki, 2008 #52} via interactions with a variety of proteins including growth factors, cytokines and chemokines {Sasisekharan, 2006 #55; Gandhi, 2008 #54}. The interactions are dependent upon the disaccharide composition and patterns of GAGs, which play a significant role in regulating various biological processes. The heterogeneity of HS-GAGs is determined by the expression patterns of a variety of linked genes and multiple HS-GAGs-editing enzymes under different pathological conditions {Lindahl, 1998 #56; Nakato, 2002 #57; Sasisekharan, 2002 #60; Bengtsson, 2003 #59; Gesslbauer, 2007 #58}. Inflammatory processes and diseases such as mucopolysaccharidoses, osteoarthritis and myeloma cancer have been reported to correlate to the different disaccharide structures of GAGs. Therefore, evaluating the variations (i.e. presence and quantity) of GAGs has a great potential for diagnosis and prognosis of diseases.

(97) The discovery that promoting GAG interaction significantly improves the efficiency of PTD-tagged biologically active, macromolecular cargo will open up new avenues for the treatment and experimental investigation of disease.

(98) Experimental Procedures

(99) Expression and Purification of Recombinant Proteins

(100) cDNA was obtained for mRFP1 (mR) as a kind gift from Prof. R. Y. Tsien (University of California, USA) {Campbell, 2002 #12}. 8R, TAT, 8K, 8RQ, P21, Cre, NANOG, MYOD and NEO cDNAs were synthesized de novo (Eurofins MWG Operon). cDNAs were cloned into the pGEX6-P1 expression vector (Novagen) to create in-frame fusions and expressed proteins in BL21 (DE21) pLysS Escherichia coli (Novagen). Exponentially growing LB cultures (OD.sub.600=0.4) shaken at 220 rpm at 37° C. were induced using 1 mM IPTG for 24 hours at 25° C. Bacterial pellets were lysed and sonicated (7 amplitudes, 1 minute, 5 times) in 1×STE extraction buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA containing 1 mM DTT, 0.2 mg/ml lysozyme, and 1× protease inhibitor cocktail).

(101) Insoluble protein was retrieved using the Rapid GST inclusion body solubilisation and renaturation kit (AKR-110; Cell Biolabs, Inc., San Diego, Calif.). Recombinant proteins were purified by affinity chromatography using Glutathione-Sepharose resin (GE Healthcare). GST-tags were removed and eluted from resin by PreScission™ Protease cleavage (GE healthcare) in 1× cleavage buffer (50 mM Tris-HCl pH 7.0, 150 mM NaCl, 1 mM EDTA and 1 mM DTT). Protein concentration was determined using a BCA-based protein assay (BioRad) with absorbance measured at 595 nm using recombinant mR protein as a standard, Integrity and full-length protein expression was confirmed by SDS-PAGE. The fluorescence of recombinant proteins (excitation: 584 nm; emission: 607 nm) was determined with all preparations <10% intensity difference between samples (fluorescence/μg). Standards and samples were analysed using the TECAN infinite 200PRO multimode reader. Aliquots were stored at −80° C.

(102) Cell Culture

(103) NIH3T3 mouse fibroblast cells, [HEK293T human embryonic kidney cells, C2C12 mouse myoblast cells, iIMSC immortalised human mesenchymal stem cells (created as described {Okamoto, 2002 #8}) and MEF murine embryonic fibroblasts (harvested as described {Anderson, 2007 #9}) were maintained in DMEM with 10% (v/v) fetal calf serum (FCS; Sigma) media supplemented with 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin). CGR-8 mouse embryonic stem cells (mESCs) and EXT1-7-mESCs (a kind gift from Dr. D. E. Wells, University of Houston, USA; {Lin, 2000 #78}) were maintained in DMEM, 20% (v/v) FCS, 1000 units/ml leukaemia inhibitory factor (LIF), non-essential amino acids, 100 μM β-mecaptoethanol (Sigma) 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin). HL1 mouse cardiomyocyte cells were maintained as described {Claycomb, 1998 #7}. HUES7 human embryonic and IPS2 induced pluripotent stem cells were cultured as previously described {Dick, 2011 #10}. HUES7fib human fibroblasts derived from HUES7 cells were generated and cultured as previously described {Dick, 2011 #10}. All cells were cultured at 37° C. under 5% CO.sub.2

(104) Flow Cytometry and Microscopy

(105) For flow cytometry, cells were trypsinized (unless otherwise stated), fixed in 4% (w/v) PFA, resuspended in PBS (pH7.5) and analysed on a MoFlo™ DP (DAKO) Flow Cytometer using a 488 nm green laser. (50,000 cells; gated on live cells by forward/side scatter). Median fluorescence was used for statistical analyses with background from unlabelled/transduced cells subtracted and values taken as ratios to the experimental control. Data shown are three experiments of triplicate samples. For microscopy, cultures were rinsed twice with PBS and imaged with inverted fluorescence microscope (Nikon Eclipse TS100).

(106) Fluorescence Delivery Assay

(107) For testing multiple cell lines we plated 2=10.sup.5 cells/well (in 12-well plates) onto the surface relevant to the tested cell line, attached cells for 2 hours and transduced with recombinant proteins in cell-type specific growth media. After transduction cells were washed with PBS, trypsinized and fixed in 4% PFA for flow cytometry. For membrane localization, intracellular localisation or both we plated cells as above, but cultures pre-incubated in serum-free media for 1 hour before transduction. Membrane localization to assess cell interaction was achieved by a short transduction of 1 hour in serum-free media. Intracellular localization to assess transduction efficiency was achieved by a short transduction of 1 hour followed by 5 hour incubation in serum-free media only. Cell association (membrane and intracellular levels) were assessed by transducing cells for 6 hours in serum-free medium. For flow cytometry cells were trypsinized, washed and fixed in 4% PFA and for microscopy cells were imaged live after washing in PBS. For trypsin depletion of cell-surface proteins, cells were treated with trypsin/EDTA (Invitrogen) or EDTA-based cell dissociation solution (CDS) (Sigma) for 15 minutes at 37° C., followed by washes with PBS and 1× soybean trypsin inhibitor (10 mg/ml in PBS; Sigma). Cells were then treated with proteins for 1 hour at 37° C. in serum-free medium. For detergent depletion of cell membranes, cells were treated with PBS (pH7.5) containing 0.1% (v/v) Triton-X100 (Tx100) for 10 minutes at 37° C., followed by washes with PBS. Cells were then treated with proteins for 1 hour at 37° C. in serum-free medium. For GAG-treatment cells were pre-treated with GAGs in DMEM without serum before transduction and were included in the transduction media. This included heparin and chondroitin sulphate A, B and C (0-50 μg/ml).

(108) Total Delivered Protein Analyses

(109) 5×10.sup.6 NIH3t3 cells were plated (in T25 flasks), pre-incubated cells in serum-free DMEM for 1 hour, and transduced them with mR-8R or P21-mR-8R (0-200 μg/ml; 1 ml volume) in serum-free DMEM for 6 hours. NIH3t3 cells transduced with SIN-mR lentiviruses were used as a control for the levels achieved by transgenic systems {Dixon, 2011 #15; Dick, 2011 #10}. Cells were harvested by trypsinization, fixed in 4% PFA for flow cytometry or washed several times in cold PBS with soluble protein extracted in cold HKM buffer (20 mM HEPES, pH 7.5, 5 mM KCl, 0.5 mM MgCl.sub.2 and 0.5 mM DTT with 1× complete EDTA-free protease inhibitor cocktail) for fluorometry {Medina, 2000 #14}. Extracts were sonicated, centrifuged and NaCl added to yield a final concentration of 100 mM prior to analyses. Fluorometry was used to compare soluble extracts with purified mRFP protein diluted in HKM buffer with 100 mM NaCl as standards. Flow cytometry was used to assess total delivered protein in intact cells.

(110) Media Depletion Assessment

(111) 2×10.sup.6 NIH13t3 cells or HUES7 HESCs were plated (in 6-well plates), pre-incubated cells in serum-free DMEM for 1 hour, and transduced with recombinant proteins (20 μg/ml; 1 ml volume) in serum-free DMEM for 12 hours. Media was harvested and fluorometry was used to compare the remaining fluorescence in media verses that before cell-incubation. Fluorescence of media pre-incubation was assigned as 100% fluorescence units and background of serum-free media subtracted.

(112) Heparin-Binding Assay, Heparinase Treatment and Depletion of P21-Binding Molecules from Serum

(113) For Heparin binding activity we incubated 1 ml of recombinant proteins (20 μg/ml) in DMEM with 50 μl of PBS-washed Heparin-sepharose beads (Sigma) for 1 hour at 37° C. shaking at 100 rpm. Media pre- and post-incubation was compared by fluorometry. For Heparinase treatment, we plated NIH3t3 cells at 2×10.sup.5/well (in 12-well plates) and were pre-incubated in serum-free media for 1 hour with Heparinase III (0-1 U/ml) or Heparin (0-50 μg/ml). Cells were then washed and transduced with mR or P21-mR-8R (20 μg/ml in serum-free media or media with different FCS concentrations) containing Heparinase III or Heparin for 12 hours. FCS was depleted of P21-binding material by affinity chromatography. This was achieved by incubating 50 ml FCS with 2 ml Glutathione-Sepharose resin (GE Healthcare) pre-absorbed with GST-P21 protein expressed in Escherichia coli.

(114) Macropinocytosis Assessment

(115) To measure the effects of protein transduction on general macropinocytosis, cells were incubated with 100 μg/ml FITC-70 kDa neutral dextran (Sigma), along with different recombinant proteins (0-10 μg/ml) for 1 hour at 4° C. or 37° C. Cells were trypsinized and washed in PBS before analyses by flow cytometry.

(116) Cre Recombination Assay

(117) To measure Cre Recombinase activity the NIH3t3: LSL-eGFP cell line was created using the pZ/EG plasmid transfection and G-418 selection {Novak, 2000 #6}. To confirm Cre activity efficiently led to recombination and eGFP activation cells were transduced with SIN-Cre lentiviruses (as described in Dixon et al. 2011) and >95% of cells were confirmed eGFP-positive 48 hours post-transduction. 2×10.sup.5 cells/well were plated (in 12-well plates), pre-incubated them in DMEM without serum for 1 hour and treated with Cre proteins (0-500 μg/ml) in DMEM without serum. After the Cre incubation cells were trypsinized, replated into complete media and incubated for 2 days. Cells were pre-treated with drugs for the stated time-period in DMEM without serum, were included in Cre-transduction medium and were added after replating. Pre-treatments included: heparin (0-50 μg/ml), chondroitin sulphate A, B and C (0-50 μg/ml), chloroquine (0-100 μM), cytochalasin-D (0-10 μM), amiloride (0-5 mM), methyl-β-cyclodextrin (0-5 mM), and nystatin (0-50 μg/ml). After incubations cell were trypsinized, washed, fixed in 4% PFA and % recombined cells was determined by flow cytometry. For mR-Cre-8R and P21-mR-Cre-8R comparisons concentrations of 100 μg/ml and 10 μg/ml were used, respectively and data was expressed as % maximum recombination (i.e. the % relative to the maximum recombination achieved at the stared dose of Cre).

(118) NEO Antibiotic-Resistance Assay

(119) To measure NEO activity the survival and proliferation of NIH3t3 and MEF in the presence of G-418 antibiotic selection was assessed. Cell number, viability and live/dead ratios were measured. To confirm NEO activity efficiently leads to survival and proliferation under G-418 selection of NIH3t3 and MEF cells, the cells were transduced with SIN-NEO lentiviruses (as described in Dixon et al. 2009) and confirmed that NEO transduction prevents cell death and retains viability under stringent G-418 selection. This was used in comparisons with NEO protein transductions. We plated 3×10.sup.5 MEF cells/well or 1×10.sup.5 NIH3t3 cells/well (in 12-well plates) and cultured them in DMEM with 10% FCS containing P21-mR-NEO-8R (0-100 μg/ml) for 3 days. Cells were replated at 3×10.sup.5 cells/well for MEF cells or 1×10.sup.5 cells/well for NI-13t3 cells. Cells were cultured in DMEM with 10% FCS containing P21-mR-NEO-8R (0-100 μg/ml) and G-418 sulphate (0-300 μg/ml) for a further 3 days feeding daily to kill non-resistant cells. Cells were counted, assessed for viability using trypan blue exclusion or assayed using the LIVE/DEAD staining {Bayoussef, 2012 #13}.

(120) NANOG Self-Renewal Assay

(121) To measure NANOG activity we used LIF-withdrawal from CGR-8 mESCs and measured alkaline phosphatase (AP) activity, cell numbers and assessed gene expression changes by quantitative-PCR (QPCR). To confirm NANOG activity efficiently leads to rescue of self-renewal without LIF we transduced cells with SIN-NANOG lentiviruses (as described in Dixon et al. 2009) and confirmed that CGR-8 self-renewal was efficiently rescued and this was used in comparisons with NANOG protein transductions. 2×10.sup.5 cells/well were plated (in 6-well plates), pre-incubated them in growth media with LIF for one passage/3 days containing P21-mR-NANOG-8R (0-50 μg/ml) feeding with fresh media daily. Cells were replated at 2×10.sup.5 cells/well (in 6-well plates) in growth media without LIF (and with LIF as controls) containing P21-mR-NANOG-8R (0-50 μg/ml). Cells were fed daily with this media and every 3 days were counted and passaged replating one-tenth of the cells until 3 passages. After the third passage post-LIF withdrawal cells were stained for AP activity (86R-1 kit; based on Naphthol AS-BI and fast red violet LB; Sigma)) or processed for QPCR analyses. Relative expression levels (ΔΔCT) were determined QPCR using TaqMan™ Gene Expression Master Mix and specific TaqMan™ Gene Expression Assays (Applied Biosystems).

(122) MYOD Myogenesis Assay

(123) To measure MYOD activity we used differentiation of HUES7 HESCs and assessed cell morphology, cell multinucleation, gene expression changes by quantitative-PCR (QPCR) and MYOGENIN protein expression. To confirm MYOD activity efficiently leads to myogenic differentiation of HESCs we transduced cells with SIN-MYOD lentiviruses (as described in Dixon et al. 2009) and confirmed that MYOD directs multinucleated myotube differentiation which was used in comparisons with MYOD protein transductions. 1×10.sup.6 cells/well were plated into 0.1% gelatin-coated plates (in 6-well plates) and cultured them in DMEM with 10% FCS for 1 week with one passage using trypsin. Cells were replated at 1×10.sup.6 cells/well into 0.1% gelatin-coated plates and cultured in DMEM with 10% FCS containing P21-mR-MYOD-8R (0-50 μg/ml). Cells were fed daily with this media for 7 days. The culture media was then switched to DMEM with 2% horse serum (HS) and cultures maintained for a further 7 days. Cells were then processed for QPCR or fixed in 4% PFA and immunostained {Bayoussef, 2012 #13}. Nuclei were labelled using DAPI as previously described (Dixon et al. 2009). The percentage of MYOGENIN-positive nuclei/total nuclei was quantified, with a minimum of 200 nuclei counted per condition. Relative expression levels (AACT) were determined QPCR using TaqMan® Gene Expression Master Mix and specific TaqMan™ Gene Expression Assays (Applied Biosystems).

(124) Antibody, Nucleic Acid and Nanoparticle Delivery Biotinylated-Goat anti-Rabbit and FITC-Rabbit anti-mouse antibodies (Sigma), pSIN-GFP (Dixon et al. 2014), modified nucleotide RNA (modRNA) for GFP (Miltenyi Biotech) and FAM-labelled siRNA against GAPDH (Sigma), and nanomag-D (250 nm) (MircoMod) were complexed with GET-proteins or -peptides and added to cells. For antibodies complexes were allowed to form in growth media for 20 mins before cell addition. For nucleic acids a 2:1 peptide:nucleic acid charge ratio was used for complexation. GET- or LIPO2000 (lipofectamine 2000; Invitrogen) transfection used 10 μg or 1 μg nucleic acid per transfection of 100,000 hMSCs in 12 well plates. GET-peptide substituted LIPO2000 following the exact manufacturer's instructions. For MNPs, a final concentration of 25 M peptide was used in an EDAC/NHS reaction using 2 mg MNPs according to manufacturer's instructions. Prussian blue was carried out using potassium ferrocyanide (2.5% w/v) in 2.5% w/v HCl.

(125) Statistical Analysis

(126) Statistical comparisons were carried out using the GraphPad Prism software package. Comparisons were made using Tukey-Kramer analysis of variance (ANOVA). Results were considered significant if p<0.05.

(127) Example Sequences

(128) Example HS-GAG Binding Sequences

(129) P21 amino acid sequence

(130) TABLE-US-00002 P21 amino acid sequence (SEQ ID NO. 1) KRKKKGKGLGKKRDPCLRKYK P21 nucleotide sequence (with a methione/ATG): (SEQ ID NO: 2) aagcgcaagaagaagggcaaaggcctgggcaagaagcgcgatccgtgcct gcgcaagtataag PDGF (194-211) amino acid sequence: (SEQ ID NO. 3) G R P R E S G K K R K R K R L K P T PDGF (194-211) nucleotide sequence: (SEQ ID NO: 4) ggccgcccgcgcgaaagcggcaaaaaacgcaaacgcaaacgcctgaaacc gacc FGF7B amino acid sequence: (SEQ ID NO. 5) T Y A S A K W T H N G G E M F V A L N Q. FGF7B nucleotide sequence: (SEQ ID NO: 6) Acctatgcgagcgcgaaatggacccataacggcggcgaaatgtttgtggc gctgaaccag FGF2 HBD B(247-262) amino acid sequence: (SEQ ID NO. 7) T Y R S R K Y T S W Y V A L K R. FGF2 HBD B(247-262) nucleotide sequence: (SEQ ID NO: 8) acctatcgcagccgcaaatataccagctggtatgtggcgctgaaacgc

(131) Nucleotides Encoding 8R Protein Transduction Domain Sequence:

(132) TABLE-US-00003 (SEQ ID NO: 9) CGA AGA CGC AGG AGA CGT CGA AGG

(133) Example Delivery Molecule Nucleotide Sequence (P21-Cargo-8R):

(134) TABLE-US-00004 (SEQ ID NO: 10) aagcgcaagaagaagggcaaaggcctgggcaagaagcgcgatccgtgcct gcgcaagtataagNcgaagacgcaggagacgtcgaagg

(135) N=cargo nucleic acid sequence of various length (i.e. the number of nucleotide residues may vary), or another molecular entity.

(136) Two versions of each of the nanobody variants of the ScFv antibodies were made; one with identical sequence to the ScFv vHH domain (Frame domain1-CDR1-Frame domain 2-CDR2-Frame domain 3-CDR3-IgA Hinge domain/Frame domain 4) and one in which the CDR1, 2 and 3 domains were grafted into a generic vHH domain sequence. Both versions have comparable activity and the grafting version was created to prove that simply grafting the CDR domains onto a generic antibody also works.

(137) Below are the sequences of the HS4C3, and AO4B08 ScFv vHH and grafted vHH:

(138) TABLE-US-00005 HS4C3 ScFv vHH (SEQ ID NO: 11) EVQLVESGGGLVQPRGSLRLSCAASGFTVSSNEMSWIRQAPGKGLEWVSS ISGGSTYYADSRKGRFTISRDNSKNTLYLQMNNLRAEGTAAYYCGRRLKD PSTPPTPSPSTPPTPSPS CDR1 GFTVSSNE CDR2 ISGGST CDR3 GRRLKD H54C3 ScFv vHH nucleotide sequence (SEQ ID NO: 12) gaagtgcagctggtggaaagcggcggcggcctggtgcagccgcgcggcag cctgcgcctgagctgcgcggcgagcggctttaccgtgagcagcaacgaaa tgagctggattcgccaggcgccgggcaaaggcctggaatgggtgagcagc attagcggcggcagcacctattatgcggatagccgcaaaggccgctttac cattagccgcgataacagcaaaaacaccctgtatctgcagatgaacaacc tgcgcgcggaaggcaccgcggcgtattattgcggccgccgcctgaaagat ccgagcaccccgccgaccccgagcccgagcaccccgccgaccccgagccc gagc H54C3 grafted vHH (SEQ ID NO: 13) QVQLVESGGGSVQAGGSLRLSCTASGFTVSSNELGWFRQAPGQERWAVAA ISGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTAIYYCGRRLKD WGQGTQVTVSSPSTPPTPSPSTPPTPSPS CDR1 GFTVSSNE CDR2 ISGGST CDR3 GRRLKD H54C3 grafted vHH nucleotide (SEQ ID NO: 14) caggtgcagctggtggaaagcggcggcggcagcgtgcaggcgggcggcag cctgcgcctgagctgcaccgcgagcggctttaccgtgagcagcaacgaac tgggctggtttcgccaggcgccgggccaggaacgctgggcggtggcggcg attagcggcggcagcacctattatgcggatagcgtgaaaggccgctttac cattagccgcgataacgcgaaaaacaccgtgaccctgcagatgaacaacc tgaaaccggaagataccgcgatttattattgcggccgccgcctgaaagat tggggccagggcacccaggtgaccgtgagcagcccgagcaccccgccgac cccgagcccgagcaccccgccgaccccgagcccgagc A04B08 ScFv vHH (SEQ ID NO: 15) EDQLVESGGGLVQPGGSLRPSCAASGFAFSSYALHWVRRAPGKGLEWVSA IGTGGDTYYADSVMGRFTISRDNAKKSLYLHMNSLIAEDMAVYYCSLRMN GWRAHQPSTPPTPSPSTPPTPSPS CDR1 (SEQ ID NO: 22) GFAFSSYA CDR2 (SEQ ID NO: 24) IGTGGDT CDR3 (SEQ ID NO: 26) SLRMNGWRAHQ AO4B08 ScFv vHH nucleotide sequence (SEQ ID NO: 16) gaagatcagctggtggaaagcggcggcggcctggtgcagccgggcggcag cctgcgcccgagctgcgcggcgagcggctttgcgtttagcagctatgcgc tgcattgggtgcgccgcgcgccgggcaaaggcctggaatgggtgagcgcg attggcaccggcggcgatacctattatgcggatagcgtgatgggccgctt taccattagccgcgataacgcgaaaaaaagcctgtatctgcatatgaaca gcctgattgcggaagatatggcggtgtattattgcagcctgcgcatgaac ggctggcgcgcgcatcagccgagcaccccgccgaccccgagcccgagcac cccgccgaccccgagcccgagc AO4B08 grafted vHH (SEQ ID NO: 17) QVQLVESGGGSVQAGGSLRLSCTASGFAFSSYALGWFRQAPGQERWAVAA IGTGGDTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTAIYYCSLRMN GWRAHQWGQGTQVTVSSPSTPPTPSPSTPPTPSPS CDR1 (SEQ ID NO: 22) GFAFSSYA CDR2 (SEQ ID NO: 24) IGTGGDT CDR3 (SEQ ID NO: 26) SLRMNGWRAHQ AO4B08 grafted vHH nucleotide sequence (SEQ ID NO: 18) caggtgcagctggtggaaagcggcggcggcagcgtgcaggcgggcggcag cctgcgcctgagctgcaccgcgagcggctttgcgtttagcagctatgcgc tgggctggtttcgccaggcgccgggccaggaacgctgggcggtggcggcg attggcaccggcggcgatacctattatgcggatagcgtgaaaggccgctt taccattagccgcgataacgcgaaaaacaccgtgaccctgcagatgaaca acctgaaaccggaagataccgcgatttattattgcagcctgcgcatgaac ggctggcgcgcgcatcagtggggccagggcacccaggtgaccgtgagcag cccgagcaccccgccgaccccgagcccgagcaccccgccgaccccgagcc cgagc

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(141) Improving the Efficiency of Iron Oxide Nanoparticle Uptake Using Cell Penetrating Peptides

(142) Background

(143) Superparamagnetic Iron Oxide nanoparticles (SPIONS) are small highly magnetised particles consisting of an iron oxide core and surface coating. SPIONS have been clinically approved for use in MRI contrast agents.sup.1, and are currently being researched for use in targeted drug delivery.sup.2, hyperthermia treatment and cell labelling.sup.3. SPIONS have been approved for uses in MRI contrast agents and commercially available products include Lumiren, Resivist and Feridex..sup.1

(144) Applications of SPIONS require an adequate concentration being internalised into cells and without the required targeting of nanoparticles it can lead to an inefficient outcome. The efficiency of cell internalisation can depend on the size, coating and additional ligands to name a few.sup.4. Literature shows that without the attachment of internalisation agents researchers are achieving a range of 15-30 μg of iron per cell.sup.5,6. The functional groups on nanoparticle coatings can be exploited to target increase cell internalisation by attaching monoclonal antibodies, cell penetrating peptides and small molecules as internalisation agents..sup.7

(145) A currently researched cell penetrating peptide is Arg-Gly-Asp (RGD). RGD was designed to target the avβ3 intergrin.sup.8. The intergrin can be found predominantly on cancer cells, so can also be used as a targeting peptide. Research found that the RGD peptide increased nanoparticle uptake by 50%..sup.9

(146) The following study focuses on a cell penetrating peptide of the invention herein, in particular P218R. The peptide has two domains, P21 binds to the heparan sulphate (HS) on the cell membrane and the 8R aids in the transduction. The aim of the study was to identify the efficiency of the P218R and to investigate its mechanism.

(147) Materials and Methods

(148) Nanoparticle Labelling

(149) A 31 mM EDAC with 0.1 M NIS dissolved in 0.5M MES buffer was added to Nanomag-D (250 nm) particles in a 1:5 ratio respectively and mixed for 1 hour. The particles are then washed in a 0.1M MES buffer and 0.2 μg/μl of the required labelling agent dissolved in the same buffer was added to give a 1:1 ratio of labelling solution and nanoparticles, an aliquot of the labelling solution was kept for testing labelling efficiency. The solution is then continuously mixed at room temperature for 3 hours. Once the particles are labelled a 25 mM glycine solution is added to the particles then further incubated for 30 minutes. An aliquot of the labelling solution is kept for comparison with the earlier aliquot and the particles washed in 0.1% BSA in PBS. Particles are finally diluted in 0.1% BSA in PBS to give a 1 mg/ml solution. Both aliquots of labelling solution and some of the labelled nanoparticles were assessed for fluorescence.

(150) Cell Culture

(151) NIH 3t3 fibroblast cells were cultured in Dulbecco's modified Eagle's media (DMEM; Gibeco), supplemented with 10% (v/v) Fetal Calf Serum (FCS, Sigma), 2 mM L-glutamine and (PS) at 37° C. and 5% CO2. The cells were then cultured until confluent.

(152) Cell Labelling

(153) Confluent cells were split into 12 well plates at 200,000 cells/well and incubated for 24 hours at 37° C. After 24 hours 50 μg of Nanomag-D iron oxide nanoparticles (250 nm) and either 0, 0.01, 0.05, 0.1, 0.5, 2, 1, 5 and 10 μM of cell penetrating peptide were added to the cells with either 10% FCS DMEM or serum free DMEM media and left for 24 hours for iron nanoparticles to be internalised. After incubation cells were washed in PBS to remove excess nanoparticles then harvested for qualitative Prussian blue staining, quantitative colorimetric iron assay or fluorescence activated flow cytometry.

(154) Prussian Blue Staining

(155) Cells were labelled then fixed in 4% (w/v) PFA for 15-20 minutes at 4° C. A staining solution of 2.5% potassium ferrocyanide in 2.5% HCL was added to cells and incubated for an hour at room temperature. If nanoparticles were present a blue stain appeared which is proportional to the concentration of iron.

(156) Quantitative Colorimetric Iron Assay Cells were labelled, trypsinised and pelleted then all media removed. 40 μl of 37% HCL was added to the cells and heated at 70° C. until dissolved, then neutralised with 50 μl of NaOH. Those samples containing a high concentration of iron were diluted 1:10 then 40 μl of Quantichrom working reagent was added and the instructions in the Quantichrom iron assay followed.

(157) Flow Cytometry

(158) Cells were labelled with 50 μg of Nanomag-D particles, 1 μM P218R and either 0, 0.1, 1 and 5 μg/ml of FitC-BSA. Cells were then fixed cells and run through a Coulter Altra flow cytometer to assess the green fluorescence. Findings were then statistically analysed by Wesal software.

(159) Results

(160) Nanoparticle and Cell Labelling

(161) Nanomag-D (250 nm) particles were successfully labelled with mR, P21mR, 8RmR and P21mR8R as shown in FIG. 32. The graphs clearly show a reduction in fluorescence in the post labelling solution and an increased fluorescence on the particles hypothesising that the protein has reacted with carboxylic acid group and bonded to the surface of the Nanomag particles. The graphs show that not all of the protein has reacted with the particle surface but considering the proteins were added in excess this was not seen as an issue. Another feature of the graphs is that the P21 domain compared with the 8R has a higher affinity for binding to the particles as there is a more distinctive reduction in fluorescence post labelling and a more intense signal exhibited from the nanoparticles for the P21. Considering that the protein was added in excess of the particles binding capacity such a reduction was unexpected. The P21 domain's role is that it binds to the HS on a cell's membrane, it could also be concluded that the protein is binding to the dextran surface of the particles considering the similar structures of the two substances.

(162) To further test the hypothesis of the P21 binding to the dextran surface both particles which have been pre-labelled with P21mR8R were added to cells and particles and P218R were separately added to cells. The results of the prussian blue stain is shown in FIG. 32. For the unlabelled particle control no blue staining was seen therefore concluding a limited number of particles must have been associated with cells. However for both variables the stain had reacted producing a blue colour even when the protein was not pre-bound. The protein must have bound to the particle at some point in order for particle uptake to occur. The most likely hypothesis is that the P21 is binding to the dextran surface.

(163) Quantitative Assessment of Nanoparticle Uptake

(164) Optimisation of Protein Concentration

(165) As shown in FIG. 33, Prussian blue staining proves that the P218R is the most effective peptide for particle uptake compared to P21 and 8R alone. The iron assay results showed that when cells are incubated for 24 hours with 1 μM of P218R and 50 μg of Nanomag-D particles 100% of the particles become associated with the cells and 63 μg/cell. Therefore it was concluded that only 1 μM of P218R is needed for 100% uptake of particles.

(166) Assessing the Time Taken for Nanoparticle Cell Association

(167) All results indicated that optimum nanoparticle cell association is at 24 hours, as shown in FIG. 34. As at the 24 hour time point 100% of the particles are associated with the cells; this also fits with the results shown in FIG. 33. The graph is also clear evidence of the dramatic increase in particle uptake with the added peptide, from this information the percentage uptake increases from 7% without the added peptide to 96% with the added peptide after 24 hours.

(168) Effect of Serum Free Media

(169) Results in FIG. 35 show a 30% decrease in uptake as the percentage of serum increases to 20%. This decrease reduces to only 16% when 1 μM P218R is added. This decrease in uptake as the amount of serum increases has been attributed to the decrease in endocytosis, as serum free conditions induce endocytosis due to the decrease in nutrients. If the endocytosis decrease is reduced when the peptide is added then it could be assumed that it increases endocytosis. It was found that the addition of the peptide increased endocytosis as the amount of Fitc-BSA taken up by cells increases, whereas the nanoparticles did not affect the uptake, these results are shown in FIG. 35. The increase in uptake of FitC-BSA could be correlated to an increase in endocytosis leading to an increase in particle uptake. There is a less distinctive difference when cells are in serum free conditions as the serum free alone increases endocytosis.

(170) Competing Factors for P21 Binding

(171) It has been proven that the P21 binds to HS on cell membranes, therefore it can be hypothesised that if heparin is added to the media it will competitively inhibit the binding and therefore nanoparticle uptake. Results are shown in FIG. 35. The graph proves that heparin is an inhibitor for the binding and the extent of the inhibition proves that the P21 has a higher affinity for the heparin than the HS on the cell membrane, and therefore proving that the P21 binding is essential to the uptake mechanism of the nanoparticles. P21 may also be binding to the dextran on the surface of the nanoparticles therefore increasing concentrations of dextran was added to media. The results are shown in FIG. 35. There is a decrease in nanoparticle uptake of 18% as the dextran concentration increases. The dextran will be acting as a competitive inhibitor for particle binding and due to the limited reduction in inhibition the P21 has a higher affinity for the cell membrane.

(172) Discussion

(173) The results show that the addition of a small amount of P218R leads to 100% uptake of iron oxide nanoparticles. Microscopy and the trypsinisation of the cells indicate that the particles are being internalised. Experiments were also conducted using mesenchymal stem cells showing a 90% association of particles. The mechanism behind the uptake is dependent on the symbiotic action of the two domains of the peptide, The hypothesis is that the P21 can bind to both the HS on the cell membrane and the dextran in the coating of the nanoparticles, the peptide either has multiple binding points by which both nanoparticle and cell can both be attached to the same P21. Therefore the pre bound protein to the particle can also bind to the membrane keeping the particle in close proximity to the cell. The 8R can then aid in the transduction of the nanoparticle by endocytosis. Or the other mechanism could involve the peptide pre binding to the nanoparticle then when in close proximity to a cell membrane the HS has a higher binding efficiency so the P21 then binds to the cell. This may then lead to the particle being internalised. The advantages of using the P218R peptide is its efficiency in serum media which is more relatable to the in vivo environment and that the system does not require the use of the functional group on the nanoparticles surface coating. The free functional group means that targeting molecules or drugs can be covalently attached to the particle.

(174) Conclusion

(175) The peptide P218R has been found to cause 100% cell association of nanoparticles. This has been found to be due to a dextran binding mechanism which can be utilised for many applications for example targeting of nanoparticles for specific tissues by attaching antibodies, or drug delivery.

REFERENCES

(176) 1. Singh, A & Sahoo, S, (2013), Magnetic Nanoparticles: a novel platform for theranostics, Drug Disc Today, 2. Arruebo, M et al, (2007) Magnetic Nanoparticles for drug delivery, Nanotoday, 3, 22 3. Wang, Z and Cuschieri A, (2013) Tumor cell labelling by magnetic nanoparticles with determination of intracellular iron content and spatial distribution of the intracellular iron, Int. J. Mol. Sci, 14, 9111 4. Sun, C. Lee, J and Zhang, M, (2008), Magnetic Nanoparticles in MR Imaging and drug delivery, Adv Drug Deli Rev, 60, 1253 5. Schlorf, T et al, (2011), Biological properties of iron oxide nanoparticles for cellular and molecular magnetic resonance imaging, Int. J. Mol. Sci. 12, 12 6. Markides, I I et al. (2013) Whole body tracking of superparamagnetic iron oxide nanoparticle labelled cells—a rheumatoid arthritis mouse model, Stem cell Res & ther, 4, 126 7. Peng et al, (2008), targeted magnetic iron oxide nanoparticles for tumour imaging and therapy, Int. J. of Nanomed. 3, 311 8. Zhang, C et al, (2007), Specific Targeting of Tumor Angiogenesis by RGD-Conjugated Ultrasmall Superparamagnetic Iron Oxide Particles Using a Clinical 1.5-T Magnetic Resonance Scanner, Can. Res. 67, 1555 9. Ji, S et al. (2012), RGD-conjugated albumin nanoparticles as a novel delivery vehicle in pancreatic cancer therapy, Cancer Biology & Therapy 13:4, 206.

(177) Modified CPPs for Efficient Cell Type Specific Delivery of Therapeutic Molecules Via GET (Glycosaminoglycan (GAG)-Binding Enhanced Transduction)

(178) Introduction

(179) The first aim of this study was to investigate whether the GET-mediated synergistic increase in delivery of mRFP into cells with P21 8R could be observed when P21 was replaced by growth factor derived HS-GAG binding domains. The second aim of this study was to show cell type specific delivery in a heterogeneous population of cells by targeting a specific cell surface HS-epitope. Merry et al have demonstrated the utility of a HS-epitope binding antibody in targeting a subpopulation of cells during mesodermal differentiation [13]. The variable region of this antibody was conjugated to 8R to show an example of cell type specific delivery. The third aim of this study was to demonstrate the GET-mediated delivery of therapeutic biomolecules. The transfection of reporter gene (pSIN GFP) was optimized with P21 LK15 8R peptide and compared to a ‘gold standard’ commercial lipid based transfection reagent Lipofectamine2000.

(180) Cell Type Specific Delivery Via GET

(181) Experimental Procedures

(182) Preparation of Peptides Peptides, mRFP, mRFP 8R, P21 mRFP 8R, FGF1A mRFP, FGF1A mRFP 8R, FGF2A mRFP, FGF2A mRFP 8R, FGF4A mRFP, FGF4A mRFP 8R, FGF7A mRFP, FGF7A mRFP 8R, FGF1B mRFP, FGF1B mRFP 8R, FGF2B mRFP, FGF2B mRFP 8R, FGF4B mRFP, FGF4B mRFP 8R, FGF7B mRFP, FGF7B mRFP 8R, FGF1C mRFP, FGF1C mRFP 8R, FGF2C mRFP, FGF2C mRFP 8R, FGF4C mRFP. FGF4C mRFP 8R, FGF7C mRFP, FGF7C mRFP 8R, ATIII mRFP, ATIII mRFP 8R. PDGF mRFP, PDGF mRFP 8R, VEGF mRFP, VEGF mRFP 8R. HS4C3 mRFP and HS4C3 mRFP 8R, were cloned as cDNAs into pGEX6-PI vector (Novagen), expressed in 13121 (DE21) pLysS Escherichia coli (Novagen) and purified as previously described [12]. Integrity and full length peptide expression was confirmed by SDS-PAGE. Fluorescence of the recombinant peptides was confirmed using the TECAN infinite 200PRO multimode reader, the difference in fluorescence intensity measurements between samples was <10%.

(183) Peptide Assay

(184) Bradford assay was used to quantify protein concentration [14]. Absorbance was measured at 595 nm using recombinant mRFP protein as a standard [12]. Samples were analysed using the TECAN infinite 200PRO multimode reader.

(185) Cell Culture of NIH3T3, CGR-8 and HUES7 Cells

(186) NIH3T3, CGR-8 and HUES7 cells were grown and maintained as previously described [12]. NIH3T3 mouse fibroblast cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) fetal calf serum (FCS) supplemented with 2 mM L-glutamine and 100 ug/ml streptomycin. CGR-8 mouse embryonic stem cells were maintained in DMEM with 20% (v/v) FCS supplemented with 1000 units/ml leukaemia inhibitory factor (LIF), 100 μM β-mecaptoethanol, 2 mM L-glutamine and 100 ug/ml streptomycin. HUES7 human embryonic stem cells were cultured on gelatin coated tissue culture flask. The cells were maintained in DMEM with 20% (v/v) FCS supplemented with 1000 units/ml LIF, 100 μM β-mecaptoethanol, 2 mM L-glutamine and 100 ug/ml streptomycin. All cells were incubated at 37° C. under humidified 5% CO.sub.2 conditions.

(187) Peptide Delivery to Cells

(188) Cells were seeded at 2×10.sup.5 cells/well in 12-well plates and incubated for 2 h in 1 mL of relevant growth media (GM) at 37° C. in a 5% CO.sub.2 humidified incubator. The peptide was diluted to 20 ug/ml in 500 ul of GM. Each well of cells was aspirated, washed with phosphate buffered saline (PBS) and replaced with 500 ul of peptide solution. The cells were incubated with the peptide at 37° C. in a 5% CO.sub.2 humidified atmosphere for 20 h. Following incubation, each well of cells was washed with PBS, trypsinized and fixed with 3.7% paraformaldehyde (PFA) in preparation for flow cytometry. Each experiment was done in duplicate and repeated 3 times, n=3.

(189) Maintenance and Differentiation of Bry-GFP ES Cells, Generation of EBs

(190) Bry-GFP murine embryonic stem cell line was maintained and differentiated as previously described [13]. Bry-GFP cells were maintained on feeders in DMEM-ES (DMEM with 15% FCS supplemented with 1.5×10.sup.5 M monothioglycerol (MTG), 10 ng/ml LIF and 2 mM L-glutamine).

(191) Bry-GFP cells were differentiated as EBs. Prior to differentiation the cells were passaged twice, first onto a gelatin coated flask in DMEM-ES and second into a flask in Iscove's modified Dulbecco's medium (IMDM)-ES (IMDM with 15% FCS supplemented with 1.5×10.sup.4 M monothioglycerol (MTG), 10 ng/ml LIF and 2 mM L-glutamine). Cells were then differentiated as EBs for 2.8 days in IMDM with 15% FCS supplemented with 4×10.sup.4 M MTG, 300 ug/ml transferrin, 25 ug/ml ascorbic acid and 2 mM L-glutamine in Petri-grade dishes. 3 h before dissociation, EBs were treated with 50 ug/ml of HS4C3 mRFP or HS4C3 mRFP 8R. Following differentiation EBs were separated into single cells by 10 min incubation and agitation in cell dissociation buffer and fixed in PFA.

(192) Flow Cytometry Analysis

(193) Cells were analysed on a MoFlo™ DP (DAKO) Flow Cytometer using a 488 nm green laser and/or 633 nm red laser. (40,000 cells; gated on live cells by forward/side scatter). Median fluorescence was used for statistical analyses.

(194) Results and Discussion

(195) CPPs Modified to Include GET

(196) In this study the HS-GAG binding domains of fibroblast growth factor (FGF)-1, FGF-2, FGF-4, FGF-7, platelet derived growth factor (PDGF) and antithrombin-III (ATIII) were coupled to 8R. These growth factors play important biological roles in embryonic development and angiogenesis. They have also been shown to interact with cell surface HS-GAGs, similarly to P21. NIH 3T3 murine fibroblasts, CGR8 murine embryonic stem cells and HUES-7 human embryonic stem cells were treated with these modified CPPs to investigate whether any of the peptides demonstrated a GET-mediated increase in delivery of mRFP (FIG. 37). It was also important to explore whether any of the modified peptides would preferentially target HS epitopes that were more abundantly expressed in any of the different cell types.

(197) A panel of four modified CPPs that showed GET-mediated enhanced transduction into cells have been identified. P21 8R, FGF2B 8R, FGF7B 8R and PDGF 8R have demonstrated 30-100 fold increase in transduction of mRFP into cells over using an 8R alone (FIGS. 38 and 39). P21 8R, FGF7B 8R and PDGF 8R showed preferential transduction into HUES-7 embryonic stem cells. This demonstrates preferential transduction of CPPs into a cell-type that is considered difficult to transduce in to. FGF2B 8R showed pluripotency specific transduction into CGR8 and HUES7 embryonic stem cells. The diverse delivery profiles of the modified CPPs into the three cell types suggests the HS-GAG binding domains of different growth factors target and bind different cell surface HS-epitopes. Targeting HS-epitopes that are expressed more abundantly by specific cell types can be utilized for the selection of CPPs that are more suitable for their application.

(198) GET Mediated Delivery of Plasmid DNA

(199) Experimental Procedures

(200) Preparation of Peptides

(201) P21-LK15-8R peptide was synthesised using solid phase t-Boc chemistry (Novabiochem (Beeston, Nottinghamshire, UK)).

(202) Cell Culture

(203) NIH3T3 mouse fibroblast cells were maintained in DMEM with 10% (v/v) fetal calf serum (FCS) media supplemented with 2 mM L-glutamine and 100 ug/ml streptomyocin. The cells were incubated at 37° C. under humidified 5% CO.sub.2 conditions.

(204) Preparation of Plasmid DNA

(205) DNA (pSIN GFP) was amplified in E. coli. The DNA was extracted and purified using a QIAGEN Plasmid Maxi kit (Qiagen). DNA was precipitated in 100% ethanol and rehydrated in dH.sub.2O. Plasmid purity was confirmed using the nanodrop.

(206) Peptide-DNA Complexation Assay

(207) 10 ug DNA was diluted in 60 ul 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered saline (10 mM HEPES, 150 mM sodium chloride (NaCl) solution. pH 7.4). 1 mM YO-PRO-1 stock solution was diluted to 0.1 mM in dimethyl sulfoxide (DMSO). 2.7 ul of the diluted YO-PRO-1 solution was made up to 60 ul in HEPES-buffered saline and added dropwise to the diluted DNA. The DNA/YO-PRO-1 solution was mixed, wrapped in foil and incubated for 5 h at room temperature. After 5 h, the DNA/YO-PRO-1 solution was made up to 1 ml in HEPES-buffered saline and 100 ul aliquots were pipetted into eppindorf tubes per treatment condition. Peptide amounts corresponding to the desired (+/−) charge ratios were added to each eppindorf (Appendix 1). Peptide/DNA/YO-PRO-1 solutions were mixed and incubated at room temperature for 10 min. Fluorescence measurements were then analysed using the TECAN infinite 200PRO multimode reader. Similarly, a no DNA control was made by diluting 2.7 ul of the diluted YO-PRO-1 solution in 120 ul HEPES-buffered saline and following the procedure above.

(208) Design and Optimization of Transfection Experiment

(209) Cells were seeded at 80,000 cells per well on 12-well plates and incubated overnight in 1 mL of 10% GM at 37° C. in a 5% CO.sub.2 humidified incubator. For each well of cells to be transfected. DNA was diluted in 100 ul Opti-MEMR and mixed. The peptide was added directly to the diluted DNA at the optimal (˜+/−) charge ratio. The solution was then mixed and incubated for 25 min at room temperature. The cells were aspirated, washed with PBS and replaced with 400 ul Opti-MEM®. Each well of cells was treated with 100 ul of Peptide/DNA complex and incubated 37° C. in a 5% CO.sub.2 humidified atmosphere. Appendix 2 shows the different treatment conditions used for each well. Following incubation, the cells were washed with PBS and replaced with 1 ml GM. After 48 h, each well of cells was washed with PBS, trypsinized and fixed with 3.7% PFA. Experiments were repeated 3 times.

(210) Lipofectamine2000 transfection optimization was carried out as described in the manufacturers guide (Invitrogen). Cells were seeded at 80,000 cells per well on 12-well plates and incubated overnight in 1 mL of GM at 37° C. in a 5% CO.sub.2 humidified incubator. For each well of cells to be transfected, DNA was diluted in 100 ul Opti-MEM® and mixed. 1.5 ul of Lipofectamine2000 was added directly to the diluted DNA. The mixture was then mixed and incubated for 25 mins at room temperature. The cells were aspirated, washed with PBS and replaced with 400 ul Opti-MEM®. Each well of cells was treated with 100 ul of Lipofectamine2000/DNA complex and incubated 37° C. in a 5% CO.sub.2 humidified atmosphere. After 6 h, the cells was washed with PBS, trypsinized and fixed with 3.7% PFA. This was repeated with varying volumes of Lipofectamine2000 (3 ul and 4.5 ul) to find the optimal ratio of Lipofectamine2000 to DNA. Experiments were repeated 3 times.

(211) Flow Cytometry Analysis

(212) Cells were analysed on a MoFlo™ DP (DAKO) Flow Cytometer using a 633 nm red laser. (40.000 cells: gated on live cells by forward/side scatter). Median fluorescence was used for statistical analyses.

(213) Results and Discussion

(214) Peptide to DNA Binding

(215) YO-PRO-1 assay can be used to investigate the DNA condensation ability of a DNA binding peptide. YO-PRO-1 is a cyanine dye that binds DNA to form a fluorescent DNA/dye complex. Different (+/−) charge ratios of peptide can be added to the fluorescent DNA/dye complex. As the peptide out competes the dye by binding the DNA a reduction in fluorescence intensity is observed. In this study, LK15 was fused to P21 8R transduction protein to improve the DNA binding ability of the modified cell penetrating peptide. Fusion of LK15 peptide to TAT has been shown to significantly improve transfection of pDNA into HT29 and HT1080 cultured cells [19]. Enhanced transduction efficiency of Tat-LK15 over Tat is thought to be due to the improved condensation ability of the peptide and DNA, and better transduction of the DNA across the cell membrane [20].

(216) A graph of (+/−) charge ratio was plotted against % fluorescence to investigate the optimum ratio of P21 LK15 8R to pSIN GFP (FIG. 40). Results showed that the optimal (+/−) charge ratio of P21 LK15 8R to pSIN GFP was 2:1, respectively. This ratio was used in the transfection experiments.

(217) Transfection of pDNA Reporter Gene Via P21 LK15 8R

(218) The phospholipid bilayer of the cell membrane acts as an impenetrable barrier to nucleic acids and thus pDNA will be conjugated to a modified CPP to facilitate its transport into the cell [2]. In this study the GET-mediated transfection of the reporter gene pSIN GFP into NIII 3T3 murine fibroblast cells was optimized in terms of transfection time (3, 6 or 24 h), transfection media (with or without serum) and amount of DNA (1, 4 or 10 ug). The reporter gene pSIN GFP was transfected into cells using P21 LK15 8R where P21 targets and binds cell surface HS-GAGs, LK15 complexes pSIN GFP and 8R transduces pSIN GFP across the cell membrane. The transfection efficiency of pSIN GFP with P21 LK15 8R was compared to the transfection efficiency of commercially used lipid-based transfection reagent lipofectamine2000. Cells were fixed at 48 h following transfection to allow time for the transient expression of GFP to be captured and transfection efficiencies were quantified by flow cytometry (FIG. 41).

(219) Gene carrier systems must be serum resistant for efficacious in-vivo applications, however most gene carries, including lipofectamine2000, have demonstrated steep decreases in transfection efficiency in serum containing media [21]. This is believed to be because serum molecules competitively bind the gene carrier, therefore decreasing free gene carriers available to bind the DNA [22]. The transfection efficiency of P21 LK15 8R was characterised in serum and serum free transfection media. The optimal transfection conditions were when cells were transfected with 10 ug DNA for 24 h in serum conditions where transfection efficiency reached 17.9±4.8%. (FIG. 42). This is 3 fold lower than the optimized transfection efficiency observed for lipofectamine2000 in serum free conditions (54.7±10.3%, Appendix 3) however, the serum-resistance of P21 LK15 8R transfection is advantageous for any sort of clinical/in-vivo delivery of therapeutic biomolecules. In addition, it is well documented that endosomal escape strategies greatly increase the efficiencies of CPP-mediated transfections.

(220) Conclusions

(221) A panel of CPPs that have been modified to include growth factor derived cell surface HS-GAG binding domains have shown 30-100 fold increase in transduction into cells, compared to unmodified CPPs. The hypothesis is that the GET-mediated delivery of these peptides is due to the dual functionality of the peptide in i) increasing interaction with the cell membrane via the HS-GAG binding domain, and ii) transducing protein across cell membrane via 8R. The modified CPPs showed preferential delivery profiles of mRFP into different cell types, this is due the HS-GAG binding domains targeting specific HS-epitopes that are more abundantly expressed in different cell types. Future work is to modify CPPs to include specific antibody-derived HS-epitope binding domains. HS-epitope binding libraries of antibodies can be utilized for the cell type specific delivery of therapeutic molecules via GET.

(222) To demonstrate the utility of these peptides for the delivery of therapeutic molecules P21 LK15 8R was used to deliver the reporter gene pSIN GFP into cells. Results showed GET-mediated transfection efficiencies of up to 17.9±4.8% in serum conditions, without any endosomal escape strategy. CPPs modified to include HS-GAG binding domains show great promise as alternatives to using viral and lipid based delivery vehicles for the in-vivo delivery of therapeutic biomolecules.

REFERENCES

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(224) TABLE-US-00006 APPENDIX 1 Table showing amounts of P21 LK15 8R added to 1 ug of pSIN GFP at different (+/−) charge ratios Peptide/DNA (+/−) Charge Concentration Ratio of peptide (uM) Volume of peptide (ul) 1:5 0.49 0.05 1:3 0.82 0.07 1:2 1.23 0.12 1:1 2.47 0.24 2:1 4.94 0.49 3:1 7.41 0.74 5:1 12.35 1.23 10:1  24.7 2.46

(225) TABLE-US-00007 APPENDIX 2 Table showing the different treatment conditions for the optimization of the transfection of pSIN GFP into NIH 3T3 cells by P21 LK15 8R. Transfection Media (Optimem, Amount of DNA OptiMEM + 10% Transfection Well (ug) Serum) Time (h) 1 1 Opti-MEM ® 3 h 2 1 Opti-MEM ® + 3 h 10% serum 3 1 Opti-MEM ® 6 h 4 1 Opti-MEM ® + 6 h 10% serum 5 1 Opti-MEM ® 24 h  6 1 Opti-MEM ® + 24 h  10% serum 7 4 Opti-MEM ® 3 h 8 4 Opti-MEM ® + 3 h 10% serum 9 4 Opti-MEM ® 6 h 10 4 Opti-MEM ® + 6 h 10% serum 11 4 Opti-MEM ® 24 h  12 4 Opti-MEM ® + 24 h  10% serum 13 10 Opti-MEM ® 3 h 14 10 Opti-MEM ® + 3 h 10% serum 15 10 Opti-MEM ® 6 h 16 10 Opti-MEM ® + 6 h 10% serum 17 10 Opti-MEM ® 24 h  18 10 Opti-MEM ® + 24 h  10% serum