CONSTRUCT FOR THE DELIVERY OF A MOLECULE INTO THE CYTOPLASM OF A CELL

Abstract

Described is a construct comprising (a) a targeting moiety; (b) a fusogenic moiety consisting one or more fusogenic sequence(s) derived from dengue virus glycoprotein E comprising the sequence DRGWGNGCGLFGKGGI (SEQ ID NO:1) or a sequence which shows 1 to 8 substitutions, deletions, or insertions in comparison to SEQ ID NO:1; and (c) a molecule which is to be delivered into the cytoplasm of a cell. Moreover, described is a pharmaceutical composition comprising the construct according to the invention and optionally a pharmaceutical acceptable carrier. Further, described is a kit comprising one or more fusogenic sequence(s) derived from dengue virus glycoprotein E comprising the sequence as shown in SEQ ID NO:1 or a sequence which shows 1 to 8 substitutions, deletions, or insertions in comparison to SEQ ID NO:1. Further, described is the use of one or more fusogenic sequence(s) derived from dengue virus glycoprotein E for use in delivery of a therapeutic moiety, a detectable moiety, a nucleic acid molecule, preferably an siRNA, a carrier molecule, preferably a nanoparticle, a liposome and a viral vector into the cytoplasm of a cell.

Claims

1. A construct comprising (a) a targeting moiety; (b) a fusogenic moiety consisting of one or more fusogenic sequence(s) derived from dengue virus glycoprotein E comprising the sequence DRGWGNGCGLFGKGGI (SEQ ID NO:1) or a sequence which shows 1 to 8 substitutions, deletions, or insertions in comparison to SEQ ID NO:1; and (c) a molecule which is to be delivered into the cytoplasm of a cell.

2. The construct according to claim 1, wherein the targeting moiety is an antibody, an antibody fragment, a cytokine or a ligand.

3. The construct according to claim 1, wherein the targeting moiety is a F(ab′).sub.2, F(ab).sub.2, Fab′, a Fv antibody fragment a scFv a Fab, a VH, an scFv-Fc, an sdFv, a diabody, a triabody, a tetrabody, a minibody, a tandem-scFv, a tandem-scFv-Fc, an scFv-Fc-scFv, a Fab-scFv, a Fab.sub.3, an IgG-scFv, an scFv-IgG, an IgG-VH or a single domain antibody, preferably a sdAb, a V.sub.HH fragment from camelids, or a V.sub.NAR fragment from cartilaginous fishes.

4. The construct according to claim 1, wherein the targeting moiety targets an antigen specific for cancer, an antigen specific for infectious diseases or an antigen specific for autoimmune diseases.

5. The construct according to claim 1, wherein the targeting moiety is a humanized anti-EGFR antibody single chain Fv fragment (scFv).

6. The construct according to claim 1, wherein the molecule which is to be delivered into the cytoplasm is selected from the group consisting of a therapeutic moiety, a detectable moiety, a nucleic acid molecule, preferably an siRNA, a carrier molecule, preferably a nanoparticle, a liposome and a viral vector.

7. The construct according claim 6, wherein the therapeutic moiety is selected from the group consisting of a cytotoxic moiety, an antibody, an antibody fragment, a drug, a chemotherapeutic agent, a small molecule, an enzyme, a hormone, an antisense oligonucleotide, siRNA, RNAi, a radionuclide, a boron compound, a photoactive agent, an anti-angiogenic agent and a pro-apoptotic agent.

8. The construct according to claim 7, wherein the cytotoxic moiety is selected from the group consisting of RNase A family members, ricin, abrin, alpha toxin, saporin, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

9. The construct according to claim 8, wherein the RNase A family member is an RNase derived from Rana pipines.

10. The construct according to claim 1, wherein the fusogenic moiety consists of one or more fusogenic sequence(s) selected from the group consisting of TABLE-US-00015 (SEQ ID NO: 5) DRGWGNGCGLFGKGSI; (SEQ ID NO: 6) DRGWGNGCGLFGKGSL; (SEQ ID NO: 7) DRGWGNGCGLFGKGGV; (SEQ ID NO: 8) DRGWHNGCGLFGKGSI; (SEQ ID NO: 9) DRGWHNGCGFFGKGSI; (SEQ ID NO: 10) DRGWGNGCGLFGKGSM; (SEQ ID NO: 11) DRGWGNGCGLFGKGSY; (SEQ ID NO: 12) DRGWNNGCGLFGKGSL; (SEQ ID NO: 13) NRGWNNGCGLFGKGDI; (SEQ ID NO: 14) DRGWGNHCGLFGKGSI; (SEQ ID NO: 15) DRGWGNNCGLFGKGSI; (SEQ ID NO: 16) DRGWGNGCALFGKGSI; (SEQ ID NO: 17) DRGWGNHCGFFGKGSI; (SEQ ID NO: 18) DRGWDSGCFIFGKGEV; (SEQ ID NO: 19) NRGWGTGCFKWGIGFV and (SEQ ID NO: 20) NRGWGTGCFEWGLGQV.

11. The construct according to claim 1, wherein the targeting moiety, the fusogenic moiety consisting of one or more fusogenic sequence(s) and the molecule which is to be delivered into the cytoplasm of a cell are chemically coupled in a covalent linkage.

12. The construct according to claim 1, wherein the construct is a fusion protein.

13. A nucleic acid molecule encoding the fusion protein according claim 12.

14. A vector comprising the nucleic acid molecule of claim 13.

15. A host cell comprising the vector of claim 14.

16. A pharmaceutical composition comprising the construct according to claim 1 and optionally a pharmaceutical acceptable carrier.

17. The pharmaceutical composition of claim 16 for use in treating cancer, a cardiovascular disease, a viral infection, an immune dysfunction, an autoimmune disease, a neurologic disorder, an inherited metabolic disorders or a genetic disorder.

18. A kit comprising the fusogenic moiety of claim 1.

19. Use of the fusogenic moiety of claim 1 to deliver a therapeutic moiety, a detectable moiety, a nucleic acid molecule, preferably an siRNA, a carrier molecule, preferably a nanoparticle, a liposome and a viral vector into the cytoplasm of a cell.

20. The use according to claim 19, wherein said fusogenic moiety consists of one or more fusogenic sequence(s) derived from dengue virus glycoprotein E comprising the sequence as shown in SEQ ID NO:1.

21. The use according to claim 20, wherein said one or more fusogenic sequence(s) is a sequence which shows 1 to 8 substitutions, deletions, or insertions in comparison to SEQ ID NO:1.

Description

[0195] FIG. 1: shows a schematic presentation of scFv (A), Ranpirnase-GS-scFv (B) and Ranpirnase-DEN-scFv (C). Ranpirnase was fused to the N-terminus of the scFv fragment either by a glycine-serine linker (B) or by a fusogenic peptide from dengue virus whose sequence is indicated (C). V.sub.H and V.sub.L: variable domain of the heavy and light chain, respectively, of a humanized scFv fragment derived from Cetuximab. A C-terminal c-myc tag (c-myc) and a hexahistidine tag (Hiss) was used for detection and purification purposes, respectively.

[0196] FIG. 2: shows an assessment of specific antitumor activity in vitro. Cell viability of EGFR-positive (A) and EGFR-negative (B) tumor cell lines was determined after 72 h of treatment with indicated concentrations of scFv, Ranpirnase, Ranpirnase-GS-scFv or Ranpirnase-DEN-scFv. Results are expressed relative to buffer-treated control cells. Data depict the mean values ±SE from one representative experiment performed in triplicates.

[0197] FIG. 3: shows the antigen-specific cytotoxicity of Ranpirnase-DEN-scFv. HNO211 cells were treated with Ranpirnase-DEN-scFv (20 nM) either in the presence or absence of a 50-fold molar excess of anti-EGFR scFv (1000 nM). For control, cells were treated with anti-EGFR scFv (1000 nM) alone. Cell viability was determined after 72 h by MTT-Assay. Data depict the mean values ±SE from one representative experiment performed in triplicates.

[0198] Other aspects and advantages of the invention will be described in the following examples, which are given for purposes of illustration and not by way of limitation. Each publication, patent, patent application or other document cited in this application is hereby incorporated by reference in its entirety.

EXAMPLES

[0199] Materials and Methods

[0200] 1. Cloning of Ranpirnase-GS-scFv and Ranpirnase-DEN-scFv Fusion Proteins

[0201] The gene of Ranpirnase (UniProtKB/Swiss-Prot: P22069.2) with an appending gene segment for a C-terminal (G.sub.4S).sub.3 linker and flanking restriction sites was synthesized for optimized expression in mammalian cells (Entelechon, Bad Abbach, Germany) and cloned as ApaLI/PvuII fragment into the subcloning vector pMJA-1B (Krauss et al., 2005a). For the generation of the immunoRNase Ranpirnase-GS-scFv gene, the DNA sequence of the humanized anti-epidermal growth factor receptor (EGFR) scFv IZI08 (Seifert et al., 2012) generated from the antibody C225 (Goldstein et al., 1995) was amplified by polymerase chain reaction (PCR) using ESR-1s (5′-TATAGAAGTGCAGCTGGTTGAAAGC-3′) forward primer introducing a PvuII restriction site at the 5′ end of the V.sub.H domain DNA sequence and ESR-2as (5′-TATAGGATCCACGTTTAATTTCCAG-3′) reverse primer that appends a BamHI restriction site at the 3′ end of the VL domain DNA sequence. Prior to insertion of the IZI08 gene into the subcloning vector containing the Ranpimase gene silent mutations for disrupting additional internal PvuII and BamHI sites were introduced. The anti-EGFR scFv IZI08 gene was subsequently cloned as PvuII/BamHI fragment downstream of the Ranpimase gene into subcloning vector pMJA-1B containing the DNA sequences encoding for a c-myc tag and a hexahistidine tag. The DNA sequence for the standard (G.sub.4S).sub.3 linker sequence between the RNase and antibody moiety was exchanged by the DNA sequence coding for amino acids 96-114 of glycoprotein E of dengue virus serotype 2 by overlap extension PCR resulting in Ranpimase-DEN-scFv cassette subcloning vector pDEN14. Ranpimase-GS-scFv and Ranpimase-DEN-scFv fusion protein encoding genes were digested with EcoRI and cloned into mammalian cell expression vector pEE12.4 (Lonza Biologics, Slough, UK). The correct orientation of the DNA insert was confirmed by restriction digest using BamHI.

[0202] 2. Cell Lines and Proteins

[0203] The cell lines A431 (human epidermoid carcinoma), MCF7 (human breast adenocarcinoma) and Raji (human Burkitt's lymphoma) were purchased from ATCC (Manassas, Va., USA). The human head and neck squamous cell carcinoma (HNSCC) cell lines HNO97 (oral cavitiy), HNO211 (oropharynx) and HNO410 (hypopharyngeal lymph node metastasis) were established from surgical specimens of HNSCC patients after informed consent and approval by the ethics committee of the Faculty of Medicine, Heidelberg University. A431, MCF7, and HNSCC cell lines were cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich, Taufkirchen, Germany) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich) in a humidified incubator with 5% CO.sub.2 at 37° C. Raji cells were cultivated in RPMI1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin under identical conditions. HEK293-6E cells (licensed from National Research Council, Biotechnological Research Institute, Montreal, Canada) were cultured in F17 medium (Invitrogen, Life Technologies, Darmstadt, Germany) supplemented with 0.1% Kolliphor P188 (Sigma-Aldrich), 4 mM glutamine (Invitrogen) and 25 μg/ml G418 (Carl Roth, Karlsruhe, Germany) in shaker incubators at 37° C., 5% CO.sub.2 and 120 rpm.

[0204] For controls Ranpimase was kindly provided by Kuslima Shogen, Alfacell Corporation.

3. Expression and Purification of scFv and ImmunoRNases

[0205] Soluble expression of the anti-EGFR scFv into the periplasm of E. coli TG1 cells (Stratagene, Agilent Technologies, Santa Clara, Calif., USA) using vector pAB1 (Müller et al., 2007) was performed as described previously (Diebolder et al., 2014). The scFv-containing periplasmic extract was thoroughly dialyzed against SP10 buffer (20 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 10 mM imidazole, pH 8.0) at 4° C. for further purification by immobilized metal ion affinity chromatography (IMAC). RNase fusion proteins were transiently expressed in suspension growing HEK293-6E cells in shaker flasks (Falcon, Becton Dickinson, Heidelberg, Germany). At a cell density of 1.7- to 2×10.sup.6 cells/ml 1 μg endofree plasmid DNA and 2 μg polyethylenimine (Polysciences, Warrington, Pa., USA) per ml final culture volume were prepared separately in 1/20 of final culture volume in F17 medium without G418, mixed, incubated at room temperature for 3 min and added to HEK293-6E cells. One day after transfection, cells were supplemented with 0.5% (w/v) tryptone TN1 (Organotechnie S.A.S, La Courneuve, France). After 5 days post transfection, protein-containing supernatants were collected and dialyzed either against SP20 (20 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 20 mM imidazole, pH 8.0) for Ranpimase-GS-scFv or Tris-HCl buffer (25 mM Tris, 500 mM NaCl, pH 8.2) for Ranpimase-DEN-scFv, respectively, using the SARTOFLOW® Slice 200 benchtop crossflow system (Sartorius, Goettingen, Germany). Purification of recombinant proteins by IMAC was conducted using Ni-NTA columns (GE Healthcare, Muenchen, Germany), equilibrated with the corresponding buffer. After extensive washing with buffer, bound proteins were eluted with a multiple-step gradient of imidazole containing buffer. Fractions containing the recombinant protein (determined by SDS-PAGE and Simply Blue Safe Stain (Invitrogen) and western blot) were pooled and dialyzed against PBS overnight at 4° C. Final purification and separation of monomeric scFv fragments and immunoRNases from higher molecular weight species was done by size exclusion chromatography in PBS buffer using a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare).

4. Determination of Ribonucleolytic Activity

[0206] Ribonucleolytic activity of Ranpimase and immunoRNases was measured by monitoring cleavage of the fluorogenic substrate 6-Carboxyfluorescein-dArUdGdA-Black-Hole-Quencher-1 (6-FAM-dArUdGdA-BHQ-1) (biomers.net, Ulm, Germany) over time. The fluorescence intensity was measured in 96-well black microtiter plates using an Infinite F200Pro microplate reader (Tecan, Maennedorf, Switzerland) with a 485/535 nm (excitation/emission) filter set. The reaction was carried out in 100 mM MES-NaOH buffer (pH 6.0) containing 100 mM NaCl and 6-FAM-dArUdGdA-BHQ-1 (5 nM) at 25° C. in a total reaction volume of 200 μl per well. Buffer without RNase served as negative control and an excess concentration of RNase A was used as positive control. At least three independent assays each containing triplicates were performed.

[0207] Values of k.sub.cat/K.sub.M were calculated using the equation:

[00001]$k_{\mathrm{cat}}/K_M=\frac{\left(\Delta .Math..Math.F/\Delta .Math..Math.t\right)}{\left(F_{\max }-F_{\min }\right).Math.\left[E\right]}$

[0208] In this equation, ΔF/Δt represents the initial reaction velocity, F.sub.min is the initial fluroescence intensity before addition of RNase, F.sub.max is the fluorescence intensity after complete cleavage of the substrate by excess RNase A and [E] is the RNase concentration.

[0209] 5. Antibody Binding

[0210] For determination of equilibrium-binding curves A431 cells were incubated in triplicates with serial dilutions of either purified anti-EGFR scFv, Ranpimase-GS-scFv or Ranpimase-DEN-scFv. Detection of bound antibody or immunoRNase was performed using murine anti-c-myc monoclonal antibody clone 9E10 (Roche, Penzberg, Germany) and goat-anti-mouse fluorescein isothiocyanate (FITC) conjugate (Jackson ImmunoResearch, Suffolk, UK). Fluorescence of stained cells was measured on a FACS Canto II flow cytometer (Becton Dickinson) using the FACS Diva Software (Becton Dickinson). Background fluorescence was substracted from measured median fluorescence and relative affinities were calculated by nonlinear regression using GraphPad Prism 5.0 (GraphPad Software, La Jolla, Calif., USA).

[0211] 6. Cell Viability Assays and Competition Analysis

[0212] In order to assess the antitumor efficacy of the recombinant proteins, cells were seeded in a 96-well flat-bottom plate (Falcon) and incubated with different concentrations of protein or buffer as control at 37° C., 5% CO.sub.2 for 72 h in a total volume of 110 μl. For adherent cell lines (A431, HNO97, HNO211, HNO410, MCF7), cell viability was determined by addition of 20 μl of a 5 mg/ml solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) diluted in PBS. After incubation with MTT at 37° C., 5% CO.sub.2 for 4 h medium was removed and cells were lysed in 100 μl lysis solution (10% SDS (w/v) and 0.6% acetic acid (v/v) in dimethyl sulfoxide) per well. Plates were incubated at room temperature for 5 min followed by gentle agitation for 5 min to dissolve released formazan crystals. Formazan concentration was determined by measuring absorbance at 570 nm (reference: 620 nm) using an Infinite F200Pro microplate reader (Tecan). For the determination of viability of the suspension cell line Raji, cells were incubated with 10 μl alamarBlue® (ThermoScientific, Rockford, Ill., USA) per well at 37° C., 5% CO.sub.2 for 4 h without subsequent cell lysis. Absorbance was directly measured using the same wavelengths and instrumentation as for MTT-treated cells.

[0213] Cell viability was expressed as percentage of viable cells treated with protein related to buffer control. IC.sub.50 was defined as the concentration at which cell viability was reduced by 50% related to buffer control. Each assay was performed at least in duplicate with each assay containing triplicates.

[0214] For competition analysis, HNO211 cells were seeded in a 96-well flat-bottom plate (Falcon) and pre-incubated for 3 h with the anti-EGFR scFv IZI08 at a concentration of 1000 nM before Ranpimase-DEN-scFv (20 nM) was added.

[0215] For controls cell were either incubated with Ranpimase-DEN-scFv (20 nM) or the anti-EGFR scFv alone (1000 nM). After incubation for 72 h at 37° C., 5% CO.sub.2 cell viability was determined using the MTT assay as described above. All reactions were performed in triplicates.

Example 1: Generation of Ranpirnase-GS-scFv and Ranpirnase-DEN-scFv

[0216] For targeted killing of EGFR-expressing cancer cells, Ranpimase was fused to the N-terminus of a humanized scFv fragment with specificity identical to the clinically established mAb Cetuximab (Erbitux®) by a flexible (G.sub.4S).sub.3 linker (Ranpimase-GS-scFv). Alternatively, Ranpimase was fused to the scFv fragment by a linker composed of a viral sequence including the putative viral fusion peptide of dengue virus serotype 2 reported to be involved in the endosomal escape mechanism of the virus (Melo et al., 2009; Zaitseva et al., 2010), resulting in the immunoRNase Ranpimase-DEN-scFv. A schematic overview of investigated proteins is shown in FIG. 1.

Example 2: Expression and Purification of scFv and ImmunoRNases

[0217] The scFv fragment was expressed in E. coli and isolated from the periplasmic space whereas immunoRNases Ranpimase-GS-scFv and Ranpimase-DEN-scFv were produced in HEK293-6E cells and secreted into the cell culture supernatant by employment of an IgV.sub.H leader peptide (Krauss et al., 2005a). Proteins were purified by Ni-NTA columns followed by size exclusion chromatography, yielding homogeneous protein preparations with >95% purity. Production yields after complete purification were 1.9 mg/l for scFv, 0.9 mg/l for Ranpimase-GS-scFv and 3.2 mg/l for Ranpimase-DEN-scFv, respectively.

Example 3: Functional Analysis of ImmunoRNases

[0218] Cell binding and apparent equilibrium dissociation constants (K.sub.D) of scFv, Ranpimase-GS-scFv and Ranpimase-DEN-scFv were determined by flow cytometry on EGFR-expressing A431 cells. As shown in Table I both immunoRNases bound to the target antigen with high affinity similar to the scFv alone.

TABLE-US-00012 TABLE I Affinity of the antibody constructs for binding to A431 cells as analyzed by flow cytometry. K.sub.D ± SE (nM) scFv  9.6 ± 1.2 Ranpirnase-GS-scFv 14.7 ± 1.2 Ranpirnase-DEN-scFv 11.2 ± 0.8

[0219] Ribonucleolytic activity of Ranpimase, Ranpimase-GS-scFv and Ranpimase-DEN-scFv was measured by their ability to cleave a fluorogenic RNA substrate matching the nucleobase specificity of Ranpimase (Lee and Raines, 2003). Fusion of Ranpimase to the N-terminus of the scFv fragment by a flexible glycine-serine linker slightly reduced its catalytic activity when compared to wild-type Ranpimase (Table II).

TABLE-US-00013 TABLE II Ribonucleolytic activity of Ranpirnase and Ranpirnase fusion proteins k.sub.cat/K.sub.M (10.sup.3 M.sup.−1s.sup.−1)* Ranpirnase 11.3 ± 1.0  Ranpirnase-GS-scFv 8.7 ± 0.4 Ranpirnase-DEN-scFv 4.2 ± 0.4 *values of k.sub.cat/K.sub.M (±SE) for cleavage of 6-FAM-dArUdGdA-BHQ-1 were determined as described in Material and Methods.

[0220] The catalytic activity of Ranpimase-DEN-scFv was about 2-fold lower in comparison to Ranpimase-GS-scFv, indicating that linker-dependent alterations in folding and conformation of Ranpimase or steric hindrances with substrate interaction may have occurred.

Example 4: In Vitro Cytotoxicity

[0221] For evaluating the specific toxicity towards EGFR-expressing cancer cells the scFv fragment, Ranpimase-GS-scFv, Ranpimase-DEN-scFv and Ranpimase were incubated with the EGFR-overexpressing epidermoid carcinoma cell line A431 and several EGFR-positive primary cell lines derived from resected tumors of head and neck cancer patients (HNO97, HNO211, HNO410). The EGFR-negative breast cancer cell line MCF7 and Burkitt's lymphoma cell line Raji served as negative controls. As shown in FIG. 2 and Table III the anti-EGFR scFv fragment alone had almost no effect on cell viability.

TABLE-US-00014 TABLE III Antitumor activity of tested constructs towards EGFR- positive cell lines in vitro. IC.sub.50 values are indicated as mean ± SE and are derived from at least two independent experiments each performed in triplicates. IC.sub.50 (nM) A431 HNO97 HNO211 HNO410 scFv >3618 >3618 >3618 >3618 Ranpirnase-GS- >3618 1262 ± 59  1423 ± 712 2524 ± 168 scFv Ranpirnase-DEN- 38 ± 5 411 ± 55 18 ± 8 308 ± 85 scFv Ranpirnase 107 ± 29 255 ± 38  4 ± 1 119 ± 48

[0222] As Ranpimase is capable to enter cells in an EGFR-independent manner (Rodriguez et al., 2007), Ranpimase alone exerted cytotoxicity towards both EGFR-negative and EGFR-positive cells (FIG. 2). Fusion of Ranpimase to the N-terminus of the scFv fragment by a flexible glycine-serine linker (Ranpimase-GS-scFv) resulted in significantly diminished cytotoxicity when compared with Ranpimase as single agent. In contrast, the immunoRNase containing the fusogenic peptide derived from dengue virus (Ranpimase-DEN-scFv) exhibited similar antitumor efficacy as Ranpimase alone towards EGFR-expressing cells yet did not affect EGFR-negative cells even at very high concentrations (FIG. 2B). Ranpimase-DEN-scFv exhibited strongest cytotoxicity towards the primary HNO211 cell line with an average IC.sub.50 value of 18 nM corresponding to a 79-fold increase in potency compared to Ranpimase-GS-scFv.

[0223] To prove that the introduced viral linker sequence has no negative impact on the specificity of Ranpimase-DEN-scFv, we performed competition assays on the HNO211 cell line. As shown in FIG. 3 cytotoxicity of Ranpimase-DEN-scFv could be completely abrogated in the presence of a 50-fold molar excess of scFv, confirming that both compounds bind to the same targeted epitope and that the cell entry of Ranpimase-DEN-scFv is EGFR-specific and not mediated by unspecific interaction of the fusogenic peptide with the plasma membrane.

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