Identification of folate receptor α (FRα) binding oligopeptides and their evaluation for targeted virotherapy applications

Oncolytic virotherapies (OV) based on human adenoviral (HAdV) vectors hold significant promise for the treatment of advanced ovarian cancers where local, intraperitoneal delivery to tumour metastases is feasible, bypassing many complexities associated with intravascular delivery. The efficacy of HAdV-C5-based OV is hampered by a lack of tumour selectivity, where the primary receptor, hCAR, is commonly downregulated during malignant transformation. Conversely, folate receptor alpha (FRα) is highly expressed on ovarian cancer cells, providing a compelling target for tumour selective delivery of virotherapies. Here, we identify high-affinity FRα-binding oligopeptides for genetic incorporation into HAdV-C5 vectors. Biopanning identified a 12-mer linear peptide, DWSSWVYRDPQT, and two 7-mer cysteine-constrained peptides, CIGNSNTLC and CTVRTSAEC that bound FRα in the context of the phage particle. Synthesised lead peptide, CTVRTSAEC, bound specifically to FRα and could be competitively inhibited with folic acid. To assess the capacity of the elucidated FRα-binding oligopeptides to target OV to FRα, we genetically incorporated the peptides into the HAdV-C5 fiber-knob HI loop including in vectors genetically ablated for hCAR interactions. Unfortunately, the recombinant vectors failed to efficiently target transduction via FRα due to defective intracellular trafficking following entry via FRα, indicating that whilst the peptides identified may have potential for applications for targeted drug delivery, they require additional refinement for targeted virotherapy applications

Adenovirus Gene Transfer to Amelogenesis Imperfecta Ameloblast-Like Cells

To explore gene therapy strategies for amelogenesis imperfecta (AI), a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5) vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR) on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including “pK7” and/or “RGD” motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3) fiber “knob” domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold) of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both αvβ3/αvβ5 integrins and heparan sulfate proteoglycans (HSPGs) highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI

Development of a low-seroprevalence, αvβ6 integrin-selective virotherapy based on human adenovirus type 10

Oncolytic virotherapies (OV) hold immense clinical potential. OV based on human adenoviruses (HAdV) derived from HAdV with naturally low rates of pre- existing immunity will be beneficial for future clinical translation. We generated a low- seroprevalence HAdV-D10 serotype vector incorporating an αvβ6 integrin-selective peptide, A20, to target αvβ6-positive tumor cell types. HAdV-D10 has limited natural tropism. Structural and biological studies of HAdV-D10 knob protein highlighted low-affinity engagement with native adenoviral receptors CAR and sialic acid. HAdV-D10 fails to engage blood coagulation factor X, potentially eliminating “off-target” hepatic sequestration in vivo. We engineered an A20 peptide that selectively binds αvβ6 integrin into the DG loop of HAdV-D10 fiber knob. Assays in αvβ6+ cancer cell lines demonstrated significantly increased transduction mediated by αvβ6-targeted variants compared with controls, confirmed microscopically. HAdV-D10.A20 resisted neutralization by neutralizing HAdV-C5 sera. Systemic delivery of HAdV-D10.A20 resulted in significantly increased GFP expression in BT20 tumors. Replication-competent HAdV-D10.A20 demonstrated αvβ6 integrin-selective cell killing in vitro and in vivo. HAdV-D10 possesses characteristics of a promising virotherapy, combining low seroprevalence, weak receptor interactions, and reduced off-target uptake. Incorporation of an αvβ6 integrin-selective peptide resulted in HAdV-D10.A20, with significant potential for clinical translation

Gene therapy for monogenic liver diseases: clinical successes, current challenges and future prospects

Over the last decade, pioneering liver-directed gene therapy trials for haemophilia B have achieved sustained clinical improvement after a single systemic injection of adeno-associated virus (AAV) derived vectors encoding the human factor IX cDNA. These trials demonstrate the potential of AAV technology to provide long-lasting clinical benefit in the treatment of monogenic liver disorders. Indeed, with more than ten ongoing or planned clinical trials for haemophilia A and B and dozens of trials planned for other inherited genetic/metabolic liver diseases, clinical translation is expanding rapidly. Gene therapy is likely to become an option for routine care of a subset of severe inherited genetic/metabolic liver diseases in the relatively near term. In this review, we aim to summarise the milestones in the development of gene therapy, present the different vector tools and their clinical applications for liver-directed gene therapy. AAV-derived vectors are emerging as the leading candidates for clinical translation of gene delivery to the liver. Therefore, we focus on clinical applications of AAV vectors in providing the most recent update on clinical outcomes of completed and ongoing gene therapy trials and comment on the current challenges that the field is facing for large-scale clinical translation. There is clearly an urgent need for more efficient therapies in many severe monogenic liver disorders, which will require careful risk-benefit analysis for each indication, especially in paediatrics

Photophysical characterization and the self-assembly properties of mono- and bis-pyrene derivatives for cell imaging applications

A series of fluorescent mono-(1-4) and bis-pyrene derivatives (5-7) were synthesized and their photo-physical and self-aggregation properties in solution were fully investigated by UV-vis and fluorescence spectroscopies and dynamic light scattering (DLS). While in dilute DMSO mono-pyrene derivatives 1-4 show the characteristic features of monomer emission, bis-pyrene derivatives 5-7 display both monomer and excimer emissions, the latter attributable to a dynamic process. The gelation behaviour of these compounds was also studied, and only mono-pyrene leucine derivative 1 was found to display efficient gelation in aromatic solvents, such as toluene and p-xylene, and in a polar solvent mixture, water/DMSO (4 : 1 v/v). Finally, mono-pyrenes 1-4 showed no apparent cytotoxic impact on A549 and RD cell lines; derivatives 1-5 showed accumulation within both live and fixed cells, and macropinocytosis was found to be the main pathway for 5 to enter into the cells

Talin2 and KANK2 functionally interact to regulate microtubule dynamics, paclitaxel sensitivity and cell migration in the MDA-MB-435S melanoma cell line

Abstract Background Focal adhesions (FAs) are integrin-containing, multi-protein structures that link intracellular actin to the extracellular matrix and trigger multiple signaling pathways that control cell proliferation, differentiation, survival and motility. Microtubules (MTs) are stabilized in the vicinity of FAs through interaction with the components of the cortical microtubule stabilizing complex (CMSC). KANK (KN motif and ankyrin repeat domains) family proteins within the CMSC, KANK1 or KANK2, bind talin within FAs and thus mediate actin-MT crosstalk. We previously identified in MDA-MB-435S cells, which preferentially use integrin αVβ5 for adhesion, KANK2 as a key molecule enabling the actin-MT crosstalk. KANK2 knockdown also resulted in increased sensitivity to MT poisons, paclitaxel (PTX) and vincristine and reduced migration. Here, we aimed to analyze whether KANK1 has a similar role and to distinguish which talin isoform binds KANK2. Methods The cell model consisted of human melanoma cell line MDA-MB-435S and stably transfected clone with decreased expression of integrin αV (3αV). For transient knockdown of talin1, talin2, KANK1 or KANK2 we used gene-specific siRNAs transfection. Using previously standardized protocol we isolated integrin adhesion complexes. SDS-PAGE and Western blot was used for protein expression analysis. The immunofluorescence analysis and live cell imaging was done using confocal microscopy. Cell migration was analyzed with Transwell Cell Culture Inserts. Statistical analysis using GraphPad Software consisted of either one-way analysis of variance (ANOVA), unpaired Student’s t-test or two-way ANOVA analysis. Results We show that KANK1 is not a part of the CMSC associated with integrin αVβ5 FAs and its knockdown did not affect the velocity of MT growth or cell sensitivity to PTX. The talin2 knockdown mimicked KANK2 knockdown i.e. led to the perturbation of actin-MT crosstalk, which is indicated by the increased velocity of MT growth and increased sensitivity to PTX and also reduced migration. Conclusion We conclude that KANK2 functionally interacts with talin2 and that the mechanism of increased sensitivity to PTX involves changes in microtubule dynamics. These data elucidate a cell-type-specific role of talin2 and KANK2 isoforms and we propose that talin2 and KANK2 are therefore potential therapeutic targets for improved cancer therapy. Graphical Abstrac

Additional file 1 of Talin2 and KANK2 functionally interact to regulate microtubule dynamics, paclitaxel sensitivity and cell migration in the MDA-MB-435S melanoma cell line

Additional file 1: Fig. S1. KANK1 localization is not affected by talin1 or talin2 knockdown. (A, B) Talin2, but not talin1, knockdown slightly affects KANK1 appearance. Forty-eight hours after transfection with either control, talin1 or talin2-specific siRNA, MDA-MB-435S cells were methanol fixed and stained with anti-talin1 or anti-talin2 antibody followed by Alexa-Fluor 546-conjugated antibody (red) or Alexa-Fluor 488-conjugated antibody (green), respectively. KANK1 was further visualized by anti-KANK1 antibody followed by Alexa-Fluor 555-conjugated antibody or Alexa-Fluor 488-conjugated antibody (shown in magenta). Finally, vinculin was visualized using conjugated anti-vinculin Alexa Fluor 647 antibody (shown in grey) and IRM images were taken. Analysis was performed using TCS SP8 Leica. Scale bar = 10 µm. (C) KANK1 knockdown does not affect sensitivity to PTX. Sensitivity of cells transfected with either control or KANK1-specific siRNA to PTX was measured by MTT assay. Twenty-four hours upon transfection, cells were seeded in 96-well plates and 24 h later treated with different concentrations of PTX. Data were analyzed by two-way analysis of variance (ANOVA) with Šídák’s multiple comparisons test, with a single pooled variance; ns, not significant; *P  30 cells (n = 2). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Fig. S3. The CMSC protein liprin-β1 loses its organization upon KANK2, but not upon talin2 knockdown. (A, B) Forty-eight hours after transfection with talin2 or KANK2-specific siRNA, MDA-MB-435S cells were fixed with methanol and stained with anti-liprin-β1 antibody followed by Alexa-Flour 546-conjugated antibody, anti-talin2 antibody followed by Alexa-Fluor IgG2b 488-conjugated antibody (green) and anti-KANK2 antibody followed by Alexa-Flour 647-conjugated antibody and IRM images were taken. Analysis was performed using TCS SP8 Leica. Scale bar = 10 µm. Fig. S4. Verification of the MDA-MB-435S and 3αV cell model expressing fluorescently labelled EB3. (A, B) Clone 3αV-EB3 shows decreased expression of talin1, integrin β5 and KANK2 as compared to MDA-MB-435S-EB3 cells. Forty-eight hours after seeding cells were methanol fixed and stained with anti-talin1 followed by Alexa-Fluor 546-conjugated antibody (red), anti-KANK2 antibody or anti-β5 antibody followed by Alexa-Fluor 647-conjugated antibody (magenta) and IRM images were taken. Analysis was performed using TCS SP8 Leica. Scale bar = 10 µm. (C) Clone 3αV-EB3 demonstrates increased sensitivity to PTX as compared to parental MDA-MB-435S-EB3 cells. Twenty-four hours upon seeding in 96-well plates cells were treated with different concentrations of PTX. Cytotoxicity was measured by MTT assay. Data were analyzed by two-way analysis of variance (ANOVA) with Šídák’s multiple comparisons test, with a single pooled variance; ns denotes not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 (n = 3). Fig. S5. Knockdown of KANK2 increases talin2-positive FAs size. (A) Forty-eight hours after transfection with either control or KANK2-specific siRNA, MDA-MB-435S cells were methanol fixed and stained with anti-talin2 antibody followed by Alexa-Fluor IgG2b 488-conjugated antibody (green) and anti-KANK2 antibody followed by Alexa-Fluor 647-conjugated antibody (magenta) IRM images were taken. (B) Quantification of data presented in (a). Scatter plot with median marked in red represents measurements of ≥ 30 cells, (n = 2). Data were analyzed by unpaired Student’s t-test. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Fig. S6. Full images of the blots in Fig. 2A. Images were obtained using Uvitec Alliance Q9 mini, which directly scanned membranes developed with ECL reagents. Fig. S7. Full images of the blots in Fig. 3C. Images were obtained using Uvitec Alliance Q9 mini, which directly scanned membranes developed with ECL reagents

Identification of folate receptor α (FRα) binding oligopeptides and their evaluation for targeted virotherapy applications

Oncolytic virotherapies (OV) based on human adenoviral (HAdV) vectors hold significant promise for the treatment of advanced ovarian cancers where local, intraperitoneal delivery to tumour metastases is feasible, bypassing many complexities associated with intravascular delivery. The efficacy of HAdV-C5-based OV is hampered by a lack of tumour selectivity, where the primary receptor, hCAR, is commonly downregulated during malignant transformation. Conversely, folate receptor alpha (FRα) is highly expressed on ovarian cancer cells, providing a compelling target for tumour selective delivery of virotherapies. Here, we identify high-affinity FRα-binding oligopeptides for genetic incorporation into HAdV-C5 vectors. Biopanning identified a 12-mer linear peptide, DWSSWVYRDPQT, and two 7-mer cysteine-constrained peptides, CIGNSNTLC and CTVRTSAEC that bound FRα in the context of the phage particle. Synthesised lead peptide, CTVRTSAEC, bound specifically to FRα and could be competitively inhibited with folic acid. To assess the capacity of the elucidated FRα-binding oligopeptides to target OV to FRα, we genetically incorporated the peptides into the HAdV-C5 fiber-knob HI loop including in vectors genetically ablated for hCAR interactions. Unfortunately, the recombinant vectors failed to efficiently target transduction via FRα due to defective intracellular trafficking following entry via FRα, indicating that whilst the peptides identified may have potential for applications for targeted drug delivery, they require additional refinement for targeted virotherapy applications

KANK2 links αVβ5 focal adhesions to microtubules and regulates sensitivity to microtubule poisons and cell migration

Integrins are heterodimeric glycoproteins that bind cells to extracellular matrix. Upon integrin clustering, multimolecular integrin adhesion complexes (IACs) are formed, creating links to the cell cytoskeleton. We have previously observed decreased cell migration and increased sensitivity to microtubule (MT) poisons, paclitaxel and vincristine, in the melanoma cell line MDA-MB-435S upon transfection with integrin alpha V specific siRNA, suggesting a link between adhesion and drug sensitivity. To elucidate the underlying mechanism, we determined alpha V-dependent changes in IAC composition. Using mass spectrometry (MS)-based proteomics, we analyzed the components of isolated IACs of MDA-MB-435S cells and two MDA-MB-435S-derived integrin alpha V specific shRNA-expressing cell clones with decreased expression of integrin alpha V. MS analysis showed that cells preferentially use integrin alpha V beta 5 for the formation of IACs. The differential analysis between MDA-MB-435S cells and clones with decreased expression of integrin alpha V identified key components of integrin alpha V beta 5 adhesion complexes as talins 1 and 2, alpha-actinins 1 and 4, filamins A and B, plectin and vinculin. The data also revealed decreased levels of several components of the cortical microtubule stabilization complex, which recruits MTs to adhesion sites (notably liprins alpha and beta, ELKS, LL5b, MACF1, KANK1, and KANK2), following alpha V knockdown. KANK2 knockdown in MDA-MB-435S cells mimicked the effect of integrin alpha V knockdown and resulted in increased sensitivity to MT poisons and decreased migration. Taken together, we conclude that KANK2 is a key molecule linking integrin alpha V beta 5 IACs to MTs, and enabling the actin-MT crosstalk that is important for both sensitivity to MT poisons and cell migration

Additional file 8 of Talin2 and KANK2 functionally interact to regulate microtubule dynamics, paclitaxel sensitivity and cell migration in the MDA-MB-435S melanoma cell line

Additional file 8: Fig. S8. Still images of Additional file 2–Additional file 8: Movie S1–S6. Images were obtained using Image J manual tracking tool. Each arrow represent position of one microtubule tip through 104 s. Images were captured every 26 s